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Sample records for autophagic machinery activated

  1. Autophagic machinery activated by dengue virus enhances virus replication

    SciTech Connect

    Lee, Y.-R.; Lei, H.-Y.; Liu, M.-T.; Wang, J.-R.; Chen, S.-H.; Jiang-Shieh, Y.-F.; Lin, Y.-S.; Yeh, T.-M.; Liu, C.-C.; Liu, H.-S.

    2008-05-10

    Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.

  2. The Autophagic Machinery in Viral Exocytosis

    PubMed Central

    Münz, Christian

    2017-01-01

    The discovery of the molecular machinery of autophagy, namely Atg proteins, was awarded with the Nobel prize in physiology and medicine to Yoshinori Ohsumi in 2016. While this machinery was originally identified by its ability to allow cells to survive starvation via lysosomal degradation to recycle cellular components, it has recently become apparent that it also is used by cells to secrete cytoplasmic constituents. Furthermore, viruses have learned to use this Atg supported exocytosis to exit cells, acquire envelopes in the cytosol and select lipids into their surrounding membranes that might allow for increased robustness of their virions and altered infection behavior. Along these lines, picornaviruses exit infected cells in packages wrapped into autophagic membranes, herpesviruses recruit autophagic membranes into their envelopes and para- as well as orthomyxoviruses redirect autophagic membranes to the cell membrane, which increases the robustness of their envelope that they acquire at this site. These recent findings open a new exciting field on the regulation of degradation vs. release of autophagic membranes and will be discussed in this minireview. PMID:28270807

  3. The Autophagic Machinery in Enterovirus Infection.

    PubMed

    Lai, Jeffrey K F; Sam, I-Ching; Chan, Yoke Fun

    2016-01-27

    The Enterovirus genus of the Picornaviridae family comprises many important human pathogens, including polioviruses, rhinovirus, enterovirus A71, and enterovirus D68. They cause a wide variety of diseases, ranging from mild to severe life-threatening diseases. Currently, no effective vaccine is available against enteroviruses except for poliovirus. Enteroviruses subvert the autophagic machinery to benefit their assembly, maturation, and exit from host. Some enteroviruses spread between cells via a process described as autophagosome-mediated exit without lysis (AWOL). The early and late phases of autophagy are regulated through various lipids and their metabolizing enzymes. Some of these lipids and enzymes are specifically regulated by enteroviruses. In the present review, we summarize the current understanding of the regulation of autophagic machinery by enteroviruses, and provide updates on recent developments in this field.

  4. The Autophagic Machinery in Enterovirus Infection

    PubMed Central

    Lai, Jeffrey K. F.; Sam, I-Ching; Chan, Yoke Fun

    2016-01-01

    The Enterovirus genus of the Picornaviridae family comprises many important human pathogens, including polioviruses, rhinovirus, enterovirus A71, and enterovirus D68. They cause a wide variety of diseases, ranging from mild to severe life-threatening diseases. Currently, no effective vaccine is available against enteroviruses except for poliovirus. Enteroviruses subvert the autophagic machinery to benefit their assembly, maturation, and exit from host. Some enteroviruses spread between cells via a process described as autophagosome-mediated exit without lysis (AWOL). The early and late phases of autophagy are regulated through various lipids and their metabolizing enzymes. Some of these lipids and enzymes are specifically regulated by enteroviruses. In the present review, we summarize the current understanding of the regulation of autophagic machinery by enteroviruses, and provide updates on recent developments in this field. PMID:26828514

  5. Regulation of the autophagic machinery in human neutrophils.

    PubMed

    Mitroulis, Ioannis; Kourtzelis, Ioannis; Kambas, Konstantinos; Rafail, Stavros; Chrysanthopoulou, Akrivi; Speletas, Matthaios; Ritis, Konstantinos

    2010-05-01

    The induction of the autophagy machinery, a process for the catabolism of cytosolic proteins and organelles, constitutes a crucial mechanism in innate immunity. However, the involvement of autophagy in human neutrophils and the possible inducers of this process have not been completely elucidated. In this study, the induction of autophagy was examined in human neutrophils treated with various activators and detected by the formation of acidified autophagosomes through monodansylcadaverine staining and via LC-3B conversion screened by immunoblotting and immunofluorescence confocal microscopy. In addition, the expression of the ATG genes was assessed by real-time RT-PCR. We provide evidence that autophagy is implicated in human neutrophils in both a phagocytosis-independent (rapamycin, TLR agonists, PMA) and phagocytosis (Escherichia coli)-dependent initiation manner. ROS activation is a positive mechanism for autophagy induction in the case of PMA, TLR activation and phagocytosis. Furthermore, LC3B gene expression was uniformly upregulated, indicating a transcriptional level of regulation for the autophagic machinery. This study provides a stepping stone toward further investigation of autophagy in neutrophil-driven inflammatory disorders.

  6. Chloroquine synergizes sunitinib cytotoxicity via modulating autophagic, apoptotic and angiogenic machineries.

    PubMed

    Abdel-Aziz, Amal Kamal; Shouman, Samia; El-Demerdash, Ebtehal; Elgendy, Mohamed; Abdel-Naim, Ashraf B

    2014-06-25

    Tyrosine kinases play a pivotal role in oncogenesis. Although tyrosine kinase inhibitors as sunitinib malate are used in cancer therapy, emerging studies report compromised cytotoxicity when used as monotherapy and thus combinations with other anti-cancer agents is recommended. Chloroquine is a clinically available anti-malarial agent which has been shown to exhibit anti-cancer activity. In the current study, we questioned whether chloroquine can modulate sunitinib cytotoxicity. We found that chloroquine synergistically augmented sunitinib cytotoxicity on human breast (MCF-7 and T-47D), cervical (Hela), colorectal (Caco-2 and HCT116), hepatocellular (HepG2), laryngeal (HEp-2) and prostate (PC3) cancer cell lines as indicated by combination and concentration reduction indices. These results were also consistent with that of Ehrlich ascites carcinoma (EAC) Swiss albino mice models as confirmed by tumor volume, weight, histopathological examination and PCNA expression. Sunitinib induced autophagy via upregulating beclin-1 expression which was blocked by chloroquine as evidenced by accumulated SQTSM1/p62 level. Furthermore, chloroquine augmented sunitinib-induced apoptosis by decreasing survivin level and increasing caspase 3 activity. Chloroquine also enhanced the antiangiogenic capacity of sunitinib as indicated by decreased CD34 expression and peritoneal/skin angiogenesis. Sunitinib when combined with chloroquine also increased reactive nitrogen species production via increasing inducible nitric oxide synthase expression and nitric oxide level whilst reduced reactive oxygen species production by increasing GSH level, activities of glutathione peroxidase and catalase and reducing lipid peroxides compared to sunitinib-only treated group. Taken together, these findings suggest that chloroquine enhanced sunitinib cytotoxicity in a synergistic manner via inducing apoptosis while switching off autophagic and angiogenic machineries. Nevertheless, further studies are

  7. The hippocampal autophagic machinery is depressed in the absence of the circadian clock protein PER1 that may lead to vulnerability during cerebral ischemia.

    PubMed

    Rami, Abdelhaq; Fekadu, Julia; Rawashdeh, Oliver

    2017-06-18

    Autophagy is an intracellular bulk self-degrading process in which cytoplasmic contents of abnormal proteins and excess or damaged organelles are sequestered into autophagosomes, and degraded upon fusion with lysosomes. Although autophagy is generally considered to be pro-survival, it also functions in cell death processes. We recently reported on the hippocampal higher vulnerability to cerebral ischemia in mice lacking the circadian clock protein PERIOD1 (PER1), a phenomenon we found to be linked to a PER1-dependent modulation of the expression patterns of apoptotic/autophagic markers. To exclude the contribution of vascular or glial factors to the innate vulnerability of Per1 knockout-mice (Per1-/--mice) to cerebral ischemia in vivo, we compared the autophagic machinery between primary hippocampal cultures from wild-type (WT)- and Per1-/--mice, using the lipophilic macrolide antibiotic, Rapamycin to induce autophagy. Development of autophagy in WT cells involved an increased LC3-II-to-LC3-I ratio (microtubule-associated protein 1 light chain 3) and an overall increase in the level of LC3-II. In addition, immunostaining of LC3 in WT cells revealed the typical transformation of LC3 localization from a diffused staining to a dot- and ring-like pattern. In contrast, Per1-/--hippocampal cells were resistant to Rapamycin induced alterations of autophagy hallmarks. Our in vitro data suggests that basal activity of autophagy seems to be modulated by PER1, and confirms the in vivo data by showing that the autophagic machinery is depressed in Per1-/--hippocampal neurons.The implication of both autophagy and circadian dysfunction in the pathogenesis of cerebral ischemia suggests that a functional connection between the two processes may exist. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. The tyrosine kinase inhibitor, sunitinib malate, induces cognitive impairment in vivo via dysregulating VEGFR signaling, apoptotic and autophagic machineries.

    PubMed

    Abdel-Aziz, Amal Kamal; Mantawy, Eman M; Said, Riham Soliman; Helwa, Reham

    2016-09-01

    Chemobrain refers to a cluster of cognitive deficits which affects almost 4-75% of chemotherapy-treated cancer patients. Sunitinib, an FDA-approved multityrosine kinase inhibitor, is currently used in treating different types of tumors. Despite being regarded as targeted therapy which blunts sustained angiogenesis in cancer milieu through inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signaling, the latter has a cardinal role in cognition. Recent clinical reports warned that sunitinib adversely affected memory processing in cancer patients. Nevertheless, the underlying mechanisms have not been investigated yet. Hence, we explored the impact of a clinically relevant dose of sunitinib on memory processing in vivo and questioned the implication of VEGFR2 signaling, autophagy and apoptosis. Strikingly, sunitinib preferentially impaired spatial cognition as evidenced in Morris water maze, T-maze and passive avoidance task. Consistently, sunitinib degenerated cortical and hippocampal neurons as assessed by histopathological examination and toluidine blue staining. Ultrastructural examination also depicted chromatin condensation, mitochondrial damage and accumulated autophagosomes. Digging deeper, central VEGF/VEGFR2/mTOR signaling was robustly suppressed. Besides, sunitinib boosted cortical and hippocampal p53 and executioner caspase-3 and decreased nuclear factor kappa B and Bcl-2 levels promoting apoptotic cell death. It also profoundly impeded neuronal autophagic flux as shown by decreased beclin-1 and Atg5 and increased p62/SQTSM1 levels. To our knowledge, this is the first study to provide molecular insights into sunitinib-induced chemofog where impeded VEGFR2 signaling and autophagic and hyperactivated apoptotic machineries act in neurodegenerative concert. Importantly, our findings shed light on potential therapeutic strategies to be exploited in the management of sunitinib-induced chemobrain. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. BH3 mimetics activate multiple pro-autophagic pathways.

    PubMed

    Malik, S A; Orhon, I; Morselli, E; Criollo, A; Shen, S; Mariño, G; BenYounes, A; Bénit, P; Rustin, P; Maiuri, M C; Kroemer, G

    2011-09-15

    The BH3 mimetic ABT737 induces autophagy by competitively disrupting the inhibitory interaction between the BH3 domain of Beclin 1 and the anti-apoptotic proteins Bcl-2 and Bcl-X(L), thereby stimulating the Beclin 1-dependent allosteric activation of the pro-autophagic lipid kinase VPS34. Here, we examined whether ABT737 stimulates other pro-autophagic signal-transduction pathways. ABT737 caused the activating phosphorylation of AMP-dependent kinase (AMPK) and of the AMPK substrate acetyl CoA carboxylase, the activating phosphorylation of several subunits of the inhibitor of NF-κB (IκB) kinase (IKK) and the hyperphosphorylation of the IKK substrate IκB, inhibition of the activity of mammalian target of rapamycin (mTOR) and consequent dephosphorylation of the mTOR substrate S6 kinase. In addition, ABT737 treatment dephosphorylates (and hence likewise inhibits) p53, glycogen synthase kinase-3 and Akt. All these effects were shared by ABT737 and another structurally unrelated BH3 mimetic, HA14-1. Functional experiments revealed that pharmacological or genetic inhibition of IKK, Sirtuin and the p53-depleting ubiquitin ligase MDM2 prevented ABT737-induced autophagy. These results point to unexpected and pleiotropic pro-autophagic effects of BH3 mimetics involving the modulation of multiple signalling pathways.

  10. Modulation of autophagic activity by extracellular pH.

    PubMed

    Xu, Teng; Su, Hang; Ganapathy, Suthakar; Yuan, Zhi-Min

    2011-11-01

    Reprogramming energy metabolism from oxidative phosphorylation to aerobic glycolysis, a common feature of human cancer, is associated with a relative acidic tumor microenvironment which can sometimes be further accentuated by hypoxia operating within most solid tumors. We found that alteration of extracellular pH induces marked and rapid changes of autophagic activity. Interestingly, acidic and basic conditions induced completely opposite effect on autophagy, with its activity suppressed at lower pH whereas stimulated at higher pH. Gene knockdown experiments indicated that pH induced-autophagy requires Beclin 1, Vps34 and Atg5, key components of the autophagy pathway. Of note, an acidic condition not only inhibits the basal but also blocks the starvation-induced autophagy activity. Significantly, examination of different areas of tumor mass revealed a lower autophagic activity within the inner region than the outer region. These findings have important implications on the connections between autophagy and cancer as well as a wide range of other physiological and pathological processes.

  11. Active Vibration Dampers For Rotating Machinery

    NASA Technical Reports Server (NTRS)

    Kascack, Albert F.; Ropchock, John J.; Lakatos, Tomas F.; Montague, Gerald T.; Palazzolo, Alan; Lin, Reng Rong

    1994-01-01

    Active dampers developed to suppress vibrations in rotating machinery. Essentially feedback control systems and reciprocating piezoelectric actuators. Similar active damper containing different actuators described in LEW-14488. Concept also applicable to suppression of vibrations in stationary structures subject to winds and earthquakes. Active damper offers adjustable suppression of vibrations. Small and lightweight and responds faster to transients.

  12. Active Vibration Dampers For Rotating Machinery

    NASA Technical Reports Server (NTRS)

    Kascack, Albert F.; Ropchock, John J.; Lakatos, Tomas F.; Montague, Gerald T.; Palazzolo, Alan; Lin, Reng Rong

    1994-01-01

    Active dampers developed to suppress vibrations in rotating machinery. Essentially feedback control systems and reciprocating piezoelectric actuators. Similar active damper containing different actuators described in LEW-14488. Concept also applicable to suppression of vibrations in stationary structures subject to winds and earthquakes. Active damper offers adjustable suppression of vibrations. Small and lightweight and responds faster to transients.

  13. Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle

    PubMed Central

    Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

    2016-01-01

    Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis. PMID:27213594

  14. Autophagic flux is highly active in early mitosis and differentially regulated throughout the cell cycle.

    PubMed

    Li, Zhiyuan; Ji, Xinmiao; Wang, Dongmei; Liu, Juanjuan; Zhang, Xin

    2016-06-28

    Mitosis is a fast process that involves dramatic cellular remodeling and has a high energy demand. Whether autophagy is active or inactive during the early stages of mitosis in a naturally dividing cell is still debated. Here we aimed to use multiple assays to resolve this apparent discrepancy. Although the LC3 puncta number was reduced in mitosis, the four different cell lines we tested all have active autophagic flux in both interphase and mitosis. In addition, the autophagic flux was highly active in nocodazole-induced, double-thymidine synchronization released as well as naturally occurring mitosis in HeLa cells. Multiple autophagy proteins are upregulated in mitosis and the increased Beclin-1 level likely contributes to the active autophagic flux in early mitosis. It is interesting that although the autophagic flux is active throughout the cell cycle, early mitosis and S phase have relatively higher autophagic flux than G1 and late G2 phases, which might be helpful to degrade the damaged organelles and provide energy during S phase and mitosis.

  15. Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

    PubMed Central

    Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Kito, Seiji; Minami, Naojiro; Kubota, Toshiro; Sato, Ken; Kokubo, Toshiaki

    2014-01-01

    Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality. PMID:24681842

  16. ATP Release from Dying Autophagic Cells and Their Phagocytosis Are Crucial for Inflammasome Activation in Macrophages

    PubMed Central

    Ayna, Gizem; Krysko, Dmitri V.; Kaczmarek, Agnieszka; Petrovski, Goran; Vandenabeele, Peter; Fésüs, László

    2012-01-01

    Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P2X7 purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses. PMID:22768222

  17. HDAC6 activity is not required for basal autophagic flux in metastatic prostate cancer cells

    PubMed Central

    Watson, Gregory W; Wickramasekara, Samanthi; Fang, Yufeng; Maier, Claudia S; Williams, David E; Dashwood, Roderick H; Perez, Viviana I

    2015-01-01

    Histone deacetylase 6 is a multifunctional lysine deacetylase that is recently emerging as a central facilitator of response to stress and may play an important role in cancer cell proliferation. The histone deacetylase 6-inhibitor tubacin has been shown to slow the growth of metastatic prostate cancer cells and sensitize cancer cells to chemotherapeutic agents. However, the proteins histone deacetylase 6 interacts with, and thus its role in cancer cells, remains poorly characterized. Histone deacetylase 6 deacetylase activity has recently been shown to be required for efficient basal autophagic flux. Autophagy is often dysregulated in cancer cells and may confer stress resistance and allow for cell maintenance and a high proliferation rate. Tubacin may therefore slow cancer cell proliferation by decreasing autophagic flux. We characterized the histone deacetylase 6-interacting proteins in LNCaP metastatic prostate cancer cells and found that histone deacetylase 6 interacts with proteins involved in several cellular processes, including autophagy. Based on our interaction screen, we assessed the impact of the histone deacetylase 6-inhibitor tubacin on autophagic flux in two metastatic prostate cancer cell lines and found that tubacin does not influence autophagic flux. Histone deacetylase 6 therefore influences cell proliferation through an autophagy-independent mechanism. PMID:26643866

  18. Featured article: autophagic activation with nimotuzumab enhanced chemosensitivity and radiosensitivity of esophageal squamous cell carcinoma.

    PubMed

    Song, Haizhu; Pan, Banzhou; Yi, Jun; Chen, Longbang

    2014-05-01

    Chemotherapy and radiotherapy are two indispensible methods for esophageal squamous cell carcinoma (ESCC), especially for those recurring and metastatic ones, but therapeutic toxicity remains a major problem to overcome. In the present study, the potential therapeutic value of nimotuzumab (an antiepidermal growth factor receptor [EGFR] monoclonal antibody) in combination with chemotherapy and radiotherapy was evaluated on Eca109 and TE-1 ESCC cells, with high and low expression of EGFR, respectively. It was shown that nimotuzumab enhanced the sensitivity of Eca109 cells to other cytotoxic agents (paclitaxel and cis-platinum) and X-ray radiation, and the cytotoxicity was associated with increased autophagy. Conversely, the chemo- and radio-sensitivity of TE-1 cells showed no improvement with addition of nimotuzumab, but could be increased by combining with rapamycin, an autophagy inducer. Therefore, it was concluded that autophagic activation mediated by nimotuzumab could promote autophagic cell death and produce additive antitumor effects.

  19. Normal autophagic activity in macrophages from mice lacking Gαi3, AGS3, or RGS19.

    PubMed

    Vural, Ali; McQuiston, Travis J; Blumer, Joe B; Park, Chung; Hwang, Il-Young; Williams-Bey, Yolanda; Shi, Chong-Shan; Ma, Dzwokai Zach; Kehrl, John H

    2013-01-01

    In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs Gαi3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-), Gpsm1(-/-), or Rgs19(-/-) mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-), and Gpsm1(-/-) macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered Gαi nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked Gαi3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while Gαi signaling may impact autophagy in some cell types it does not in macrophages.

  20. Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers

    PubMed Central

    Law, Betty Yuen Kwan; Mok, Simon Wing Fai; Chan, Wai Kit; Xu, Su Wei; Wu, An Guo; Yao, Xiao Jun; Wang, Jing Rong; Liu, Liang; Wong, Vincent Kam Wai

    2016-01-01

    Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK. PMID:26811496

  1. Hydroxychloroquine modulates metabolic activity and proliferation and induces autophagic cell death of human dermal fibroblasts.

    PubMed

    Ramser, Bettina; Kokot, Agatha; Metze, Dieter; Weiss, Nina; Luger, Thomas A; Böhm, Markus

    2009-10-01

    Hydroxychloroquine (HCQ) is a commonly used therapeutic agent in skin disorders. Some reports also suggest that HCQ can be useful in fibroblastic diseases of the skin. Here, we investigated the effects of HCQ in human dermal fibroblasts (HDFs). HCQ significantly reduced the metabolic activity and suppressed cell proliferation (IC(50) = approximately 30 microM) of HDFs. The antiproliferative effect of HCQ was associated with decreased activation of the extracellular signal-regulated kinases 1/2 but not with inhibition of the mammalian target of the rapamycin pathway or with dephosphorylation of Akt. HCQ induced a distinct type of cell death in HDFs, characterized by surface exposure of phosphatidylserine but a lack of morphological signs of apoptosis and absence of DNA fragmentation. The HCQ-treated HDFs instead showed autophagic vacuoles with double membranes and digested organelle content. These vacuoles showed light-chain 3 immunostaining, in accordance with increased protein amounts of this autophagy marker. Induction of autophagic cell death by HCQ was also paralleled by increased expression of Beclin-1, a key regulator of autophagy. Our findings indicate that HDFs are target cells of HCQ and form a rationale on the basis of which the in vivo effects of antimalarials can be studied in patients with aberrant fibroblast function.

  2. Assessing the Catalytic Activity of Transglutaminases in the Context of Autophagic Responses.

    PubMed

    D'Eletto, M; Farrace, M G; Piacentini, M; Rossin, F

    2017-01-01

    The human transglutaminases (TGases) are a widely distributed and peculiar group of enzymes that catalyze the posttranslational modification of proteins by the formation of isopeptide bonds. Tissue or type 2 transglutaminase (TG2) represents the most ubiquitous isoform belonging to TGases family. The vast array of biochemical functions catalyzed by TG2 distinguishes it from the other members of the TGase family. In the presence of high calcium levels TG2 catalyzes a vast array of protein posttranslational modifications, including protein-protein cross-linking, incorporation of primary amines into proteins, as well as glutamine deamination. In the last few years, it has become evident that TG2 is involved in the final maturation of autolysosomes. The TG2 regulation of autophagy occurs by its transamidating activity and its inhibition results in the intracellular increase of ubiquitinated protein aggregates. In this chapter, we describe the methods used in our laboratories to assess the catalytic activity of TG2 in the autophagic process.

  3. Activation of the Keap1/Nrf2 stress response pathway in autophagic vacuolar myopathies.

    PubMed

    Duleh, Steve; Wang, Xianhong; Komirenko, Allison; Margeta, Marta

    2016-10-31

    Nrf2 (nuclear factor [erythroid-derived 2]-like 2; the transcriptional master regulator of the antioxidant stress response) is regulated through interaction with its cytoplasmic inhibitor Keap1 (Kelch-like ECH-associated protein 1), which under basal conditions targets Nrf2 for proteasomal degradation. Sequestosome 1 (SQSTM1)/p62-a multifunctional adapter protein that accumulates following autophagy inhibition and can serve as a diagnostic marker for human autophagic vacuolar myopathies (AVMs)-was recently shown to compete with Nrf2 for Keap1 binding, resulting in activation of the Nrf2 pathway. In this study, we used 55 human muscle biopsies divided into five groups [normal control, hydroxychloroquine- or colchicine-treated non-AVM control, hydroxychloroquine- or colchicine-induced toxic AVM, polymyositis, and inclusion body myositis (IBM)] to evaluate whether Keap1-SQSTM1 interaction led to increased Nrf2 signaling in human AVMs. In toxic AVMs and IBM, but not in control muscle groups or polymyositis, Keap1 antibody labeled sarcoplasmic protein aggregates that can be used as an alternate diagnostic marker for both AVM types; these Keap1-positive aggregates were co-labeled with the antibody against SQSTM1 but not with the antibody against autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). In human AVM muscle, sequestration of Keap1 into the SQSTM1-positive protein aggregates was accompanied by an increase in mRNA and protein levels of Nrf2 target genes; similarly, treatment of differentiated C2C12 myotubes with autophagy inhibitor chloroquine led to an increase in the nuclear Nrf2 protein level and an increase in expression of the Nrf2-regulated genes. Taken together, our findings demonstrate that Nrf2 signaling is upregulated in autophagic muscle disorders and raise the possibility that autophagy disruption in skeletal muscle leads to dysregulation of cellular redox homeostasis.

  4. Hierarchal Autophagic Divergence of Hematopoietic System*

    PubMed Central

    Cao, Yan; Zhang, Suping; Yuan, Na; Wang, Jian; Li, Xin; Xu, Fei; Lin, Weiwei; Song, Lin; Fang, Yixuan; Wang, Zhijian; Wang, Zhen; Zhang, Han; Zhang, Yi; Zhao, Wenli; Hu, Shaoyan; Zhang, Xueguang; Wang, Jianrong

    2015-01-01

    Autophagy is integral to hematopoiesis and protects against leukemogenesis. However, the fundamentals of the required molecular machinery have yet to be fully explored. Using conditional mouse models to create strategic defects in the hematopoietic hierarchy, we have shown that recovery capacities in stem cells and somatic cells differ if autophagy is impaired or flawed. An in vivo Atg7 deletion in hematopoietic stem cells completely ablates the autophagic response, leading to irreversible and ultimately lethal hematopoiesis. However, while no adverse phenotype is manifested in vivo by Atg7-deficient myeloid cells, they maintain active autophagy that is sensitive to brefeldin A, an inhibitor targeting Golgi-derived membranes destined for autophagosome formation in alternative autophagy. Removing Rab9, a key regulatory protein, in alternative autophagy, disables autophagy altogether in Atg7-deficient macrophages. Functional analysis indicates that ATG7-dependent canonical autophagy is physiologically active in both hematopoietic stem cells and in terminally differentiated hematopoietic cells; however, only terminally differentiated cells such as macrophages are rescued by alternative autophagy if canonical autophagy is ineffective. Thus, it appears that hematopoietic stem cells rely solely on ATG7-dependent canonical autophagy, whereas terminally differentiated or somatic cells are capable of alternative autophagy in the event that ATG7-mediated autophagy is dysfunctional. These findings offer new insight into the transformational trajectory of hematopoietic stem cells, which in our view renders the autophagic machinery in stem cells more vulnerable to disruption. PMID:26245898

  5. Endurance exercise training induces fat depot-specific differences in basal autophagic activity

    SciTech Connect

    Tanaka, Goki; Kato, Hisashi; Izawa, Tetsuya

    2015-10-23

    The purpose of this study was to uncover the effect of exercise training on the expression of autophagy marker proteins in epididymal white adipose tissue (eWAT), inguinal WAT (iWAT), and the stromal vascular fraction (SVF) collected from eWAT. Male Wistar rats aged 4–5 weeks were randomly divided into two groups, sedentary control (n = 7) and exercise-trained (n = 7). Rats in the exercise-trained group were exercised on a treadmill set at a 5° incline 5 days/week for 9 weeks. We determined that the expression levels of an autophagosome-associating form of microtubule-associated protein 1 light chain 3 (LC3)-II and of p62 were significantly higher in eWAT from exercise-trained than from control rats, while those of adipose-specific deletion of autophagy-related protein (ATG7) and lysosomal-associated membrane protein type 2A (LAMP2a) showed no difference between groups. However, in iWAT, the expression levels of LC3-II and ATG7 were significantly higher in exercise-trained than in control rats. The expression of p62 was highly correlated with that of peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and lipid metabolism, in both WAT types (eWAT, r = 0.856, P < 0.05; iWAT, r = 0.762, P < 0.05), whereas LC3-II and PPARγ levels were highly correlated in eWAT (r = 0.765, P < 0.05) but not in iWAT (r = −0.306, ns). In SVF, the expression levels of LC3II, ATG7, and LAMP2a were significantly higher in exercise-trained than in control rats. These results suggest that exercise training suppresses basal autophagy activity in eWAT, but that this activity is enhanced in iWAT and SVF collected from eWAT. Thus, the adaptation of basal autophagic activity following exercise training exhibits fat depot-specific differences. - Highlights: • Autophagy has been associated with obesity and associated diseases. • We examined exercise-associated rat white adipose tissue (WAT) autophagy markers. • Exercise increased

  6. Elements of active vibration control for rotating machinery

    NASA Technical Reports Server (NTRS)

    Ulbrich, Heinz

    1990-01-01

    The success or failure of active vibration control is determined by the availability of suitable actuators, modeling of the entire system including all active elements, positioning of the actuators and sensors, and implementation of problem-adapted control concepts. All of these topics are outlined and their special problems are discussed in detail. Special attention is given to efficient modeling of systems, especially for considering the active elements. Finally, design methods for and the application of active vibration control on rotating machinery are demonstrated by several real applications.

  7. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

    PubMed

    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  8. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms

    PubMed Central

    Mansilla Pareja, Maria Eugenia; Colombo, Maria I.

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance. PMID:24137567

  9. AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation.

    PubMed

    Han, Xiaojuan; Tai, Haoran; Wang, Xiaobo; Wang, Zhe; Zhou, Jiao; Wei, Xiawei; Ding, Yi; Gong, Hui; Mo, Chunfen; Zhang, Jie; Qin, Jianqiong; Ma, Yuanji; Huang, Ning; Xiang, Rong; Xiao, Hengyi

    2016-06-01

    AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress-induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide-induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP-RFP-LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD(+) levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD(+) synthesis. In addition, the mechanistic relationship of autophagic flux and NAD(+) synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress-induced senescence by improving autophagic flux and NAD(+) homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD(+) homeostasis, and it is also valuable in the development of innovative strategies to combat aging.

  10. Autophagic Signaling and Proteolytic Enzyme Activity in Cardiac and Skeletal Muscle of Spontaneously Hypertensive Rats following Chronic Aerobic Exercise

    PubMed Central

    McMillan, Elliott M.; Paré, Marie-France; Baechler, Brittany L.; Graham, Drew A.; Rush, James W. E.; Quadrilatero, Joe

    2015-01-01

    Hypertension is a cardiovascular disease associated with deleterious effects in skeletal and cardiac muscle. Autophagy is a degradative process essential to muscle health. Acute exercise can alter autophagic signaling. Therefore, we aimed to characterize the effects of chronic endurance exercise on autophagy in skeletal and cardiac muscle of normotensive and hypertensive rats. Male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were assigned to a sedentary condition or 6 weeks of treadmill running. White gastrocnemius (WG) of hypertensive rats had higher (p<0.05) caspase-3 and proteasome activity, as well as elevated calpain activity. In addition, skeletal muscle of hypertensive animals had elevated (p<0.05) ATG7 and LC3I protein, LAMP2 mRNA, and cathepsin activity, indicative of enhanced autophagic signaling. Interestingly, chronic exercise training increased (p<0.05) Beclin-1, LC3, and p62 mRNA as well as proteasome activity, but reduced (p<0.05) Beclin-1 and ATG7 protein, as well as decreased (p<0.05) caspase-3, calpain, and cathepsin activity. Left ventricle (LV) of hypertensive rats had reduced (p<0.05) AMPKα and LC3II protein, as well as elevated (p<0.05) p-AKT, p-p70S6K, LC3I and p62 protein, which collectively suggest reduced autophagic signaling. Exercise training had little effect on autophagy-related signaling factors in LV; however, exercise training increased (p<0.05) proteasome activity but reduced (p<0.05) caspase-3 and calpain activity. Our results suggest that autophagic signaling is altered in skeletal and cardiac muscle of hypertensive animals. Regular aerobic exercise can effectively alter the proteolytic environment in both cardiac and skeletal muscle, as well as influence several autophagy-related factors in skeletal muscle of normotensive and hypertensive rats. PMID:25799101

  11. Autophagic signaling and proteolytic enzyme activity in cardiac and skeletal muscle of spontaneously hypertensive rats following chronic aerobic exercise.

    PubMed

    McMillan, Elliott M; Paré, Marie-France; Baechler, Brittany L; Graham, Drew A; Rush, James W E; Quadrilatero, Joe

    2015-01-01

    Hypertension is a cardiovascular disease associated with deleterious effects in skeletal and cardiac muscle. Autophagy is a degradative process essential to muscle health. Acute exercise can alter autophagic signaling. Therefore, we aimed to characterize the effects of chronic endurance exercise on autophagy in skeletal and cardiac muscle of normotensive and hypertensive rats. Male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were assigned to a sedentary condition or 6 weeks of treadmill running. White gastrocnemius (WG) of hypertensive rats had higher (p<0.05) caspase-3 and proteasome activity, as well as elevated calpain activity. In addition, skeletal muscle of hypertensive animals had elevated (p<0.05) ATG7 and LC3I protein, LAMP2 mRNA, and cathepsin activity, indicative of enhanced autophagic signaling. Interestingly, chronic exercise training increased (p<0.05) Beclin-1, LC3, and p62 mRNA as well as proteasome activity, but reduced (p<0.05) Beclin-1 and ATG7 protein, as well as decreased (p<0.05) caspase-3, calpain, and cathepsin activity. Left ventricle (LV) of hypertensive rats had reduced (p<0.05) AMPKα and LC3II protein, as well as elevated (p<0.05) p-AKT, p-p70S6K, LC3I and p62 protein, which collectively suggest reduced autophagic signaling. Exercise training had little effect on autophagy-related signaling factors in LV; however, exercise training increased (p<0.05) proteasome activity but reduced (p<0.05) caspase-3 and calpain activity. Our results suggest that autophagic signaling is altered in skeletal and cardiac muscle of hypertensive animals. Regular aerobic exercise can effectively alter the proteolytic environment in both cardiac and skeletal muscle, as well as influence several autophagy-related factors in skeletal muscle of normotensive and hypertensive rats.

  12. IL-10 restricts dendritic cell (DC) growth at the monocyte-to-monocyte-derived DC interface by disrupting anti-apoptotic and cytoprotective autophagic molecular machinery.

    PubMed

    Martin, Carla; Espaillat, Mel Pilar; Santiago-Schwarz, Frances

    2015-12-01

    An evolving premise is that cytoprotective autophagy responses are essential to monocyte-macrophage differentiation. Whether autophagy functions similarly during the monocyte-to-dendritic cell (DC) transition is unclear. IL-10, which induces apoptosis in maturing human DCs, has been shown to inhibit starvation-induced autophagy in murine macrophage cell lines. Based on the strict requirement that Bcl-2-mediated anti-apoptotic processes are implemented during the monocyte-to-DC transition, we hypothesized that cytoprotective autophagy responses also operate at the monocyte-DC interface and that IL-10 inhibits both anti-apoptotic and cytoprotective autophagy responses at this critical juncture. In support of our premise, we show that levels of anti-apoptotic Bcl-2 and autophagy-associated LC3 and Beclin-1 proteins are coincidentally upregulated during the monocyte-to-DC transition. Autophagy was substantiated by increased autophagosome visualization after bafilomycin treatment. Moreover, the autophagy inhibitor 3-MA restricted DC differentiation by prompting apoptosis. IL-10 implemented apoptosis that was coincidentally associated with reduced levels of Bcl-2 and widespread disruption of the autophagic flux. During peak apoptosis, IL-10 produced the death of newly committed DCs. However, cells surviving the IL-10 apoptotic schedule were highly phagocytic macrophage-like cells displaying reduced capacity to stimulate allogeneic naïve T cells in a mixed leukocyte reaction, increased levels of LC3, and mature autophagosomes. Thus, IL-10's negative control of DC-driven adaptive immunity at the monocyte-DC interface includes disruption of coordinately regulated molecular networks involved in pro-survival autophagy and anti-apoptotic responses.

  13. The autophagy machinery restrains iNKT cell activation through CD1D1 internalization.

    PubMed

    Keller, Christian W; Loi, Monica; Ewert, Svenja; Quast, Isaak; Theiler, Romina; Gannagé, Monique; Münz, Christian; De Libero, Gennaro; Freigang, Stefan; Lünemann, Jan D

    2017-03-15

    Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT-cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4(+) T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4(+) T cell stimulation.

  14. Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia

    PubMed Central

    Liu, Hui; Li, Lu; Meng, Haitao; Qian, Qijun

    2013-01-01

    An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis. PMID:23765161

  15. Suppression of autophagic activation in the mouse uterus by estrogen and progesterone.

    PubMed

    Choi, Soyoung; Shin, Hyejin; Song, Haengseok; Lim, Hyunjung Jade

    2014-04-01

    Autophagy is a major cellular catabolic pathway tightly associated with cell survival. The involvement of autophagy in the prolonged survival of blastocysts in the uterus is well established, and it was assumed that ovarian steroid hormones - progesterone (P4) and estrogens - have important roles in the regulation of autophagy. However, information is scarce regarding whether these hormones regulate autophagy in certain hormone-responsive cellular systems. In this study, we investigated the effects of estrogen and P4 on autophagic response in the uteri of pregnant mice and in ovariectomized (OVX) mice treated with hormones. During pregnancy, autophagic response is high on days 1 and 2 when the uterus shows an inflammatory response to mating, but it subsides around the time of implantation. Dexamethasone treatment to day 1 pregnant mice reduced autophagy in the uterus. In OVX mouse uteri, estrogen or P4 reduces autophagic response within 6 h. Glycogen content in OVX uteri was increased by 3-methyladenine treatment, suggesting that autophagy is involved in glycogen breakdown in the hormone-deprived uterus. The classical nuclear receptor antagonists, ICI 182 780 or mifepristone, lead to the recovery of the autophagic response in OVX uteri. The suppression of autophagy by 17β-estradiol is inversely correlated with the accumulation of phospho-mouse target of rapamycin, and rapamycin treatment is moderately effective in the upregulation of autophagic response in OVX mouse uteri. Collectively, this study establishes that the uterine autophagy is induced in hormone-derived environment and is suppressed by hormone treatment. Uterine autophagy may have multiple functions as a responsive mechanism to acute inflammation and as an energy provider by breaking down glycogen under hormone deprivation.

  16. Effect of the pituitary adenylate cyclase-activating polypeptide on the autophagic activation observed in in vitro and in vivo models of Parkinson's disease.

    PubMed

    Lamine-Ajili, Asma; Fahmy, Ahmed M; Létourneau, Myriam; Chatenet, David; Labonté, Patrick; Vaudry, David; Fournier, Alain

    2016-04-01

    Parkinson's disease (PD) is a neurodegenerative disorder that leads to destruction of the midbrain dopaminergic (DA) neurons. This phenomenon is related to apoptosis and its activation can be blocked by the pituitary adenylate cyclase-activating polypeptide (PACAP). Growing evidence indicates that autophagy, a self-degradation activity that cleans up the cell, is induced during the course of neurodegenerative diseases. However, the role of autophagy in the pathogenesis of neuronal disorders is yet poorly understood and the potential ability of PACAP to modulate the related autophagic activation has never been significantly investigated. Hence, we explored the putative autophagy-modulating properties of PACAP in in vitro and in vivo models of PD, using the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively, to trigger alterations of DA neurons. In both models, following the toxin exposure, PACAP reduced the autophagic activity as evaluated by the production of LC3 II, the modulation of the p62 protein levels, and the formation of autophagic vacuoles. The ability of PACAP to inhibit autophagy was also observed in an in vitro cell assay by the blocking of the p62-sequestration activity produced with the autophagy inducer rapamycin. Thus, the results demonstrated that autophagy is induced in PD experimental models and that PACAP exhibits not only anti-apoptotic but also anti-autophagic properties.

  17. Detection of Apoptotic Versus Autophagic Cell Death by Flow Cytometry.

    PubMed

    Sica, Valentina; Maiuri, M Chiara; Kroemer, Guido; Galluzzi, Lorenzo

    2016-01-01

    Different modes of regulated cell death (RCD) can be initiated by distinct molecular machineries and their morphological manifestations can be difficult to discriminate. Moreover, cells responding to stress often activate an adaptive response centered around autophagy, and whether such a response is cytoprotective or cytotoxic cannot be predicted based on morphological parameters only. Molecular definitions are therefore important to understand various RCD subroutines from a mechanistic perspective. In vitro, various forms of RCD including apoptosis and autophagic cell death can be easily discriminated from each other with assays that involve chemical or pharmacological interventions targeting key components of either pathway. Here, we detail a straightforward method to discriminate apoptosis from autophagic cell death by flow cytometry, based on the broad-spectrum caspase inhibitor Z-VAD-fmk and the genetic inhibition of ATG5.

  18. Arsenic Inhibits Autophagic Flux, Activating the Nrf2-Keap1 Pathway in a p62-Dependent Manner

    PubMed Central

    Lau, Alexandria; Zheng, Yi; Tao, Shasha; Wang, Huihui; Whitman, Samantha A.; White, Eileen

    2013-01-01

    The Nrf2-Keap1 signaling pathway is a protective mechanism promoting cell survival. Activation of the Nrf2 pathway by natural compounds has been proven to be an effective strategy for chemoprevention. Interestingly, a cancer-promoting function of Nrf2 has recently been observed in many types of tumors due to deregulation of the Nrf2-Keap1 axis, which leads to constitutive activation of Nrf2. Here, we report a novel mechanism of Nrf2 activation by arsenic that is distinct from that of chemopreventive compounds. Arsenic deregulates the autophagic pathway through blockage of autophagic flux, resulting in accumulation of autophagosomes and sequestration of p62, Keap1, and LC3. Thus, arsenic activates Nrf2 through a noncanonical mechanism (p62 dependent), leading to a chronic, sustained activation of Nrf2. In contrast, activation of Nrf2 by sulforaphane (SF) and tert-butylhydroquinone (tBHQ) depends upon Keap1-C151 and not p62 (the canonical mechanism). More importantly, SF and tBHQ do not have any effect on autophagy. In fact, SF and tBHQ alleviate arsenic-mediated deregulation of autophagy. Collectively, these findings provide evidence that arsenic causes prolonged activation of Nrf2 through autophagy dysfunction, possibly providing a scenario similar to that of constitutive activation of Nrf2 found in certain human cancers. This may represent a previously unrecognized mechanism underlying arsenic toxicity and carcinogenicity in humans. PMID:23589329

  19. Enhanced autophagic activity of artocarpin in human hepatocellular carcinoma cells through improving its solubility by a nanoparticle system.

    PubMed

    Tzeng, Cheng-Wei; Tzeng, Wen-Sheng; Lin, Liang-Tzung; Lee, Chiang-Wen; Yen, Feng-Lin; Lin, Chun-Ching

    2016-05-15

    Hepatocellular carcinoma (HCC) is the most common liver cancer worldwide, with poor prognosis and resistance to chemotherapy. This gives novel cancer treatment methods an overwhelming significance. Natural products offer great resources of developing new and effective chemopreventive or chemotherapeutic agents. Artocarpus communis extracts and its active constituent, prenylated flavonoid artocarpin induce human hepatocellular carcinoma cell death. However, the poor water solubility drawbacks of artocarpin restrict its clinical application and bioavailability. This study developed the artocarpin nanoparticle system to overcome the poor water solubility drawbacks and investigated the improvement of therapeutic efficacy of artocarpin by adopting novel nanoparticle delivery strategy. Antiproliferative activity of artocarpin was evaluated by MTT assay. Cell morphology observation by microscope, DNA fragmentation assay, cell cycle analysis, Annexin V apoptosis cell staining, monodansylcadaverine and acridine orange staining and immunoblot analysis were used to evaluate the induction of autophagy by artocarpin. The determination of particle size, amorphous transformation, hydrogen-bond formation, yield, encapsulation efficiency and the solubility study were used to investigate the solubility enhancement mechanism of artocarpin. The present study demonstrates that the anticancer effect of artocarpin in HepG2 and PLC/PRF/5 hepatoma cells is mediated through the autophagic cell death mechanism. Results also demonstrated that artocarpin nanoparticles enhanced the solubility of artocarpin by reducing particle size, transforming high energy amorphous state, and forming hydrogen bond with excipients. Additionally, ArtN exhibited better autophagic cytotoxicity compared to free artocarpin. This work reveals the antihepatoma activity of artocarpin by inducing autophagic cell death and the improvement of therapeutic efficacy of artocarpin by adopting novel nanoparticle delivery

  20. Ceria nanoparticles stabilized by organic surface coatings activate the lysosome-autophagy system and enhance autophagic clearance.

    PubMed

    Song, Wensi; Soo Lee, Seung; Savini, Marzia; Popp, Lauren; Colvin, Vicki L; Segatori, Laura

    2014-10-28

    Cerium oxide nanoparticles (nanoceria) are widely used in a variety of industrial applications including UV filters and catalysts. The expanding commercial scale production and use of ceria nanoparticles have inevitably increased the risk of release of nanoceria into the environment as well as the risk of human exposure. The use of nanoceria in biomedical applications is also being currently investigated because of its recently characterized antioxidative properties. In this study, we investigated the impact of ceria nanoparticles on the lysosome-autophagy system, the main catabolic pathway that is activated in mammalian cells upon internalization of exogenous material. We tested a battery of ceria nanoparticles functionalized with different types of biocompatible coatings (N-acetylglucosamine, polyethylene glycol and polyvinylpyrrolidone) expected to have minimal effect on lysosomal integrity and function. We found that ceria nanoparticles promote activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and induce upregulation of genes of the lysosome-autophagy system. We further show that the array of differently functionalized ceria nanoparticles tested in this study enhance autophagic clearance of proteolipid aggregates that accumulate as a result of inefficient function of the lysosome-autophagy system. This study provides a mechanistic understanding of the interaction of ceria nanoparticles with the lysosome-autophagy system and demonstrates that ceria nanoparticles are activators of autophagy and promote clearance of autophagic cargo. These results provide insights for the use of nanoceria in biomedical applications, including drug delivery. These findings will also inform the design of engineered nanoparticles with safe and precisely controlled impact on the environment and the design of nanotherapeutics for the treatment of diseases with defective autophagic function and accumulation of lysosomal storage material.

  1. Antiproliferative, Apoptotic, and Autophagic Activity of Ranibizumab, Bevacizumab, Pegaptanib, and Aflibercept on Fibroblasts: Implication for Choroidal Neovascularization

    PubMed Central

    Lytvynchuk, Lyubomyr; Sergienko, Andrii; Lavrenchuk, Galina; Petrovski, Goran

    2015-01-01

    Purpose. Choroidal neovascularization (CNV) is one of the most common complications of retinal diseases accompanied by elevated secretion of vascular endothelial growth factor (VEGF). Intravitreal anti-VEGFs (ranibizumab, bevacizumab, pegaptanib, and aflibercept) can suppress neovascularization, decrease vascular permeability and CNV size, and, thereby, improve visual function. The antiproliferative, apoptotic, and autophagic effect of anti-VEGF drugs on fibroblasts found in CNVs has not been yet explored. Methods. Concentration-dependent cellular effects of the four anti-VEGFs were examined in L929 fibroblasts over a 5-day period. The cell survival, mitotic and polykaryocytic indices, the level of apoptosis and autophagy, and the cellular growth kinetics were all assessed. Results. The anti-VEGFs could inhibit the survival, mitotic activity, and proliferation as well as increase the cellular heterogeneity, apoptosis, and autophagy of the fibroblasts in a dose-dependent manner. Cellular growth kinetics showed ranibizumab to be less aggressive, but three other anti-VEGFs showed higher antiproliferative and apoptotic activity and expressed negative cellular growth kinetics. Conclusions. The antiproliferative, apoptotic, and autophagic activity of anti-VEGFs upon fibroblasts may explain the cellular response and the etiology of CNV involution in vivo and serve as a good study model for CNV in vitro. PMID:26491557

  2. Zoledronate induces autophagic cell death in human umbilical vein endothelial cells via Beclin-1 dependent pathway activation

    PubMed Central

    Lu, Yong; Wang, Zhiyong; Han, Wei; Li, Hao

    2016-01-01

    Zoledronate has been reported to exhibit pro-apoptotic and anti-angiogenic effects in endothelial cells, which partially contributes to bisphosphonate-associated osteonecrosis of the jaw (BP-ONJ). Zoledronate can also induce autophagic cell death. The present study hypothesized that Zoledronate may activate autophagy to exert pro-apoptotic effects in endothelial cells and aimed to investigate the effect of Zoledronate on human umbilical vein endothelial cells (HUVECs) and explore the underlying mechanisms. The current study demonstrated that Zoledronate induced autophagy in HUVECs in a dose-dependent manner, as demonstrated by increased levels of microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) and Beclin-1, and decreased levels of sequestome 1 (SQSTM1). In addition, treatment with chloroquine further increased LC3B-II and increased SQSTM1 levels, indicating that Zoledronate induces autophagy by increasing autophagic activity. Flow cytometry and Hoechst 33258 staining revealed that inhibition of autophagy with 3-methyladenine markedly attenuated Zoledronate-induced apoptosis. Furthermore, genetic knockdown of Beclin-1 significantly inhibited autophagy and apoptosis induced by Zoledronate. The present study therefore demonstrated that Zoledronate may promote Beclin-1-mediated autophagy to induce endothelial cell apoptosis, and suggests that blocking autophagy may represent a novel approach for the prevention of BP-ONJ in patients receiving Zoledronate. PMID:27748838

  3. Reversine induces autophagic cell death through the AMP-activated protein kinase pathway in urothelial carcinoma cells.

    PubMed

    Fang, Chiung-Yao; Chen, Jeng-Sheng; Chang, Shun-Kai; Shen, Cheng-Huang

    2017-10-03

    Urothelial carcinoma is one of the most common malignancies of the urinary tract. Effective treatment of advanced urothelial carcinoma remains a clinical challenge with poor outcomes in these patients. Previous reports have shown that the expression of aurora kinase is associated with clinical stage and prognosis; hence, aurora kinases are potential targets in urothelial carcinoma therapy. Reversine, an aurora kinase inhibitor, was analyzed for its cytotoxicity in this study. Cell proliferation, flow cytometry, western blotting, and immunofluorescent assay were used to determine the effect of reversine on urothelial carcinoma cells. The results showed that reversine significantly inhibits the growth of urothelial carcinoma cell lines. Reversine induced cell cycle arrest at the G2/M phase, leading to autophagic cell death by activating the AMP-activated protein kinase pathway. Reversine induced significant cell death in urothelial carcinoma cells. Our results suggest that reversine may be a suitably small molecule for treating urothelial carcinoma in the future.

  4. An autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity.

    PubMed

    Pereira, Lilian Cristina; Duarte, Filipe Valente; Varela, Ana Teresa Inácio Ferreira; Rolo, Anabela Pinto; Palmeira, Carlos Manuel Marques; Dorta, Daniel Junqueira

    2017-02-01

    To reduce flammability and meet regulatory requirements, Brominated Flame Retardants (BFRs) are added to a wide variety of consumer products including furniture, textiles, electronics, and construction materials. Exposure to polybrominated phenyl ethers (PBDEs) adversely affects the human health. Bearing in mind that (i) PBDEs are potentially toxic, (ii) the mechanism of PBDE toxicity is unclear, and (iii) the importance of the autophagy to the field of toxicology is overlooked, this study investigates whether an autophagic process is activated in HepG2 cells (human hepatoblastoma cell line) to mediate BDE-100-induced toxicity. HepG2 cells were exposed with BDE-100 at three concentrations (0.1, 5, and 25μM), selected from preliminary toxicity tests, for 24 and 48h. To assess autophagy, immunocytochemistry was performed after exposure of HepG2 cells to BDE-100. Labeling of HepG2 cells with 100nM LysoTracker Red DND-99 aided examination of lysosome distribution. Proteins that are key to the autophagic process (p62 and LC3) were evaluated by western blotting. DNA was isolated and quantified to assess mitochondrial DNA copy number by qPCR on the basis of the number of DNA copies of a mitochondrial encoded gene normalized against a nuclear encoded gene. Conversion of LC3-I to LC3-II increased in HepG2 cells. Pre-addition of 100nM wortmannin decreased the amount of LC3 in the punctuate form and increased nuclear fragmentation (apoptotic feature). HepG2 cells exposed to BDE-100 presented increased staining with the lysosomal dye and had larger LC3 and p62 content after pre-treatment with ammonium chloride. The mitochondrial DNA copy number decreased, which probably constituted an attempt of the cell to manage mitochondrial damage by selective mitochondrial degradation (mitophagy). In conclusion, an autophagic process is activated in HepG2 cells to mediate BDE-100-induced toxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. 5'-Monophosphate-activated protein kinase (AMPK) improves autophagic activity in diabetes and diabetic complications.

    PubMed

    Yao, Fan; Zhang, Ming; Chen, Li

    2016-01-01

    Diabetes mellitus (DM), an endocrine disorder, will be one of the leading causes of death world-wide in about two decades. Cellular injuries and disorders of energy metabolism are two key factors in the pathogenesis of diabetes, which also become the important causes for the process of diabetic complications. AMPK is a key enzyme in maintaining metabolic homeostasis and has been implicated in the activation of autophagy in distinct tissues. An increasing number of researchers have confirmed that autophagy is a potential factor to affect or induce diabetes and its complications nowadays, which could remove cytotoxic proteins and dysfunctional organelles. This review will summarize the regulation of autophagy and AMPK in diabetes and its complications, and explore how AMPK stimulates autophagy in different diabetic syndromes. A deeper understanding of the regulation and activity of AMPK in autophagy would enhance its development as a promising therapeutic target for diabetes treatment.

  6. miR-34a Modulates Angiotensin II-Induced Myocardial Hypertrophy by Direct Inhibition of ATG9A Expression and Autophagic Activity

    PubMed Central

    Huang, He; Ye, Jing; Pan, Wei; Zhong, Yun; Cheng, Chuanfang; You, Xiangyu; Liu, Benrong; Xiong, Longgen; Liu, Shiming

    2014-01-01

    Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3′-UTR, but not to the mutated 3′-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity. PMID:24728149

  7. Tiron and trolox potentiate the autophagic cell death induced by magnolol analog Ery5 by activation of Bax in HL-60 cells.

    PubMed

    Kumar, Suresh; Kumar, Ajay; Pathania, Anup Singh; Guru, Santosh Kumar; Jada, Srinivas; Sharma, Parduman Raj; Bhushan, Shashi; Saxena, Ajit Kumar; Kumar, H M Sampath; Malik, Fayaz

    2013-05-01

    This study describes the mechanism of trolox and tiron induced potentiation of cytotoxicity caused by Ery5, an analog of magnolol, in human myeloid leukemia HL-60 cells. Ery5 induced cytotoxicity in HL-60 cells by involving activation of bax and cleavage of caspase 3, which contributed towards activation of both apoptotic and autophagic pathways. Trolox and tiron, even at non-toxic concentrations, contributed to the cytotoxicity of Ery5 by activation of autophagic proteins like ATG7, ATG12 and LC3-II. Z-VAD-fmk mediated reduction in the cytotoxicity and expression of autophagic proteins, further suggested that autophagy induced by Ery5 is largely dependent upon caspases. Interestingly, Ery5 induced autophagy was accompanied by the downregulation of PI3K/AKT pathway whereas, trolox and tiron strongly enhanced this effect. In addition to that treatment of cells with Ery5, trolox and tiron individually, displayed a marked upregulation of Bax. The involvement of Bax in trolox and tiron induced enhancement of the cytotoxicity of Ery5 was confirmed, when siRNA induced silencing of Bax led to increased viability of the cells and exerted a strong inhibitory effect on LC3-II accumulation and p62 degradation in case of cells treated by the combination of Ery5 with trolox or tiron. Additionally, an important role of PARP in Ery5 mediated cell death has been suggested by PARP silencing experiments, however, potentiation of autophagic cytotoxicity by trolox and tiron did not seem to be dependent on PARP-1. Therefore, Bax seems to play a vital role in trolox and tiron mediated potentiation of autophagic cell death by Ery5 in HL-60 cells.

  8. Autophagic/lysosomal dysfunction in Alzheimer’s disease

    PubMed Central

    2013-01-01

    Autophagy serves as the sole catabolic mechanism for degrading organelles and protein aggregates. Increasing evidence implicates autophagic dysfunction in Alzheimer’s disease (AD) and other neurodegenerative diseases associated with protein misprocessing and accumulation. Under physiologic conditions, the autophagic/lysosomal system efficiently recycles organelles and substrate proteins. However, reduced autophagy function leads to the accumulation of proteins and autophagic and lysosomal vesicles. These vesicles contain toxic lysosomal hydrolases as well as the proper cellular machinery to generate amyloid-beta, the major component of AD plaques. Here, we provide an overview of current research focused on the relevance of autophagic/lysosomal dysfunction in AD pathogenesis as well as potential therapeutic targets aimed at restoring autophagic/lysosomal pathway function. PMID:24171818

  9. Rotenone induces apoptosis in human lung cancer cells by regulating autophagic flux.

    PubMed

    Hu, Wensi; Tian, Hui; Yue, Weiming; Li, Lin; Li, Shuhai; Gao, Cun; Si, Libo; Qi, Lei; Lu, Ming; Hao, Bin; Shan, Shuyu

    2016-05-01

    Reactive oxygen species (ROS) are at the center of many physiological and pathological processes. ROS generated due to oxidative stress can potentiate both cancer initiation and progression. Rotenone, which is an inhibitor of the mitochondrial electron transport chain complex I, results in the activation of NOX2 and release of ROS, and has been shown to display anticancer activity through the induction of apoptosis in various cancer cells. The mechanistic link between rotenone-dependent activation of NOX2 and induction of apoptosis is still elusive. In this study, we used the human lung cancer A549 cells to study the molecular mechanism(s) involved between rotenone-dependent activation of NOX2 and impairment of autophagic machinery. We report that acute exposure to rotenone induced mild NOX2-dependnet oxidative stress, which impaired the autophagic flux, resulting in cytosolic accumulation of LC3 and p62/STSQM1. We further show that this induction occurs through the PI3K/Akt/mTORC1 signaling pathway. We furthermore show that chronic exposure to rotenone lead to excessive NOX2-dependent ROS generation, increases autophagy, and decreases p62 level via increased-autophagic flux. Taken together, this study is the first mechanistic elucidation of how rotenone can be used to potently target cancer cells without overhauling the entire cellular machinery. © 2016 IUBMB Life 68(5):388-393, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

  10. Non-autophagic roles of autophagy-related proteins.

    PubMed

    Subramani, Suresh; Malhotra, Vivek

    2013-02-01

    Autophagy and autophagy-related processes are fundamentally important in human health and disease. These processes are viewed primarily as cellular degradative pathways that recycle macromolecules and dysfunctional or redundant organelles into amino acids, sugars and lipids, especially during starvation. However, the ubiquitin-like autophagy proteins and other components of the autophagic machinery additionally participate in cellular reprogramming. We highlight these non-autophagic roles of autophagy proteins with the aim of drawing attention to this growing, but unexplored, research topic. We focus on the non-autophagic functions of autophagy proteins in cell survival and apoptosis, modulation of cellular traffic, protein secretion, cell signalling, transcription, translation and membrane reorganization.

  11. Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells.

    PubMed

    Ha, Shinwon; Jeong, Seol-Hwa; Yi, Kyungrim; Chung, Kyung Min; Hong, Caroline Jeeyeon; Kim, Seong Who; Kim, Eun-Kyoung; Yu, Seong-Woon

    2017-08-18

    In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

    SciTech Connect

    Park, Jeong Su; Kang, Dong Hoon; Lee, Da Hyun; Bae, Soo Han

    2015-10-23

    p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

  13. Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways

    PubMed Central

    Xiong, Xin-xin; Liu, Ju-mei; Qiu, Xin-yao; Pan, Feng; Yu, Shang-bin; Chen, Xiao-qian

    2015-01-01

    Aim: To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action. Methods: Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting. Results: PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC50 less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity. Conclusion: Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways. PMID:25619389

  14. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity

    PubMed Central

    Pi, Huifeng; Li, Min; Tian, Li; Yang, Zhiqi; Yu, Zhengping; Zhou, Zhou

    2017-01-01

    Cadmium (Cd), a highly ubiquitous heavy metal, is a well-known inducer of neurotoxicity. However, the mechanism underlying cadmium-induced neurotoxicity remains unclear. In this study, we found that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function by reducing the levels of lysosomal-associated membrane proteins, inhibiting lysosomal proteolysis and altering lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to nerve cell death. In addition, Cd decreases transcription factor EB (TFEB) expression at both the mRNA and protein levels. Furthermore, Cd induces the nuclear translocation of TFEB and TFEB target-gene expression, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Notably, restoration of the levels of lysosomal-associated membrane protein, lysosomal proteolysis, lysosomal pH and autophagic flux through Tfeb overexpression protects against Cd-induced neurotoxicity, and this protective effect is incompletely dependent on TFEB nuclear translocation. Moreover, gene transfer of the master autophagy regulator TFEB results in the clearance of toxic proteins and the correction of Cd-induced neurotoxicity in vivo. Our study is the first to demonstrate that Cd disrupts lysosomal function and autophagic flux and manipulation of TFEB signalling may be a therapeutic approach for antagonizing Cd-induced neurotoxicity. PMID:28240313

  15. Loss of C9orf72 Enhances Autophagic Activity via Deregulated mTOR and TFEB Signaling

    PubMed Central

    Conchina, Karen; Chu, Justin; Nirujogi, Raja Sekhar; Brady, Nathan R.; Hamacher-Brady, Anne

    2016-01-01

    The most common cause of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia is a hexanucleotide repeat expansion in C9orf72. Here we report a study of the C9orf72 protein by examining the consequences of loss of C9orf72 functions. Deletion of one or both alleles of the C9orf72 gene in mice causes age-dependent lethality phenotypes. We demonstrate that C9orf72 regulates nutrient sensing as the loss of C9orf72 decreases phosphorylation of the mTOR substrate S6K1. The transcription factor EB (TFEB), a master regulator of lysosomal and autophagy genes, which is negatively regulated by mTOR, is substantially up-regulated in C9orf72 loss-of-function animal and cellular models. Consistent with reduced mTOR activity and increased TFEB levels, loss of C9orf72 enhances autophagic flux, suggesting that C9orf72 is a negative regulator of autophagy. We identified a protein complex consisting of C9orf72 and SMCR8, both of which are homologous to DENN-like proteins. The depletion of C9orf72 or SMCR8 leads to significant down-regulation of each other’s protein level. Loss of SMCR8 alters mTOR signaling and autophagy. These results demonstrate that the C9orf72-SMCR8 protein complex functions in the regulation of metabolism and provide evidence that loss of C9orf72 function may contribute to the pathogenesis of relevant diseases. PMID:27875531

  16. Synaptic activity protects against AD and FTD-like pathology via autophagic-lysosomal degradation.

    PubMed

    Akwa, Y; Gondard, E; Mann, A; Capetillo-Zarate, E; Alberdi, E; Matute, C; Marty, S; Vaccari, T; Lozano, A M; Baulieu, E E; Tampellini, D

    2017-07-11

    Changes in synaptic excitability and reduced brain metabolism are among the earliest detectable alterations associated with the development of Alzheimer's disease (AD). Stimulation of synaptic activity has been shown to be protective in models of AD beta-amyloidosis. Remarkably, deep brain stimulation (DBS) provides beneficial effects in AD patients, and represents an important therapeutic approach against AD and other forms of dementia. While several studies have explored the effect of synaptic activation on beta-amyloid, little is known about Tau protein. In this study, we investigated the effect of synaptic stimulation on Tau pathology and synapses in in vivo and in vitro models of AD and frontotemporal dementia (FTD). We found that chronic DBS or chemically induced synaptic stimulation reduced accumulation of pathological forms of Tau and protected synapses, while chronic inhibition of synaptic activity worsened Tau pathology and caused detrimental effects on pre- and post-synaptic markers, suggesting that synapses are affected. Interestingly, degradation via the proteasomal system was not involved in the reduction of pathological Tau during stimulation. In contrast, chronic synaptic activation promoted clearance of Tau oligomers by autophagosomes and lysosomes. Chronic inhibition of synaptic activity resulted in opposite outcomes, with build-up of Tau oligomers in enlarged auto-lysosomes. Our data indicate that synaptic activity counteracts the negative effects of Tau in AD and FTD by acting on autophagy, providing a rationale for therapeutic use of DBS and synaptic stimulation in tauopathies.Molecular Psychiatry advance online publication, 11 July 2017; doi:10.1038/mp.2017.142.

  17. UVB-Induced Senescence of Human Dermal Fibroblasts Involves Impairment of Proteasome and Enhanced Autophagic Activity.

    PubMed

    Cavinato, Maria; Koziel, Rafal; Romani, Nikolaus; Weinmüllner, Regina; Jenewein, Brigitte; Hermann, Martin; Dubrac, Sandrine; Ratzinger, Gudrun; Grillari, Johannes; Schmuth, Matthias; Jansen-Dürr, Pidder

    2017-05-01

    In the current study, we have extended previous findings aiming at a better understanding of molecular mechanisms underlying UVB-induced senescence of diploid human dermal fibroblasts (HDFs), an experimental model to study the process of photoaging in the skin. We provide evidence that the inhibition of proteasomal degradation of damaged proteins and the activation of autophagosome formation are early events in UVB-induced senescence of HDFs, dependent on UVB-induced accumulation of reactive oxygen species. Our data suggest that autophagy is required for the establishment of the senescent phenotype in UVB-treated HDFs and that inhibition of autophagy is sufficient to change the cell fate from senescence to cell death by apoptosis. Studies in reconstructed skin equivalents revealed that UVB irradiation triggers hallmarks of autophagy induction in the dermal layer. These findings have potential implications for fundamental as well as translational research into skin aging, in particular photoaging. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Piezoelectric pushers for active vibration control of rotating machinery

    NASA Technical Reports Server (NTRS)

    Palazzolo, A. B.; Kascak, A. F.; Lin, R. R.; Montague, J.; Alexander, R. M.

    1989-01-01

    The active control of rotordynamic vibrations and stability by magnetic bearings and electromagnetic shakers was discussed extensively in the literature. These devices, though effective, are usually large in volume and add significant weight to the stator. The use of piezoelectric pushers may provide similar degrees of effectiveness in light, compact packages. Analyses are contained which extend quadratic regulator, pole placement and derivative feedback control methods to the prescribed displacement character of piezoelectric pushers. The structural stiffness of the pusher is also included in the theory. Tests are currently being conducted at NASA Lewis Research Center with piezoelectric pusher-based active vibration control. Results performed on the NASA test rig as preliminary verification of the related theory are presented.

  19. Autophagy: machinery and regulation

    PubMed Central

    Yin, Zhangyuan; Pascual, Clarence; Klionsky, Daniel J.

    2016-01-01

    Macroautophagy/autophagy is an evolutionarily conserved cellular degradation process that targets cytoplasmic materials including cytosol, macromolecules and unwanted organelles. The discovery and analysis of autophagy-related (Atg) proteins have unveiled much of the machinery of autophagosome formation. Although initially autophagy was regarded as a survival response to stress, recent studies have revealed its significance in cellular and organismal homeostasis, development and immunity. Autophagic dysfunction and dysregulation are implicated in various diseases. In this review, we briefly summarize the physiological roles, molecular mechanism, regulatory network, and pathophysiological roles of autophagy. PMID:28357331

  20. Centrifugally activated bearing for high-speed rotating machinery

    DOEpatents

    Post, Richard F.

    1994-01-01

    A centrifugally activated bearing is disclosed. The bearing includes an annular member that extends laterally and radially from a central axis. A rotating member that rotates about the central axis relative to the annular member is also included. The rotating member has an interior chamber that surrounds the central axis and in which the annular member is suspended. Furthermore, the interior chamber has a concave shape for retaining a lubricant therein while the rotating member is at rest and for retaining a lubricant therein while the rotating member is rotating. The concave shape is such that while the rotating member is rotating a centrifugal force causes a lubricant to be forced away from the central axis to form a cylindrical surface having an axis collinear with the central axis. This centrifugally displaced lubricant provides restoring forces to counteract lateral displacement during operation.

  1. Centrifugally activated bearing for high-speed rotating machinery

    DOEpatents

    Post, R.F.

    1994-02-15

    A centrifugally activated bearing is disclosed. The bearing includes an annular member that extends laterally and radially from a central axis. A rotating member that rotates about the central axis relative to the annular member is also included. The rotating member has an interior chamber that surrounds the central axis and in which the annular member is suspended. Furthermore, the interior chamber has a concave shape for retaining a lubricant therein while the rotating member is at rest and for retaining a lubricant therein while the rotating member is rotating. The concave shape is such that while the rotating member is rotating a centrifugal force causes a lubricant to be forced away from the central axis to form a cylindrical surface having an axis collinear with the central axis. This centrifugally displaced lubricant provides restoring forces to counteract lateral displacement during operation. 4 figures.

  2. Activation of autophagic flux by epigallocatechin gallate mitigates TRAIL-induced tumor cell apoptosis via down-regulation of death receptors

    PubMed Central

    Park, Sang-Youel

    2016-01-01

    Epigallocatechin gallate (EGCG) is a major polyphenol in green tea. Recent studies have reported that EGCG can inhibit TRAIL-induced apoptosis and activate autophagic flux in cancer cells. However, the mechanism behind these processes is unclear. The present study found that EGCG prevents tumor cell death by antagonizing the TRAIL pathway and activating autophagy flux. Our results indicate that EGCG dose-dependently inhibits TRAIL-induced apoptosis and decreases the binding of death receptor 4 and 5 (DR4 and 5) to TRAIL. In addition, EGCG activates autophagy flux, which is involved in the inhibition of TRAIL cell death. We confirmed that the protective effect of EGCG can be reversed using genetic and pharmacological tools through re-sensitization to TRAIL. The inhibition of autophagy flux affects not only the re-sensitization of tumor cells to TRAIL, but also the restoration of death receptor proteins. This study demonstrates that EGCG inhibits TRAIL-induced apoptosis through the manipulation of autophagic flux and subsequent decrease in number of death receptors. On the basis of these results, we suggest further consideration of the use of autophagy activators such as EGCG in combination anti-tumor therapy with TRAIL. PMID:27582540

  3. Rhus coriaria induces senescence and autophagic cell death in breast cancer cells through a mechanism involving p38 and ERK1/2 activation

    PubMed Central

    El Hasasna, Hussain; Athamneh, Khawlah; Al Samri, Halima; Karuvantevida, Noushad; Al Dhaheri, Yusra; Hisaindee, Soleiman; Ramadan, Gaber; Al Tamimi, Nedaa; AbuQamar, Synan; Eid, Ali; Iratni, Rabah

    2015-01-01

    Here, we investigated the anticancer effect of Rhus coriaria on three breast cancer cell lines. We demonstrated that Rhus coriaria ethanolic extract (RCE) inhibits the proliferation of these cell lines in a time- and concentration-dependent manner. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, downregulation of cyclin D1, p27, PCNA, c-myc, phospho-RB and expression of senescence-associated β-galactosidase activity. No proliferative recovery was detected after RCE removal. Annexin V staining and PARP cleavage analysis revealed a minimal induction of apoptosis in MDA-MB-231 cells. Electron microscopy revealed the presence of autophagic vacuoles in RCE-treated cells. Interestingly, blocking autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death and senescence. RCE was also found to activate p38 and ERK1/2 signaling pathways which coincided with induction of autophagy. Furthermore, we found that while both autophagy inhibitors abolished p38 phosphorylation, only CQ led to significant decrease in pERK1/2. Finally, RCE induced DNA damage and reduced mutant p53, two events that preceded autophagy. Our findings provide strong evidence that R. coriaria possesses strong anti-breast cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against breast cancer. PMID:26263881

  4. Rhus coriaria induces senescence and autophagic cell death in breast cancer cells through a mechanism involving p38 and ERK1/2 activation.

    PubMed

    El Hasasna, Hussain; Athamneh, Khawlah; Al Samri, Halima; Karuvantevida, Noushad; Al Dhaheri, Yusra; Hisaindee, Soleiman; Ramadan, Gaber; Al Tamimi, Nedaa; AbuQamar, Synan; Eid, Ali; Iratni, Rabah

    2015-08-12

    Here, we investigated the anticancer effect of Rhus coriaria on three breast cancer cell lines. We demonstrated that Rhus coriaria ethanolic extract (RCE) inhibits the proliferation of these cell lines in a time- and concentration-dependent manner. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, downregulation of cyclin D1, p27, PCNA, c-myc, phospho-RB and expression of senescence-associated β-galactosidase activity. No proliferative recovery was detected after RCE removal. Annexin V staining and PARP cleavage analysis revealed a minimal induction of apoptosis in MDA-MB-231 cells. Electron microscopy revealed the presence of autophagic vacuoles in RCE-treated cells. Interestingly, blocking autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death and senescence. RCE was also found to activate p38 and ERK1/2 signaling pathways which coincided with induction of autophagy. Furthermore, we found that while both autophagy inhibitors abolished p38 phosphorylation, only CQ led to significant decrease in pERK1/2. Finally, RCE induced DNA damage and reduced mutant p53, two events that preceded autophagy. Our findings provide strong evidence that R. coriaria possesses strong anti-breast cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against breast cancer.

  5. Pin1 mediates neural-specific activation of the mitochondrial apoptotic machinery.

    PubMed

    Becker, Esther B E; Bonni, Azad

    2006-03-02

    Apoptosis of neurons plays fundamental roles in brain development and disease. Although neurons share with other cell types components of the mitochondrial apoptotic machinery, how this machinery is specifically activated in neurons remains poorly understood. Remarkably, phosphorylation of the BH3-only protein BIMEL at Ser65 triggers apoptosis in neurons but suppresses cell death in non-neural cells. Here, we report that the prolyl isomerase Pin1 interacts with Ser65-phosphorylated BIMEL in neurons. Pin1 is enriched at the mitochondrial membrane in neurons, where it forms a physical complex with the neuron-specific JNK scaffold protein JIP3. Activation of JNK signaling induces the dissociation of Pin1 from JIP3 and concomitantly promotes Pin1 binding to phosphorylated BIMEL. The interaction of Pin1 with phosphorylated BIMEL stabilizes BIMEL and thereby activates neuronal apoptosis. These findings define a neural-specific mechanism of cell death whereby Pin1 couples phosphorylation of BH3-only proteins to activation of the mitochondrial apoptotic machinery.

  6. Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington's disease.

    PubMed

    Liu, Yanying; Hettinger, Casey L; Zhang, Dong; Rezvani, Khosrow; Wang, Xuejun; Wang, Hongmin

    2014-05-01

    The ubiquitin proteasome system (UPS) is impaired in Huntington's disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFP UPS reporter (GFPu). We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether sulforaphane promotes mutant huntingtin (mHtt) degradation, we treated Huntington's disease cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane-mediated mHtt degradation was mainly through the UPS pathway as the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for Huntington's disease and other intractable disorders. Accumulation of mutant huntingtin (mHtt) protein causes Huntington's disease (HD). Sulforaphane (SFN), a naturally occurring compound, increased proteasome and autophagy activities in vivo and enhanced mHtt turnover and cell survival in HD cell models. SFN-mediated mHtt degradation is mainly through the proteasome pathway. These data suggest that SFN can be a therapeutic reagent for treating HD and other intractable disorders. © 2014 International Society for Neurochemistry.

  7. Aqueous Extract of Solanum nigrum Leaf Activates Autophagic Cell Death and Enhances Docetaxel-Induced Cytotoxicity in Human Endometrial Carcinoma Cells

    PubMed Central

    Tai, Cheng-Jeng; Wang, Chien-Kai; Chang, Yu-Jia; Lin, Chi-Shian; Tai, Chen-Jei

    2012-01-01

    Chemotherapy is the main approach in dealing with advanced and recurrent endometrial cancer. An effective complementary ingredient can be helpful in improving the clinical outcome. Aqueous extract of Solanum nigrum leaf (AE-SN) is a principal ingredient for treating cancer patients in traditional Chinese medicinal practice but lacks sufficient evidence to verify its tumor suppression efficacy. This study evaluated the antitumor effects of AE-SN and also assessed the synergistic effects of AE-SN with docetaxel On the human endometrial cancer cell lines, HEC1A, HEC1B, and KLE. The activation of apoptotic markers, caspase-3 and poly-ADP-ribose polymerase, and autophagic marker, microtubule-associated protein 1 light chain 3 A/B, wAS determined to clarify the cell death pathways responsible for AE-SN induced tumor cell death. Results indicated that AE-SN-treatment has significant cytotoxicity on the tested endometrial cancer cells with accumulation of LC3 A/B II and demonstrated a synergistic effect of AE-SN and docetaxel in HEC1A and HEC1B cells, but not KLE cells. In conclusion, AE-SN treatment was effective in suppressing endometrial cancer cells via the autophagic pathway and was also capable of enhancing the cytotoxicity of docetaxel in human endometrial cancer cells. Our results provide meaningful evidence for integrative cancer therapy in the future. PMID:23304219

  8. Autophagic subpopulation sorting by sedimentation field-flow fractionation.

    PubMed

    Naves, Thomas; Battu, Serge; Jauberteau, Marie-Odile; Cardot, Philippe J P; Ratinaud, Marie-Hélène; Verdier, Mireille

    2012-10-16

    The development of hypoxic areas often takes place in solid tumors and leads cells to undergo adaptive signalization like autophagy. This process is responsible for misfolded or aggregated proteins and nonfunctional organelle recycling, allowing cells to maintain their energetic status. However, it could constitute a double-edged pathway leading to both survival and cell death. So, in response to stress such as hypoxia, autophagic and apoptotic cells are often mixed. To specifically study and characterize autophagic cells and the process, we needed to develop a method able to (1) isolate autophagic subpopulation and (2) respect apoptotic and autophagic status. Sedimentation field-flow fractionation (SdFFF) was first used to monitor physical parameter changes due to the hypoxia mimetic CoCl(2) in the p53 mutated SKNBE2(c) human neuroblastoma cell line. Second, we showed that "hyperlayer" elution is able to prepare autophagic enriched populations, fraction (F3), overexpressing autophagic markers (i.e., LC3-II accumulation and punctiform organization of autophagosomes as well as cathepsin B overactivity). Conversely, the first eluted fraction exhibited apoptotic markers (caspase-3 activity and Bax increased expression). For the first time, SdFFF was employed as an analytical tool in order to discriminate apoptotic and autophagic cells, thus providing an enriched autophagic fraction consecutively to a hypoxic stress.

  9. Gangliosides induce autophagic cell death in astrocytes

    PubMed Central

    Hwang, Jaegyu; Lee, Shinrye; Lee, Jung Tae; Kwon, Taeg Kyu; Kim, Deok Ryong; Kim, Ho; Park, Hae-Chul; Suk, Kyoungho

    2010-01-01

    Background and purpose: Gangliosides, sialic acid-containing glycosphingolipids, abundant in brain, are involved in neuronal function and disease, but the precise molecular mechanisms underlying their physiological or pathological activities are poorly understood. In this study, the pathological role of gangliosides in the extracellular milieu with respect to glial cell death and lipid raft/membrane disruption was investigated. Experimental approach: We determined the effect of gangliosides on astrocyte death or survival using primary astrocyte cultures and astrocytoma/glioma cell lines as a model. Signalling pathways of ganglioside-induced autophagic cell death of astrocytes were examined using pharmacological inhibitors and biochemical and genetic assays. Key results: Gangliosides induced autophagic cell death in based on the following observations. Incubation of the cells with a mixture of gangliosides increased a punctate distribution of fluorescently labelled microtubule-associated protein 1 light chain 3 (GFP-LC3), the ratio of LC3-II/LC3-I and LC3 flux. Gangliosides also increased the formation of autophagic vacuoles as revealed by monodansylcadaverine staining. Ganglioside-induced cell death was inhibited by either a knockdown of beclin-1/Atg-6 or Atg-7 gene expression or by 3-methyladenine, an inhibitor of autophagy. Reactive oxygen species (ROS) were involved in ganglioside-induced autophagic cell death of astrocytes, because gangliosides induced ROS production and ROS scavengers decreased autophagic cell death. In addition, lipid rafts played an important role in ganglioside-induced astrocyte death. Conclusions and implications: Gangliosides released under pathological conditions may induce autophagic cell death of astrocytes, identifying a neuropathological role for gangliosides. PMID:20067473

  10. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1

    NASA Astrophysics Data System (ADS)

    Yang, Shilong; Yang, Fan; Wei, Ningning; Hong, Jing; Li, Bowen; Luo, Lei; Rong, Mingqiang; Yarov-Yarovoy, Vladimir; Zheng, Jie; Wang, Kewei; Lai, Ren

    2015-09-01

    The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx-TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery.

  11. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1.

    PubMed

    Yang, Shilong; Yang, Fan; Wei, Ningning; Hong, Jing; Li, Bowen; Luo, Lei; Rong, Mingqiang; Yarov-Yarovoy, Vladimir; Zheng, Jie; Wang, KeWei; Lai, Ren

    2015-09-30

    The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx-TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery.

  12. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1

    PubMed Central

    Yang, Shilong; Yang, Fan; Wei, Ningning; Hong, Jing; Li, Bowen; Luo, Lei; Rong, Mingqiang; Yarov-Yarovoy, Vladimir; Zheng, Jie; Wang, KeWei; Lai, Ren

    2015-01-01

    The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx–TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery. PMID:26420335

  13. Melatonin antagonizes cadmium-induced neurotoxicity by activating the transcription factor EB-dependent autophagy-lysosome machinery in mouse neuroblastoma cells.

    PubMed

    Li, Min; Pi, Huifeng; Yang, Zhiqi; Reiter, Russel J; Xu, Shangcheng; Chen, Xiaowei; Chen, Chunhai; Zhang, Lei; Yang, Min; Li, Yuming; Guo, Pan; Li, Gaoming; Tu, Manyu; Tian, Li; Xie, Jia; He, Mindi; Lu, Yonghui; Zhong, Min; Zhang, Yanwen; Yu, Zhengping; Zhou, Zhou

    2016-10-01

    Cadmium (Cd), a highly ubiquitous heavy metal, induces neurotoxicity. Melatonin, a major secretory product of the pineal gland, protects against Cd-induced neurotoxicity. However, the mechanism that accounts for this protection remains to be elucidated. Herein, we exposed mouse neuroblastoma cells (Neuro-2a cells) to different concentrations of cadmium chloride (CdCl2 ) (12.5, 25, and 50 μ mol L(-1) ) for 24 hours. We showed that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function, subsequently leading to nerve cell death. In addition, Cd decreases the level of transcription factor EB (TFEB) but induces the nuclear translocation of TFEB, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Moreover, compared to the 50-μ mol L(-1) Cd group, administration of 1 μ mol L(-1) melatonin increased "TFEB-responsive genes" (P<.05) and the levels of lysosomal-associated membrane protein (0.57±0.06 vs 1.00±0.11, P<.05), preserved lysosomal protease activity (0.52±0.01 vs 0.90±0.02, P<.05), maintained the lysosomal pH level (0.50±0.01 vs 0.87±0.05, P<.01), and enhanced autophagosome-lysosome fusion (0.05±0.00 vs 0.21±0.01, P<.01). Notably, melatonin enhanced TFEB expression (0.37±0.04 vs 0.72±0.07, P<.05) and nuclear translocation (2.81±0.08 vs 3.82±0.05, P<.05). Tfeb siRNA blocked the melatonin-mediated elevation in autophagy-lysosome machinery in Cd-induced neurotoxicity (P<.01). Taken together, these results uncover a potent role for TFEB-mediated autophagy in the pathogenesis of Cd-induced neurotoxicity, suggesting that control of the autophagic pathway by melatonin might provide an important clue for exploring potential targets for novel therapeutics of Cd-induced neurotoxicity. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Test and theory for piezoelectric actuator-active vibration control of rotating machinery

    NASA Technical Reports Server (NTRS)

    Palazzolo, A. B.; Lin, R. R.; Alexander, R. M.; Kascak, A. F.; Montague, J.

    1989-01-01

    The application of piezoelectric actuators for active vibration control (AVC) of rotating machinery is examined. Theory is derived and the resulting predictions are shown to agree closely with results of tests performed on an air turbine driven-overhung rotor. The test results show significant reduction in unbalance, transient and sub-synchronous responses. Results from a 30-hour endurance test support the AVD system reliability. Various aspects of the electro-mechanical stability of the control system are also discussed and illustrated. Finally, application of the AVC system to an actual jet engine is discussed.

  15. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery

    PubMed Central

    Chakravarthy, Harini; Ormsbee, Briana D.; Mallanna, Sunil K.; Rizzino, Angie

    2011-01-01

    The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.—Chakravarthy, H., Ormsbee, B. D., Mallanna, S. K., Rizzino, A. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery. PMID:20876214

  16. Reduced Activity of Antioxidant Machinery Is Correlated with Suppression of Totipotency in Plant Protoplasts1

    PubMed Central

    Papadakis, Anastasia K.; Siminis, Charalambos I.; Roubelakis-Angelakis, Kalliopi A.

    2001-01-01

    We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197–205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O2.− and H2O2 and 7-fold lower levels of O2.− and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2.−, resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O2.− and H2O2 were still found in NT- compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery. PMID:11351105

  17. Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery.

    PubMed

    Chakravarthy, Harini; Ormsbee, Briana D; Mallanna, Sunil K; Rizzino, Angie

    2011-01-01

    The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.

  18. Ferroptosis is an autophagic cell death process.

    PubMed

    Gao, Minghui; Monian, Prashant; Pan, Qiuhui; Zhang, Wei; Xiang, Jenny; Jiang, Xuejun

    2016-09-01

    Ferroptosis is an iron-dependent form of regulated necrosis. It is implicated in various human diseases, including ischemic organ damage and cancer. Here, we report the crucial role of autophagy, particularly autophagic degradation of cellular iron storage proteins (a process known as ferritinophagy), in ferroptosis. Using RNAi screening coupled with subsequent genetic analysis, we identified multiple autophagy-related genes as positive regulators of ferroptosis. Ferroptosis induction led to autophagy activation and consequent degradation of ferritin and ferritinophagy cargo receptor NCOA4. Consistently, inhibition of ferritinophagy by blockage of autophagy or knockdown of NCOA4 abrogated the accumulation of ferroptosis-associated cellular labile iron and reactive oxygen species, as well as eventual ferroptotic cell death. Therefore, ferroptosis is an autophagic cell death process, and NCOA4-mediated ferritinophagy supports ferroptosis by controlling cellular iron homeostasis.

  19. Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells.

    PubMed

    Hieke, Nora; Löffler, Antje S; Kaizuka, Takeshi; Berleth, Niklas; Böhler, Philip; Drießen, Stefan; Stuhldreier, Fabian; Friesen, Olena; Assani, Kaivon; Schmitz, Katharina; Peter, Christoph; Diedrich, Britta; Dengjel, Jörn; Holland, Petter; Simonsen, Anne; Wesselborg, Sebastian; Mizushima, Noboru; Stork, Björn

    2015-01-01

    Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.

  20. Characterization of Autophagic Responses in Drosophila melanogaster.

    PubMed

    Xu, T; Kumar, S; Denton, D

    2017-01-01

    Drosophila is an excellent model system for studying autophagy during animal development due to the availability of genetic reagents and opportunity for in vivo cell biological analysis. The regulation and mechanism of autophagy are highly evolutionarily conserved and the role of autophagy has been characterized during various stages of Drosophila development as well as following starvation. Studies in Drosophila have revealed novel insights into the role of distinct components of the autophagy machinery. This chapter describes protocols for examining autophagy during Drosophila development. A crucial step in the induction of autophagy is the incorporation of Atg8a into the autophagosome. This can be measured as autophagic puncta using live fluorescent imaging, immunostaining, or immunoblot analysis of LC3/Atg8a processing. The level of autophagy can also be examined using other specific components of the autophagy pathway as markers detected by immunofluorescent imaging. Based on the distinct morphology of autophagy, it can also be examined by transmission electron microscopy. In addition, one of the advantages of using Drosophila as a model is the ability to undertake genetic analysis of individual components of the autophagy machinery. Current approaches that can be used to monitor autophagy, including the overall flux and individual steps in Drosophila melanogaster, will be discussed. © 2017 Elsevier Inc. All rights reserved.

  1. Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy

    PubMed Central

    Jang, Y H; Choi, K Y; Min, D S

    2014-01-01

    Autophagy is a catabolic process in which cell components are degraded to maintain cellular homeostasis by nutrient limitations. Defects of autophagy are involved in numerous diseases, including cancer. Here, we demonstrate a new role of phospholipase D (PLD) as a regulator of autophagy. PLD inhibition enhances autophagic flux via ATG1 (ULK1), ATG5 and ATG7, which are essential autophagy gene products critical for autophagosome formation. Moreover, PLD suppresses autophagy by differentially modulating phosphorylation of ULK1 mediated by mTOR and adenosine monophosphate-activated protein kinase (AMPK), and by suppressing the interaction of Beclin 1 with vacuolar-sorting protein 34 (Vps34), indicating that PLD coordinates major players of the autophagic pathway, AMPK-mTOR-ULK1 and Vps34/Beclin 1. Ultimately, PLD inhibition significantly sensitized in vitro and in vivo cancer regression via genetic and pharmacological inhibition of autophagy, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PLD inhibition. Collectively, we show a novel role for PLD in the molecular machinery regulating autophagy. PMID:24317201

  2. Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux.

    PubMed

    Rossi, Mario; Munarriz, Eliana Rosa; Bartesaghi, Stefano; Milanese, Marco; Dinsdale, David; Guerra-Martin, Maria Azucena; Bampton, Edward T W; Glynn, Paul; Bonanno, Giambattista; Knight, Richard A; Nicotera, Pierluigi; Melino, Gerry

    2009-09-15

    Alterations in the autophagic pathway are associated with the onset and progression of various diseases. However, despite the therapeutic potential for pharmacological modulators of autophagic flux, few such compounds have been characterised. Here we show that clomipramine, an FDA-approved drug long used for the treatment of psychiatric disorders, and its active metabolite desmethylclomipramine (DCMI) interfere with autophagic flux. Treating cells with DCMI caused a significant and specific increase in autophagosomal markers and a concomitant blockage of the degradation of autophagic cargo. This observation might be relevant in therapy in which malignant cells exploit autophagy to survive stress conditions, rendering them more susceptible to the action of cytotoxic agents. In accordance, DCMI-mediated obstruction of autophagic flux increased the cytotoxic effect of chemotherapeutic agents. Collectively, our studies describe a new function of DCMI that can be exploited for the treatment of pathological conditions in which manipulation of autophagic flux is thought to be beneficial.

  3. Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux

    PubMed Central

    Rossi, Mario; Munarriz, Eliana Rosa; Bartesaghi, Stefano; Milanese, Marco; Dinsdale, David; Guerra-Martin, Maria Azucena; Bampton, Edward T. W.; Glynn, Paul; Bonanno, Giambattista; Knight, Richard A.; Nicotera, Pierluigi; Melino, Gerry

    2009-01-01

    Summary Alterations in the autophagic pathway are associated with the onset and progression of various diseases. However, despite the therapeutic potential for pharmacological modulators of autophagic flux, few such compounds have been characterised. Here we show that clomipramine, an FDA-approved drug long used for the treatment of psychiatric disorders, and its active metabolite desmethylclomipramine (DCMI) interfere with autophagic flux. Treating cells with DCMI caused a significant and specific increase in autophagosomal markers and a concomitant blockage of the degradation of autophagic cargo. This observation might be relevant in therapy in which malignant cells exploit autophagy to survive stress conditions, rendering them more susceptible to the action of cytotoxic agents. In accordance, DCMI-mediated obstruction of autophagic flux increased the cytotoxic effect of chemotherapeutic agents. Collectively, our studies describe a new function of DCMI that can be exploited for the treatment of pathological conditions in which manipulation of autophagic flux is thought to be beneficial. PMID:19706685

  4. Evidence of biological activity of Mentha species extracts on apoptotic and autophagic targets on murine RAW264.7 and human U937 monocytic cells.

    PubMed

    Brahmi, Fatiha; Hadj-Ahmed, Samia; Zarrouk, Amira; Bezine, Maryem; Nury, Thomas; Madani, Khodir; Chibane, Mohamed; Vejux, Anne; Andreoletti, Pierre; Boulekbache-Makhlouf, Lila; Lizard, Gérard

    2017-12-01

    Mints (Lamiaceae) are used as traditional remedies for the treatment of several diseases. Their extracts are recognized as anti-inflammatory compounds. This study characterized the cytotoxic effects of Mentha spicata L. (MS), Mentha pulegium L. (MP) and Mentha rotundifolia (L). Huds (MR) on macrophage cells (RAW264.7; U937) and determined their impact on apoptosis and autophagy, which can play a role in controlling inflammation. The extracts were prepared in culture medium and tested from 25 to 400 μg/mL after 24-48 h of treatment. To show the effect of the aqueous ethanol (50%) extracts on apoptosis and authophagy, the presence of cleaved caspase-3, and the conversion of LC3-I to LC3-II was evaluated by Western blotting. Compared with the MTT assay, crystal violet showed a pronounced decrease in the number of cells with all extracts at 48 h. Calculated IC50 values were 257.31, 207.82 and 368.02 μg/mL for MS, MP and MR, respectively. A significant increase in PI positive cells was observed with all extracts at 200-400 μg/mL. Mitochondrial dysfunctions and nuclear morphological changes were detected with MS and MR extracts at 400 μg/mL. At this concentration, no cleaved caspase-3 was found whereas stabilized caspase-3 in its dimeric form was identified. MS and MR extracts also favour LC3-I to LC3-II conversion which is a criterion of autophagy. The cytotoxic profiles depend on the extracts considered; MS extract showed the strong activity. However, all the mint extracts studied interact with the apoptotic and autophagic pathways at elevated concentrations.

  5. Fisetin stimulates autophagic degradation of phosphorylated tau via the activation of TFEB and Nrf2 transcription factors.

    PubMed

    Kim, Sunhyo; Choi, Ki Ju; Cho, Sun-Jung; Yun, Sang-Moon; Jeon, Jae-Pil; Koh, Young Ho; Song, Jihyun; Johnson, Gail V W; Jo, Chulman

    2016-04-26

    The neuronal accumulation of phosphorylated tau plays a critical role in the pathogenesis of Alzheimer's disease (AD). Here, we examined the effect of fisetin, a flavonol, on tau levels. Treatment of cortical cells or primary neurons with fisetin resulted in significant decreases in the levels of phosphorylated tau. In addition, fisetin decreased the levels of sarkosyl-insoluble tau in an active GSK-3β-induced tau aggregation model. However, there was no difference in activities of tau kinases and phosphatases such as protein phosphatase 2A, irrespective of fisetin treatment. Fisetin activated autophagy together with the activation of transcription factor EB (TFEB) and Nrf2 transcriptional factors. The activation of autophagy including TFEB is likely due to fisetin-mediated mammalian target of rapamycin complex 1 (mTORC1) inhibition, since the phosphorylation levels of p70S6 kinase and 4E-BP1 were decreased in the presence of fisetin. Indeed, fisetin-induced phosphorylated tau degradation was attenuated by chemical inhibitors of the autophagy-lysosome pathway. Together the results indicate that fisetin reduces levels of phosphorylated tau through the autophagy pathway activated by TFEB and Nrf2. Our result suggests fisetin should be evaluated further as a potential preventive and therapeutic drug candidate for AD.

  6. Fisetin stimulates autophagic degradation of phosphorylated tau via the activation of TFEB and Nrf2 transcription factors

    PubMed Central

    Kim, Sunhyo; Choi, Ki Ju; Cho, Sun-Jung; Yun, Sang-Moon; Jeon, Jae-Pil; Koh, Young Ho; Song, Jihyun; Johnson, Gail V. W.; Jo, Chulman

    2016-01-01

    The neuronal accumulation of phosphorylated tau plays a critical role in the pathogenesis of Alzheimer’s disease (AD). Here, we examined the effect of fisetin, a flavonol, on tau levels. Treatment of cortical cells or primary neurons with fisetin resulted in significant decreases in the levels of phosphorylated tau. In addition, fisetin decreased the levels of sarkosyl-insoluble tau in an active GSK-3β-induced tau aggregation model. However, there was no difference in activities of tau kinases and phosphatases such as protein phosphatase 2A, irrespective of fisetin treatment. Fisetin activated autophagy together with the activation of transcription factor EB (TFEB) and Nrf2 transcriptional factors. The activation of autophagy including TFEB is likely due to fisetin-mediated mammalian target of rapamycin complex 1 (mTORC1) inhibition, since the phosphorylation levels of p70S6 kinase and 4E-BP1 were decreased in the presence of fisetin. Indeed, fisetin-induced phosphorylated tau degradation was attenuated by chemical inhibitors of the autophagy-lysosome pathway. Together the results indicate that fisetin reduces levels of phosphorylated tau through the autophagy pathway activated by TFEB and Nrf2. Our result suggests fisetin should be evaluated further as a potential preventive and therapeutic drug candidate for AD. PMID:27112200

  7. Apelin-13 impedes foam cell formation by activating Class III PI3K/Beclin-1-mediated autophagic pathway.

    PubMed

    Yao, Feng; Lv, Yun-Cheng; Zhang, Min; Xie, Wei; Tan, Yu-Lin; Gong, Duo; Cheng, Hai-Peng; Liu, Dan; Li, Liang; Liu, Xiao-Yan; Zheng, Xi-Long; Tang, Chao-Ke

    2015-10-30

    Apelin-13, an adipokine, promotes cholesterol efflux in macrophages with antiatherosclerotic effect. Autophagy, an evolutionarily ancient response to cellular stress, has been involved in atherosclerosis. Therefore, the purpose of this study was to investigate whether apelin-13 regulates macrophage foam cell cholesterol metabolism through autophagy, and also explore the underlying mechanisms. Here, we revealed that apelin-13 decreased lipid accumulation in THP-1 derived macrophages through markedly enhancing cholesterol efflux. Our study further demonstrated that apelin-13 induced autophagy via activation of Class III phosphoinositide 3-kinase (PI3K) and Beclin-1. Inhibition of Class III PI3K and Beclin-1 suppressed the stimulatory effects of apelin-13 on autophagy activity. The present study concluded that apelin-13 reduces lipid accumulation of foam cells by activating autophagy via Class III PI3K/Beclin-1 pathway. Therefore, our results provide brand new insight about apelin-13 inhibiting foam cell formation and highlight autophagy as a promising therapeutic target in atherosclerosis.

  8. A Novel Herbal Medicine, KIOM-C, Induces Autophagic and Apoptotic Cell Death Mediated by Activation of JNK and Reactive Oxygen Species in HT1080 Human Fibrosarcoma Cells

    PubMed Central

    Kim, Aeyung; Im, Minju; Yim, Nam-Hui; Kim, Taesoo; Ma, Jin Yeul

    2014-01-01

    KIOM-C was recently demonstrated to have anti-metastatic activity in highly malignant cancer cells via suppression of NF-κB-mediated MMP-9 activity. In addition, it was reported to be effective for clearance of the influenza virus by increasing production of anti-viral cytokines, such as TNF-α and IFN-γ, and efficacious in the treatment of pigs suffering from porcine circovirus-associated disease (PCVAD). In this study, we investigated whether KIOM-C induces cancer cell death and elucidated the underlying anti-cancer mechanisms. In addition, we examined whether KIOM-C oral administration suppresses in vivo tumor growth of HT1080 cells in athymic nude mice. We initially found that KIOM-C at concentrations of 500 and 1000 µg/ml caused dose- and time-dependent cell death in cancer cells, but not normal hepatocytes, to approximately 50% of control levels. At the early stage of KIOM-C treatment (12 h), cells were arrested in G1 phase, which was accompanied by up-regulation of p21 and p27, down-regulation of cyclin D1, and subsequent increases in apoptotic and autophagic cells. Following KIOM-C treatment, the extent of caspase-3 activation, PARP cleavage, Beclin-1 expression, and LC3-II conversion was remarkably up-regulated, but p62 expression was down-regulated. Phosphorylation of AMPK, ULK, JNK, c-jun, and p53 was increased significantly in response to KIOM-C treatment. The levels of intracellular ROS and CHOP expression were also increased. In particular, the JNK-specific inhibitor SP600125 blocked KIOM-C-induced ROS generation and CHOP expression almost completely, which consequently almost completely rescued cell death, indicating that JNK activation plays a critical role in KIOM-C-induced cell death. Furthermore, daily oral administration of 85 and 170 mg/kg KIOM-C efficiently suppressed the tumorigenic growth of HT1080 cells, without systemic toxicity. These results collectively suggest that KIOM-C efficiently induces cancer cell death by both autophagy and

  9. A novel herbal medicine, KIOM-C, induces autophagic and apoptotic cell death mediated by activation of JNK and reactive oxygen species in HT1080 human fibrosarcoma cells.

    PubMed

    Kim, Aeyung; Im, Minju; Yim, Nam-Hui; Kim, Taesoo; Ma, Jin Yeul

    2014-01-01

    KIOM-C was recently demonstrated to have anti-metastatic activity in highly malignant cancer cells via suppression of NF-κB-mediated MMP-9 activity. In addition, it was reported to be effective for clearance of the influenza virus by increasing production of anti-viral cytokines, such as TNF-α and IFN-γ, and efficacious in the treatment of pigs suffering from porcine circovirus-associated disease (PCVAD). In this study, we investigated whether KIOM-C induces cancer cell death and elucidated the underlying anti-cancer mechanisms. In addition, we examined whether KIOM-C oral administration suppresses in vivo tumor growth of HT1080 cells in athymic nude mice. We initially found that KIOM-C at concentrations of 500 and 1000 µg/ml caused dose- and time-dependent cell death in cancer cells, but not normal hepatocytes, to approximately 50% of control levels. At the early stage of KIOM-C treatment (12 h), cells were arrested in G1 phase, which was accompanied by up-regulation of p21 and p27, down-regulation of cyclin D1, and subsequent increases in apoptotic and autophagic cells. Following KIOM-C treatment, the extent of caspase-3 activation, PARP cleavage, Beclin-1 expression, and LC3-II conversion was remarkably up-regulated, but p62 expression was down-regulated. Phosphorylation of AMPK, ULK, JNK, c-jun, and p53 was increased significantly in response to KIOM-C treatment. The levels of intracellular ROS and CHOP expression were also increased. In particular, the JNK-specific inhibitor SP600125 blocked KIOM-C-induced ROS generation and CHOP expression almost completely, which consequently almost completely rescued cell death, indicating that JNK activation plays a critical role in KIOM-C-induced cell death. Furthermore, daily oral administration of 85 and 170 mg/kg KIOM-C efficiently suppressed the tumorigenic growth of HT1080 cells, without systemic toxicity. These results collectively suggest that KIOM-C efficiently induces cancer cell death by both autophagy and

  10. Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways.

    PubMed

    Duan, Wen-Jun; Li, Qi-Sheng; Xia, Ming-Yu; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2011-01-01

    Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(•-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(•-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.

  11. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex.

    PubMed

    Zhong, Yun; Wang, Qing Jun; Li, Xianting; Yan, Ying; Backer, Jonathan M; Chait, Brian T; Heintz, Nathaniel; Yue, Zhenyu

    2009-04-01

    Beclin 1, a mammalian autophagy protein that has been implicated in development, tumour suppression, neurodegeneration and cell death, exists in a complex with Vps34, the class III phosphatidylinositol-3-kinase (PI(3)K) that mediates multiple vesicle-trafficking processes including endocytosis and autophagy. However, the precise role of the Beclin 1-Vps34 complex in autophagy regulation remains to be elucidated. Combining mouse genetics and biochemistry, we have identified a large in vivo Beclin 1 complex containing the known proteins Vps34, p150/Vps15 and UVRAG, as well as two newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein). Characterization of the new proteins revealed that Atg14L enhances Vps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces Vps34 activity and downregulates autophagy. We show that Beclin 1 and Atg14L synergistically promote the formation of double-membraned organelles that are associated with Atg5 and Atg12, whereas forced expression of Rubicon results in aberrant late endosomal/lysosomal structures and impaired autophagosome maturation. We hypothesize that by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of the class III PI(3)K in regulating autophagy at multiple steps.

  12. Lung autophagic response following exposure of mice to whole body irradiation, with and without amifostine

    SciTech Connect

    Zois, Christos E.; Giatromanolaki, Alexandra; Kainulainen, Heikki; Botaitis, Sotirios; Torvinen, Sira; Simopoulos, Constantinos; Kortsaris, Alexandros; Sivridis, Efthimios; Koukourakis, Michael I.

    2011-01-07

    Research highlights: {yields} We investigated the effect 6 Gy of WBI on the autophagic machinery of normal mouse lung. {yields} Irradiation induces dysfunction of the autophagic machinery in normal lung, characterized by decreased transcription of the LC3A/Beclin-1 mRNA and accumulation of the LC3A, and p62 proteins. {yields} The membrane bound LC3A-II protein levels increased in the cytosolic fraction (not in the pellet), contrasting the patterns noted after starvation-induced autophagy. {yields} Administration of amifostine, reversed all the LC3A and p62 findings, suggesting protection of the normal autophagic function. -- Abstract: Purpose: The effect of ionizing irradiation on the autophagic response of normal tissues is largely unexplored. Abnormal autophagic function may interfere the protein quality control leading to cell degeneration and dysfunction. This study investigates its effect on the autophagic machinery of normal mouse lung. Methods and materials: Mice were exposed to 6 Gy of whole body {gamma}-radiation and sacrificed at various time points. The expression of MAP1LC3A/LC3A/Atg8, beclin-1, p62/sequestosome-1 and of the Bnip3 proteins was analyzed. Results: Following irradiation, the LC3A-I and LC3A-II protein levels increased significantly at 72 h and 7 days. Strikingly, LC3A-II protein was increased (5.6-fold at 7 days; p < 0.001) only in the cytosolic fraction, but remained unchanged in the membrane fraction. The p62 protein, was significantly increased in both supernatant and pellet fraction (p < 0.001), suggesting an autophagosome turnover deregulation. These findings contrast the patterns of starvation-induced autophagy up-regulation. Beclin-1 levels remained unchanged. The Bnip3 protein was significantly increased at 8 h, but it sharply decreased at 72 h (p < 0.05). Administration of amifostine (200 mg/kg), 30 min before irradiation, reversed all the LC3A and p62 findings on blots, suggesting restoration of the normal autophagic function

  13. Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling.

    PubMed

    Wang, Yajing; Liang, Bin; Lau, Wayne Bond; Du, Yunhui; Guo, Rui; Yan, Zheyi; Gan, Lu; Yan, Wenjun; Zhao, Jianli; Gao, Erhe; Koch, Walter; Ma, Xin-Liang

    2017-08-21

    Macroautophagy/autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of ADIPOQ, a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout (adipoq(-/-)) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq(-/-) largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq(-/-) mice with AdipoRon, a novel ADIPOR (adiponectin receptor) agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P < 0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-class III PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacological AMPK inhibition or genetic Prkaa2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic

  14. A novel Rab10-EHBP1-EHD2 complex essential for the autophagic engulfment of lipid droplets

    PubMed Central

    Li, Zhipeng; Schulze, Ryan J.; Weller, Shaun G.; Krueger, Eugene W.; Schott, Micah B.; Zhang, Xiaodong; Casey, Carol A.; Liu, Jun; Stöckli, Jacqueline; James, David E.; McNiven, Mark A.

    2016-01-01

    The autophagic digestion of lipid droplets (LDs) through lipophagy is an essential process by which most cells catabolize lipids as an energy source. However, the cellular machinery used for the envelopment of LDs during autophagy is poorly understood. We report a novel function for a small Rab guanosine triphosphatase (GTPase) in the recruitment of adaptors required for the engulfment of LDs by the growing autophagosome. In hepatocytes stimulated to undergo autophagy, Rab10 activity is amplified significantly, concomitant with its increased recruitment to nascent autophagic membranes at the LD surface. Disruption of Rab10 function by small interfering RNA knockdown or expression of a GTPase-defective variant leads to LD accumulation. Finally, Rab10 activation during autophagy is essential for LC3 recruitment to the autophagosome and stimulates its increased association with the adaptor protein EHBP1 (EH domain binding protein 1) and the membrane-deforming adenosine triphosphatase EHD2 (EH domain containing 2) that, together, are essential in driving the activated “engulfment” of LDs during lipophagy in hepatocytes. PMID:28028537

  15. Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation.

    PubMed

    Gao, Caiji; Zhuang, Xiaohong; Cui, Yong; Fu, Xi; He, Yilin; Zhao, Qiong; Zeng, Yonglun; Shen, Jinbo; Luo, Ming; Jiang, Liwen

    2015-02-10

    Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 domain-containing protein2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.

  16. Autophagic regulation of smooth muscle cell biology.

    PubMed

    Salabei, Joshua K; Hill, Bradford G

    2015-01-01

    Autophagy regulates the metabolism, survival, and function of numerous cell types, including those comprising the cardiovascular system. In the vasculature, changes in autophagy have been documented in atherosclerotic and restenotic lesions and in hypertensive vessels. The biology of vascular smooth muscle cells appears particularly sensitive to changes in the autophagic program. Recent evidence indicates that stimuli or stressors evoked during the course of vascular disease can regulate autophagic activity, resulting in modulation of VSMC phenotype and viability. In particular, certain growth factors and cytokines, oxygen tension, and pharmacological drugs have been shown to trigger autophagy in smooth muscle cells. Importantly, each of these stimuli has a redox component, typically associated with changes in the abundance of reactive oxygen, nitrogen, or lipid species. Collective findings support the hypothesis that autophagy plays a critical role in vascular remodeling by regulating smooth muscle cell phenotype transitions and by influencing the cellular response to stress. In this graphical review, we summarize current knowledge on the role of autophagy in the biology of the smooth muscle cell in (patho)physiology. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Why should autophagic flux be assessed?

    PubMed Central

    Zhang, Xiao-jie; Chen, Sheng; Huang, Kai-xing; Le, Wei-dong

    2013-01-01

    As autophagy is involved in cell growth, survival, development and death, impaired autophagic flux has been linked to a variety of human pathophysiological processes, including neurodegeneration, cancer, myopathy, cardiovascular and immune-mediated disorders. There is a growing need to identify and quantify the status of autophagic flux in different pathological conditions. Given that autophagy is a highly dynamic and complex process that is regulated at multiple steps, it is often assessed accurately. This perspective review article will focus on the autophagic flux defects in different human disorders and update the current methods of monitoring autophagic flux. This knowledge is essential for developing autophagy-related therapeutics for treating the diseases. PMID:23474710

  18. Signalome-wide RNAi screen identifies GBA1 as a positive mediator of autophagic cell death

    PubMed Central

    Dasari, Santosh K; Bialik, Shani; Levin-Zaidman, Smadar; Levin-Salomon, Vered; Merrill, Alfred H; Futerman, Anthony H; Kimchi, Adi

    2017-01-01

    Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death. The cells displayed a strong and sustained induction of autophagic flux, cell death was prevented by knocking down autophagic genes and death occurred in the absence of apoptotic or necroptotic pathway activation. Detailed ultrastructural characterization revealed additional critical events, including a continuous increase over time in the number of autophagic vacuoles, in particular autolysosomes, occupying most of the cytoplasm at terminal stages. This was followed by loss of organelles, disruption of intracellular membranes including the swelling of perinuclear space and, occasionally, a unique type of nuclear shedding. A signalome-wide shRNA-based viability screen was applied to identify positive mediators of this type of autophagic cell death. One top hit was GBA1, the Gaucher disease-associated gene, which encodes glucocerebrosidase, an enzyme that metabolizes glucosylceramide to ceramide and glucose. Interestingly, glucocerebrosidase expression levels and activity were elevated, concomitantly with increased intracellular ceramide levels, both of which correlated in time with the appearance of the unique death characteristics. Transfection with siGBA1 attenuated the increase in glucocerebrosidase activity and the intracellular ceramide levels. Most importantly, GBA1 knockdown prevented the strong increase in LC3 lipidation, and many of the ultrastructural changes characteristic of this type of autophagic cell death, including a significant

  19. 4-Hydroxytamoxifen induces autophagic death through K-Ras degradation.

    PubMed

    Kohli, Latika; Kaza, Niroop; Coric, Tatjana; Byer, Stephanie J; Brossier, Nicole M; Klocke, Barbara J; Bjornsti, Mary-Ann; Carroll, Steven L; Roth, Kevin A

    2013-07-15

    Tamoxifen is widely used to treat estrogen receptor-positive breast cancer. Recent findings that tamoxifen and its derivative 4-hydroxytamoxifen (OHT) can exert estrogen receptor-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in estrogen receptor-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNST) where estrogen is not involved. In this study, we gained insights into the estrogen receptor-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated autophagic degradation of K-Ras, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma, and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

  20. AUTOPHAGIC VACUOLES PRODUCED IN VITRO

    PubMed Central

    Fedorko, Martha E.; Hirsch, James G.; Cohn, Zanvil A.

    1968-01-01

    Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1–2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes. PMID:4874492

  1. Improved hybrid isolator with maglev actuator integrated in air spring for active-passive isolation of ship machinery vibration

    NASA Astrophysics Data System (ADS)

    Li, Yan; He, Lin; Shuai, Chang-geng; Wang, Chun-yu

    2017-10-01

    A hybrid isolator consisting of maglev actuator and air spring is proposed and developed for application in active-passive vibration isolation system of ship machinery. The dynamic characteristics of this hybrid isolator are analyzed and tested. The stability and adaptability of this hybrid isolator to shock and swing in the marine environment are improved by a compliant gap protection technique and a disengageable suspended structure. The functions of these new engineering designs are proved by analytical verification and experimental validation of the designed stiffness of such a hybrid isolator, and also by shock adaptability testing of the hybrid isolator. Finally, such hybrid isolators are installed in an engineering mounting loaded with a 200-kW ship diesel generator, and the broadband and low-frequency sinusoidal isolation performance is tested.

  2. Activation of Short and Long Chain Fatty Acid Sensing Machinery in the Ileum Lowers Glucose Production in Vivo.

    PubMed

    Zadeh-Tahmasebi, Melika; Duca, Frank A; Rasmussen, Brittany A; Bauer, Paige V; Côté, Clémence D; Filippi, Beatrice M; Lam, Tony K T

    2016-04-15

    Evidence continues to emerge detailing the myriad of ways the gut microbiota influences host energy homeostasis. Among the potential mechanisms, short chain fatty acids (SCFAs), the byproducts of microbial fermentation of dietary fibers, exhibit correlative beneficial metabolic effects in humans and rodents, including improvements in glucose homeostasis. The underlying mechanisms, however, remain elusive. We here report that one of the main bacterially produced SCFAs, propionate, activates ileal mucosal free fatty acid receptor 2 to trigger a negative feedback pathway to lower hepatic glucose production in healthy rats in vivo We further demonstrate that an ileal glucagon-like peptide-1 receptor-dependent neuronal network is necessary for ileal propionate and long chain fatty acid sensing to regulate glucose homeostasis. These findings highlight the potential to manipulate fatty acid sensing machinery in the ileum to regulate glucose homeostasis.

  3. AUTOPHAGIC VACUOLES PRODUCED IN VITRO

    PubMed Central

    Fedorko, Martha E.; Hirsch, James G.; Cohn, Zanvil A.

    1968-01-01

    Mouse macrophages exposed to 30 µg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1–4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells. PMID:4874491

  4. Aneuploidy-induced cellular stresses limit autophagic degradation

    PubMed Central

    Santaguida, Stefano; Vasile, Eliza; White, Eileen; Amon, Angelika

    2015-01-01

    An unbalanced karyotype, a condition known as aneuploidy, has a profound impact on cellular physiology and is a hallmark of cancer. Aneuploid cells experience a number of stresses that are caused by aneuploidy-induced proteomic changes. How the aneuploidy-associated stresses affect cells and whether cells respond to them are only beginning to be understood. Here we show that autophagosomal cargo such as protein aggregates accumulate within lysosomes in aneuploid cells. This causes a lysosomal stress response. Aneuploid cells activate the transcription factor TFEB, a master regulator of autophagic and lysosomal gene expression, thereby increasing the expression of genes needed for autophagy-mediated protein degradation. Accumulation of autophagic cargo within the lysosome and activation of TFEB-responsive genes are also observed in cells in which proteasome function is inhibited, suggesting that proteotoxic stress causes TFEB activation. Our results reveal a TFEB-mediated lysosomal stress response as a universal feature of the aneuploid state. PMID:26404941

  5. Aneuploidy-induced cellular stresses limit autophagic degradation.

    PubMed

    Santaguida, Stefano; Vasile, Eliza; White, Eileen; Amon, Angelika

    2015-10-01

    An unbalanced karyotype, a condition known as aneuploidy, has a profound impact on cellular physiology and is a hallmark of cancer. Aneuploid cells experience a number of stresses that are caused by aneuploidy-induced proteomic changes. How the aneuploidy-associated stresses affect cells and whether cells respond to them are only beginning to be understood. Here we show that autophagosomal cargo such as protein aggregates accumulate within lysosomes in aneuploid cells. This causes a lysosomal stress response. Aneuploid cells activate the transcription factor TFEB, a master regulator of autophagic and lysosomal gene expression, thereby increasing the expression of genes needed for autophagy-mediated protein degradation. Accumulation of autophagic cargo within the lysosome and activation of TFEB-responsive genes are also observed in cells in which proteasome function is inhibited, suggesting that proteotoxic stress causes TFEB activation. Our results reveal a TFEB-mediated lysosomal stress response as a universal feature of the aneuploid state.

  6. Attenuation of Aβ{sub 25–35}-induced parallel autophagic and apoptotic cell death by gypenoside XVII through the estrogen receptor-dependent activation of Nrf2/ARE pathways

    SciTech Connect

    Meng, Xiangbao; Wang, Min; Sun, Guibo; Ye, Jingxue; Zhou, Yanhui; Dong, Xi; Wang, Tingting; Lu, Shan; Sun, Xiaobo

    2014-08-15

    Amyloid-beta (Aβ) has a pivotal function in the pathogenesis of Alzheimer's disease. To investigate Aβ neurotoxicity, we used an in vitro model that involves Aβ{sub 25–35}-induced cell death in the nerve growth factor-induced differentiation of PC12 cells. Aβ{sub 25–35} (20 μM) treatment for 24 h caused apoptotic cell death, as evidenced by significant cell viability reduction, LDH release, phosphatidylserine externalization, mitochondrial membrane potential disruption, cytochrome c release, caspase-3 activation, PARP cleavage, and DNA fragmentation in PC12 cells. Aβ{sub 25–35} treatment led to autophagic cell death, as evidenced by augmented GFP-LC3 puncta, conversion of LC3-I to LC3-II, and increased LC3-II/LC3-I ratio. Aβ{sub 25–35} treatment induced oxidative stress, as evidenced by intracellular ROS accumulation and increased production of mitochondrial superoxide, malondialdehyde, protein carbonyl, and 8-OHdG. Phytoestrogens have been proved to be protective against Aβ-induced neurotoxicity and regarded as relatively safe targets for AD drug development. Gypenoside XVII (GP-17) is a novel phytoestrogen isolated from Gynostemma pentaphyllum or Panax notoginseng. Pretreatment with GP-17 (10 μM) for 12 h increased estrogen response element reporter activity, activated PI3K/Akt pathways, inhibited GSK-3β, induced Nrf2 nuclear translocation, augmented antioxidant responsive element enhancer activity, upregulated heme oxygenase 1 (HO-1) expression and activity, and provided protective effects against Aβ{sub 25–35}-induced neurotoxicity, including oxidative stress, apoptosis, and autophagic cell death. In conclusion, GP-17 conferred protection against Aβ{sub 25–35}-induced neurotoxicity through estrogen receptor-dependent activation of PI3K/Akt pathways, inactivation of GSK-3β and activation of Nrf2/ARE/HO-1 pathways. This finding might provide novel insights into understanding the mechanism for neuroprotective effects of phytoestrogens or

  7. Autophagic death probed by photodynamic therapy

    PubMed Central

    Kessel, David

    2015-01-01

    The high degree of selectivity for photodamage to subcellular organelles can provide a means for evaluation of autophagic death pathways. While many current reports rely on ambiguous criteria, there are glimmers of unequivocal evidence. PMID:26313747

  8. Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux

    PubMed Central

    Kim, Hyunju; Lee, Kang Il; Jang, Minsu; Namkoong, Sim; Park, Rackhyun; Ju, Hyunwoo; Choi, Inho; Oh, Won Keun

    2016-01-01

    Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2) result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux. PMID:27257813

  9. Comparison of vibration amplitude supression vs. dynamic bearing load suppression in active vibration control of rotating machinery

    NASA Astrophysics Data System (ADS)

    Clark, William W.; Kim, J. H.; Marangoni, Roy D.

    1993-04-01

    This paper presents two optimal control methods for attenuating steady-state vibrations in rotating machinery. One method minimizes shaft displacements while the other minimizes dynamic bearing reaction forces. The two methods are applied to a model of a typical rotating machinery system, and their results are compared. It is found that displacement minimization can increase bearing loads, while bearing load minimization, on the other hand, decreases bearing loads without significant change in shaft displacements.

  10. Involvement of Antibiotic Efflux Machinery in Glutathione-Mediated Decreased Ciprofloxacin Activity in Escherichia coli.

    PubMed

    Goswami, Manish; Subramanian, Mahesh; Kumar, Ranjeet; Jass, Jana; Jawali, Narendra

    2016-07-01

    We have analyzed the contribution of different efflux components to glutathione-mediated abrogation of ciprofloxacin's activity in Escherichia coli and the underlying potential mechanism(s) behind this phenomenon. The results indicated that glutathione increased the total active efflux, thereby partially contributing to glutathione-mediated neutralization of ciprofloxacin's antibacterial action in E. coli However, the role of glutathione-mediated increased efflux becomes evident in the absence of a functional TolC-AcrAB efflux pump. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Active vibration control of rotating machinery with a hybrid piezohydraulic actuator system

    SciTech Connect

    Tang, P.; Palazzolo, A.B.; Kascak, A.F.; Montague, G.T.

    1995-10-01

    An integrated, compact piezohydraulic actuator system for active vibration control was designed and developed with a primary application for gas turbine aircraft engines. Copper tube was chosen as the transmission line material for ease of assembly. Liquid plastic, which meets incompressibility and low-viscosity requirements, was adjusted to provide optimal actuator performance. Variants of the liquid plastic have been prepared with desired properties between {minus}40 F and 400 F. The effectiveness of this hybrid actuator for active vibration control (AVC) was demonstrated for suppressing critical speed vibration through two critical speeds for various levels of intentionally placed imbalance. A high-accuracy closed-loop simulation, which combines both finite element and state space methods, was applied for the closed-loop unbalance response simulation with/without AVC. Good correlation between the simulation and test results was achieved.

  12. The ESCRT machinery regulates the secretion and long-range activity of Hedgehog.

    PubMed

    Matusek, Tamás; Wendler, Franz; Polès, Sophie; Pizette, Sandrine; D'Angelo, Gisela; Fürthauer, Maximilian; Thérond, Pascal P

    2014-12-04

    The conserved family of Hedgehog (Hh) proteins acts as short- and long-range secreted morphogens, controlling tissue patterning and differentiation during embryonic development. Mature Hh carries hydrophobic palmitic acid and cholesterol modifications essential for its extracellular spreading. Various extracellular transportation mechanisms for Hh have been suggested, but the pathways actually used for Hh secretion and transport in vivo remain unclear. Here we show that Hh secretion in Drosophila wing imaginal discs is dependent on the endosomal sorting complex required for transport (ESCRT). In vivo the reduction of ESCRT activity in cells producing Hh leads to a retention of Hh at the external cell surface. Furthermore, we show that ESCRT activity in Hh-producing cells is required for long-range signalling. We also provide evidence that pools of Hh and ESCRT proteins are secreted together into the extracellular space in vivo and can subsequently be detected together at the surface of receiving cells. These findings uncover a new function for ESCRT proteins in controlling morphogen activity and reveal a new mechanism for the transport of secreted Hh across the tissue by extracellular vesicles, which is necessary for long-range target induction.

  13. Experimental study on active structural acoustic control of rotating machinery using rotating piezo-based inertial actuators

    NASA Astrophysics Data System (ADS)

    Zhao, G.; Alujević, N.; Depraetere, B.; Pinte, G.; Swevers, J.; Sas, P.

    2015-07-01

    In this paper, two Piezo-Based Rotating Inertial Actuators (PBRIAs) are considered for the suppression of the structure-borne noise radiated from rotating machinery. As add-on devices, they can be directly mounted on a rotational shaft, in order to intervene as early as possible in the transfer path between disturbance and the noise radiating surfaces. A MIMO (Multi-Input-Multi-Output) form of the FxLMS control algorithm is employed to generate the appropriate actuation signals, relying on a linear interpolation scheme to approximate time varying secondary plants. The proposed active vibration control approach is tested on an experimental test bed comprising a rotating shaft mounted in a frame to which a noise-radiating plate is attached. The disturbance force is introduced by an electro-dynamic shaker. The experimental results show that when the shaft spins below 180 rpm, more than a 7 dB reduction can be achieved in terms of plate vibrations, along with a reduction in the same order of magnitude in terms of noise radiation.

  14. Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification

    PubMed Central

    Li, Dan L.; Wang, Zhao V.; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W.; Gillette, Thomas G.; Hill, Joseph A.

    2016-01-01

    Background The clinical use of doxorubicin is limited by cardiotoxicity. Histopathologic changes include interstitial myocardial fibrosis and appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Methods and Results Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity due to haploinsufficiency for Beclin 1. Beclin 1+/− mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals over-expressing Beclin 1 manifested an amplified cardiotoxic response. Conclusions Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. PMID:26984939

  15. Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification.

    PubMed

    Li, Dan L; Wang, Zhao V; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W; Gillette, Thomas G; Hill, Joseph A

    2016-04-26

    The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. © 2016 American Heart Association, Inc.

  16. Use of piezoelectric actuators in active vibration control of rotating machinery

    NASA Technical Reports Server (NTRS)

    Lin, Reng Rong; Palazzolo, Alan B.; Kascak, Albert F.; Montague, Gerald

    1990-01-01

    Theoretical and test results for the development of piezoelectric-actuator-based active vibration control (AVC) are presented. The evolution of this technology starts with an ideal model of the actuator and progresses to a more sophisticated model where the pushers force the squirrel cage ball bearing supports of a rotating shaft. The piezoelectric pushers consist of a stack of piezoelectric ceramic disks that are arranged on top of one another and connected in parallel electrically. This model consists of a prescribed displacement that is proportional to the input voltage and a spring that represents the stiffness of the stack of piezoelectric disks. System tests were carried out to stabilize the AVC system, verify its effectiveness in controlling vibration, and confirm the theory presented.

  17. Use of piezoelectric actuators in active vibration control of rotating machinery

    NASA Technical Reports Server (NTRS)

    Lin, Reng Rong; Palazzolo, Alan B.; Kascak, Albert F.; Montague, Gerald

    1990-01-01

    Theoretical and test results for the development of piezoelectric-actuator-based active vibration control (AVC) are presented. The evolution of this technology starts with an ideal model of the actuator and progresses to a more sophisticated model where the pushers force the squirrel cage ball bearing supports of a rotating shaft. The piezoelectric pushers consist of a stack of piezoelectric ceramic disks that are arranged on top of one another and connected in parallel electrically. This model consists of a prescribed displacement that is proportional to the input voltage and a spring that represents the stiffness of the stack of piezoelectric disks. System tests were carried out to stabilize the AVC system, verify its effectiveness in controlling vibration, and confirm the theory presented.

  18. Endothelial IL-33 Expression Is Augmented by Adenoviral Activation of the DNA Damage Machinery.

    PubMed

    Stav-Noraas, Tor Espen; Edelmann, Reidunn J; Poulsen, Lars La Cour; Sundnes, Olav; Phung, Danh; Küchler, Axel M; Müller, Fredrik; Kamen, Amine A; Haraldsen, Guttorm; Kaarbø, Mari; Hol, Johanna

    2017-04-15

    IL-33, required for viral clearance by cytotoxic T cells, is generally expressed in vascular endothelial cells in healthy human tissues. We discovered that endothelial IL-33 expression was stimulated as a response to adenoviral transduction. This response was dependent on MRE11, a sensor of DNA damage that can also be activated by adenoviral DNA, and on IRF1, a transcriptional regulator of cellular responses to viral invasion and DNA damage. Accordingly, we observed that endothelial cells responded to adenoviral DNA by phosphorylation of ATM and CHK2 and that depletion or inhibition of MRE11, but not depletion of ATM, abrogated IL-33 stimulation. In conclusion, we show that adenoviral transduction stimulates IL-33 expression in endothelial cells in a manner that is dependent on the DNA-binding protein MRE11 and the antiviral factor IRF1 but not on downstream DNA damage response signaling. Copyright © 2017 by The American Association of Immunologists, Inc.

  19. MTOR-independent, autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Zhang, Xiaojie; Chen, Sheng; Song, Lin; Tang, Yu; Shen, Yufei; Jia, Li; Le, Weidong

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1G93A mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1G93A mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1G93A mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1G93A mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1G93A mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS. PMID:24441414

  20. Quantitation of selective autophagic protein aggregate degradation in vitro and in vivo using luciferase reporters

    PubMed Central

    Ju, Jeong-Sun; Miller, Sara E.; Jackson, Erin; Cadwell, Ken; Piwnica-Worms, David; Weihl, Conrad C.

    2010-01-01

    The analysis of autophagy in cells and tissue has principally been performed via qualitative measures. These assays identify autophagosomes or measure the conversion of LC3I to LC3II. However, qualitative assays fail to quantitate the degradation of an autophagic substrate and therefore only indirectly measure an intact autophagic system. “Autophagic flux” can be measured using long-lived proteins that are degraded via autophagy. We developed a quantifiable luciferase reporter assay that measures the degradation of a long-lived polyglutamine protein aggregate, polyQ80-luciferase. Using this reporter, the induction of autophagy via starvation or rapamycin in cells preferentially decreases polyQ80-luciferase when compared with a non-aggregating polyQ19-luciferase after four hours of treatment. This response was both time- and concentration-dependent, prevented by autophagy inhibitors and absent in ATG5 knockout cells. We adapted this assay to living animals by electroporating polyQ19-luciferase and polyQ80-luciferase expression constructs into the right and left tibialis anterior (TA) muscles of mice, respectively. The change in the ratio of polyQ80-luciferase to polyQ19-luciferase signal before and after autophagic stimulation or inhibition was quantified via in vivo bioluminescent imaging. Following two days of starvation or treatment with intraperitoneal rapamycin, there was a ~35% reduction in the ratio of polyQ80:polyQ19-luciferase activity, consistent with the selective autophagic degradation of polyQ80 protein. This autophagic response in skeletal muscle in vivo was abrogated by co-treatment with chloroquine and in ATG16L1 hypomorphic mice. Our study demonstrates a method to quantify the autophagic flux of an expanded polyglutamine via luciferase reporters in vitro and in vivo. PMID:19305149

  1. New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach.

    PubMed

    Malagoli, Davide; Boraldi, Federica; Annovi, Giulia; Quaglino, Daniela; Ottaviani, Enzo

    2009-04-01

    Autophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity.

  2. Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress

    PubMed Central

    ZHANG, Si-Wei; FENG, Jiang-Nan; CAO, Yi; MENG, Li-Ping; WANG, Shu-Lin

    2015-01-01

    Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species (ROS) generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membrane-bound vacuoles characteristic of autophagy followed by autophagic cell death (referred to as cell death with autophagy) after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena. PMID:26018860

  3. Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress.

    PubMed

    Zhang, Si-Wei; Feng, Jiang-Nan; Cao, Yi; Meng, Li-Ping; Wang, Shu-Lin

    2015-05-18

    Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species (ROS) generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membrane-bound vacuoles characteristic of autophagy followed by autophagic cell death (referred to as cell death with autophagy) after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena.

  4. Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload

    PubMed Central

    Fu, Lianwu; Wei, Chih-Chang; Powell, Pamela C.; Bradley, Wayne E.; Ahmad, Sarfaraz; Ferrario, Carlos M.; Collawn, James F.; Dell’Italia, Louis J.

    2016-01-01

    Background Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. Methods and results Isolated LV fibroblasts taken from 4 and 12 week ACF Sprague–Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24 h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase’s role in autophagy induction. Conclusion In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation. PMID:26807691

  5. Autophagic Adaptations to Long-term Habitual Exercise in Cardiac Muscle.

    PubMed

    Tam, B T; Pei, X M; Yung, B Y; Yip, S P; Chan, L W; Wong, C S; Siu, P M

    2015-06-01

    Autophagy has been shown to be responsive to physical exercise. However, the effects of prolonged habitual exercise on autophagy in cardiac muscle remain unknown. The present study aimed to examine whether long-term habitual exercise alters the basal autophagic signalling in cardiac muscle. Female Sprague-Dawley rats aged 2 months were randomly assigned to control and exercise groups. Animals in exercise group were kept in cages with free access exercise wheels to perform habitual exercise for 5 months. Animals in the control group were placed in cages without exercise wheels. Ventricular muscle tissues were harvested for analysis after 5 months. Phosphorylation statuses of upstream autophagic regulatory proteins and protein expressions of downstream autophagic facts remained unchanged in the cardiac muscle of exercise animals when compared to control animals. Intriguingly, the protein abundance of microtubule-associated protein-1 light chain -3 II (LC3-II), heat shock protein 72 (HSP72) and peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α) were significantly increased in cardiac muscle of exercise rats relative to control rats. 5 months of habitual exercise causes the adaptive increase in LC3-II reserve without altering autophagic flux, which probably contributes to the elevation of cellular autophagic capacity and efficiency of cardiac muscle.

  6. Autophagic flux regulates microglial phenotype according to the time of oxygen-glucose deprivation/reperfusion.

    PubMed

    Xia, Cong-Yuan; Zhang, Shuai; Chu, Shi-Feng; Wang, Zhen-Zhen; Song, Xiu-Yun; Zuo, Wei; Gao, Yan; Yang, Peng-Fei; Chen, Nai-Hong

    2016-10-01

    Microglial phenotype alternation is a potential novel pathogenic mechanism for cerebral ischemia. Cerebral ischemia induced autophagy aggravates inflammation and neural injury. However, the effect of autophagy in the modulation of microglial phenotype is still unknown. In this study, we investigated the role of autophagic flux in the alternation of microglial phenotype following oxygen glucose deprivation/reperfusion (OGD/R) in BV-2 cells. Inhibition of autophagic flux by NH4Cl exposure significantly increased the level of microtubule-associated protein 1 light chain 3 (LC3)-II and p62 in control and OGD/R (12h, 24h and 48h) groups, but did not change their expression in OGD/R 72h group, indicating that autophagic flux was inhibited at OGD/R 72h. Once autophagic flux was inhibited at OGD/R 72h or at OGD/R 24h (with NH4Cl), BV-2 cells mainly showed M1 phenotype with increased tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and decreased M2 markers including interleukin-10 (IL-10), Arginase 1 (Arg-1), and brain derived neurotrophic factor (BDNF). Further study indicated that inhibition of autophagic flux activated NF-κB pathway and decreased the activity of cAMP-response element binding protein (CREB), which contributed to the alternation of microglial phenotype. Therefore, inhibition of autophagic flux regulated the alternation of microglial phenotype by modulating the balance between NF-κB and CREB.

  7. ATG13: just a companion, or an executor of the autophagic program?

    PubMed

    Alers, Sebastian; Wesselborg, Sebastian; Stork, Björn

    2014-06-01

    During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play.

  8. ADP-ribosylation factor 1 protein regulates trypsinogen activation via organellar trafficking of procathepsin B protein and autophagic maturation in acute pancreatitis.

    PubMed

    Orlichenko, Lidiya; Stolz, Donna B; Noel, Pawan; Behari, Jaideep; Liu, Shiguang; Singh, Vijay P

    2012-07-13

    Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.

  9. Aberrant Autophagic Response in The Muscle of A Knock-in Mouse Model of Spinal and Bulbar Muscular Atrophy.

    PubMed

    Rusmini, Paola; Polanco, Maria Josefa; Cristofani, Riccardo; Cicardi, Maria Elena; Meroni, Marco; Galbiati, Mariarita; Piccolella, Margherita; Messi, Elio; Giorgetti, Elisa; Lieberman, Andrew P; Milioto, Carmelo; Rocchi, Anna; Aggarwal, Tanya; Pennuto, Maria; Crippa, Valeria; Poletti, Angelo

    2015-10-22

    Spinal and bulbar muscular atrophy (SBMA) is characterized by loss of motoneurons and sensory neurons, accompanied by atrophy of muscle cells. SBMA is due to an androgen receptor containing a polyglutamine tract (ARpolyQ) that misfolds and aggregates, thereby perturbing the protein quality control (PQC) system. Using SBMA AR113Q mice we analyzed proteotoxic stress-induced alterations of HSPB8-mediated PQC machinery promoting clearance of misfolded proteins by autophagy. In muscle of symptomatic AR113Q male mice, we found expression upregulation of Pax-7, myogenin, E2-ubiquitin ligase UBE2Q1 and acetylcholine receptor (AchR), but not of MyoD, and of two E3-ligases (MuRF-1 and Cullin3). TGFβ1 and PGC-1α were also robustly upregulated. We also found a dramatic perturbation of the autophagic response, with upregulation of most autophagic markers (Beclin-1, ATG10, p62/SQSTM1, LC3) and of the HSPB8-mediated PQC response. Both HSPB8 and its co-chaperone BAG3 were robustly upregulated together with other specific HSPB8 interactors (HSPB2 and HSPB3). Notably, the BAG3:BAG1 ratio increased in muscle suggesting preferential misfolded proteins routing to autophagy rather than to proteasome. Thus, mutant ARpolyQ induces a potent autophagic response in muscle cells. Alteration in HSPB8-based PQC machinery may represent muscle-specific biomarkers useful to assess SBMA progression in mice and patients in response to pharmacological treatments.

  10. Aberrant Autophagic Response in The Muscle of A Knock-in Mouse Model of Spinal and Bulbar Muscular Atrophy

    PubMed Central

    Rusmini, Paola; Polanco, Maria Josefa; Cristofani, Riccardo; Cicardi, Maria Elena; Meroni, Marco; Galbiati, Mariarita; Piccolella, Margherita; Messi, Elio; Giorgetti, Elisa; Lieberman, Andrew P.; Milioto, Carmelo; Rocchi, Anna; Aggarwal, Tanya; Pennuto, Maria; Crippa, Valeria; Poletti, Angelo

    2015-01-01

    Spinal and bulbar muscular atrophy (SBMA) is characterized by loss of motoneurons and sensory neurons, accompanied by atrophy of muscle cells. SBMA is due to an androgen receptor containing a polyglutamine tract (ARpolyQ) that misfolds and aggregates, thereby perturbing the protein quality control (PQC) system. Using SBMA AR113Q mice we analyzed proteotoxic stress-induced alterations of HSPB8-mediated PQC machinery promoting clearance of misfolded proteins by autophagy. In muscle of symptomatic AR113Q male mice, we found expression upregulation of Pax-7, myogenin, E2-ubiquitin ligase UBE2Q1 and acetylcholine receptor (AchR), but not of MyoD, and of two E3-ligases (MuRF-1 and Cullin3). TGFβ1 and PGC-1α were also robustly upregulated. We also found a dramatic perturbation of the autophagic response, with upregulation of most autophagic markers (Beclin-1, ATG10, p62/SQSTM1, LC3) and of the HSPB8-mediated PQC response. Both HSPB8 and its co-chaperone BAG3 were robustly upregulated together with other specific HSPB8 interactors (HSPB2 and HSPB3). Notably, the BAG3:BAG1 ratio increased in muscle suggesting preferential misfolded proteins routing to autophagy rather than to proteasome. Thus, mutant ARpolyQ induces a potent autophagic response in muscle cells. Alteration in HSPB8-based PQC machinery may represent muscle-specific biomarkers useful to assess SBMA progression in mice and patients in response to pharmacological treatments. PMID:26490709

  11. Apigenin potentiates the antitumor activity of 5-FU on solid Ehrlich carcinoma: Crosstalk between apoptotic and JNK-mediated autophagic cell death platforms.

    PubMed

    Gaballah, Hanaa H; Gaber, Rasha A; Mohamed, Darin A

    2017-02-01

    Although 5- Fluorouracil (5-FU) has exhibited effectiveness against cancer, novel therapeutic strategies are needed to enhance its antitumor efficiency and modulate its cytotoxity. Apigenin, a flavonoid present in fruits and vegetables, is a potent dietary phytochemical effective in cancer chemoprevention. This study was undertaken to investigate the potential synergistic antitumor activity of apigenin and 5-FU on Solid Ehrlich carcinoma (SEC). Eighty Swiss albino male mice were divided into four equal groups: vehicle treated control SEC, SEC+5-FU, SEC+apigenin, SEC+ 5-FU+apigenin. Beclin-1 and caspases 3, 9 and JNK activities were estimated by ELISA; mRNA expression levels of the antiapoptotic gene Mcl-1 were estimated using quantitative real-time RT-PCR, while tissue malondialdehyde (MDA), glutathione peroxidase and total antioxidant capacity were evaluated spectrophotometrically. A part of the tumor was examined for histopathological and Ki-67 immunohistochemistry analysis. 5-FU and/or apigenin caused significant increase in tissue levels of Beclin-1, caspases 3, 9 and JNK activities, MDA with significant decrease in tumor volume, Mcl-1expression, tissue glutathione peroxidase and total antioxidant capacity and alleviated the histopathological changes with significant decrease of Ki-67 proliferation index compared to vehicle treated SEC control group. The combination of 5-FU and apigenin had a greater effect than each of 5-FU or apigenin alone against solid Ehrlich carcinoma in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways*

    PubMed Central

    Agarwal, Swati; Tiwari, Shashi Kant; Seth, Brashket; Yadav, Anuradha; Singh, Anshuman; Mudawal, Anubha; Chauhan, Lalit Kumar Singh; Gupta, Shailendra Kumar; Choubey, Vinay; Tripathi, Anurag; Kumar, Amit; Ray, Ratan Singh; Shukla, Shubha; Parmar, Devendra; Chaturvedi, Rajnish Kumar

    2015-01-01

    The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A1 increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A1) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell's compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be

  13. Mcl-1-dependent activation of Beclin 1 mediates autophagic cell death induced by sorafenib and SC-59 in hepatocellular carcinoma cells

    PubMed Central

    Tai, W-T; Shiau, C-W; Chen, H-L; Liu, C-Y; Lin, C-S; Cheng, A-L; Chen, P-J; Chen, K-F

    2013-01-01

    We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway. PMID:23392173

  14. Samsoeum, a traditional herbal medicine, elicits apoptotic and autophagic cell death by inhibiting Akt/mTOR and activating the JNK pathway in cancer cells

    PubMed Central

    2013-01-01

    Background Samsoeum (SSE), a traditional herbal formula, has been widely used to treat cough, fever, congestion, and emesis for centuries. Recent studies have demonstrated that SSE retains potent pharmacological efficiency in anti-allergic and anti-inflammatory reactions. However, the anti-cancer activity of SSE and its underlying mechanisms have not been studied. Thus, the present study was designed to determine the effect of SSE on cell death and elucidate its detailed mechanism. Methods Following SSE treatment, cell growth and cell death were measured using an MTT assay and trypan blue exclusion assay, respectively. Cell cycle arrest and YO-PRO-1 uptake were assayed using flow cytometry, and LC3 redistribution was observed using confocal microscope. The mechanisms of anti-cancer effect of SSE were investigated through western blot analysis. Results We initially found that SSE caused dose- and time-dependent cell death in cancer cells but not in normal primary hepatocytes. In addition, during early SSE treatment (6–12 h), cells were arrested in G2/M phase concomitant with up-regulation of p21 and p27 and down-regulation of cyclin D1 and cyclin B1, followed by an increase in apoptotic YO-PRO-1 (+) cells. SSE also induced autophagy via up-regulation of Beclin-1 expression, conversion of microtubule-associated protein light chain 3 (LC3) I to LC3-II, and re-distribution of LC3, indicating autophagosome formation. Moreover, the level of B-cell lymphoma 2 (Bcl-2), which is critical for cross-talk between apoptosis and autophagy, was significantly reduced in SSE-treated cells. Phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was increased, followed by suppression of the protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathway, and phosphorylation of mitogen-activated protein kinases (MAPKs) in response to SSE treatment. In particular, among MAPKs inhibitors, only the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 nearly

  15. MAPK/JNK1 activation protects cells against cadmium-induced autophagic cell death via differential regulation of catalase and heme oxygenase-1 in oral cancer cells.

    PubMed

    So, Keum-Young; Kim, Sang-Hun; Jung, Ki-Tae; Lee, Hyun-Young; Oh, Seon-Hee

    2017-10-01

    Antioxidant enzymes are related to oral diseases. We investigated the roles of heme oxygenase-1 (HO-1) and catalase in cadmium (Cd)-induced oxidative stress and the underlying molecular mechanism in oral cancer cells. Exposing YD8 cells to Cd reduced the expression levels of catalase and superoxide dismutase 1/2 and induced the expression of HO-1 as well as autophagy and apoptosis, which were reversed by N-acetyl-l-cysteine (NAC). Cd-exposed YD10B cells exhibited milder effects than YD8 cells, indicating that Cd sensitivity is associated with antioxidant enzymes and autophagy. Autophagy inhibition via pharmacologic and genetic modulations enhanced Cd-induced HO-1 expression, caspase-3 cleavage, and the production of reactive oxygen species (ROS). Ho-1 knockdown increased autophagy and apoptosis. Hemin treatment partially suppressed Cd-induced ROS production and apoptosis, but enhanced autophagy and CHOP expression, indicating that autophagy induction is associated with cellular stress. Catalase inhibition by pharmacological and genetic modulations increased Cd-induced ROS production, autophagy, and apoptosis, but suppressed HO-1, indicating that catalase is required for HO-1 induction. p38 inhibition upregulated Cd-induced phospho-JNK and catalase, but suppressed HO-1, autophagy, apoptosis. JNK suppression exhibited contrary results, enhancing the expression of phospho-p38. Co-suppression of p38 and JNK1 failed to upregulate catalase and procaspase-3, which were upregulated by JNK1 overexpression. Overall, the balance between the responses of p38 and JNK activation to Cd appears to have an important role in maintaining cellular homeostasis via the regulation of antioxidant enzymes and autophagy induction. In addition, the upregulation of catalase by JNK1 activation can play a critical role in cell protection against Cd-induced oxidative stress. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Lysine suppresses protein degradation through autophagic-lysosomal system in C2C12 myotubes.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nedachi, Taku; Nagasawa, Takashi

    2014-06-01

    Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic-lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0-3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic-lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic-lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic-lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic-lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.

  17. Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak.

    PubMed

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B H; Yue, Zhenyu; Oh, Young J

    2012-07-06

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

  18. Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak*

    PubMed Central

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B. H.; Yue, Zhenyu; Oh, Young J.

    2012-01-01

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K+/H+ ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis. PMID:22493436

  19. Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates

    PubMed Central

    Maulucci, Giuseppe; Chiarpotto, Michela; Papi, Massimiliano; Samengo, Daniela; Pani, Giovambattista; De Spirito, Marco

    2015-01-01

    Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5–6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates. PMID:26506895

  20. Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates.

    PubMed

    Maulucci, Giuseppe; Chiarpotto, Michela; Papi, Massimiliano; Samengo, Daniela; Pani, Giovambattista; De Spirito, Marco

    2015-01-01

    Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.

  1. Autophagic cell death: Loch Ness monster or endangered species?

    PubMed

    Shen, Han-Ming; Codogno, Patrice

    2011-05-01

    The concept of autophagic cell death was first established based on observations of increased autophagic markers in dying cells. The major limitation of such a morphology-based definition of autophagic cell death is that it fails to establish the functional role of autophagy in the cell death process, and thus contributes to the confusion in the literature regarding the role of autophagy in cell death and cell survival. Here we propose to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: (i) cell death occurs without the involvement of apoptosis; (ii) there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (iii) suppression of autophagy via both pharmacological inhibitors and genetic approaches is able to rescue or prevent cell death. In light of this new definition, we will discuss some of the common problems and difficulties in the study of autophagic cell death and also revisit some well-reported cases of autophagic cell death, aiming to achieve a better understanding of whether autophagy is a real killer, an accomplice or just an innocent bystander in the course of cell death. At present, the physiological relevance of autophagic cell death is mainly observed in lower eukaryotes and invertebrates such as Dictyostelium discoideum and Drosophila melanogaster. We believe that such a clear definition of autophagic cell death will help us study and understand the physiological or pathological relevance of autophagic cell death in mammals.

  2. Rab2 promotes autophagic and endocytic lysosomal degradation.

    PubMed

    Lőrincz, Péter; Tóth, Sarolta; Benkő, Péter; Lakatos, Zsolt; Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Takáts, Szabolcs; Scita, Giorgio; Juhász, Gábor

    2017-07-03

    Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. © 2017 Lőrincz et al.

  3. Astragalus polysaccharide restores autophagic flux and improves cardiomyocyte function in doxorubicin-induced cardiotoxicity

    PubMed Central

    Cao, Yuan; Shen, Tao; Huang, Xiuqing; Lin, Yajun; Chen, Beidong; Pang, Jing; Li, Guoping; Wang, Que; Zohrabian, Sylvia; Duan, Chao; Ruan, Yang; Man, Yong; Wang, Shu; Li, Jian

    2017-01-01

    Doxorubicin (adriamycin), an anthracycline antibiotic, is commonly used to treat many types of solid and hematological malignancies. Unfortunately, clinical usage of doxorubicin is limited due to the associated acute and chronic cardiotoxicity. Previous studies demonstrated that Astragalus polysaccharide (APS), the extracts of Astragalus membranaceus, had strong anti-tumor activities and anti-inflammatory effects. However, whether APS could mitigate chemotherapy-induced cardiotoxicity is unclear thus far. We used a doxorubicin-induced neonatal rat cardiomyocyte injury model and a mouse heart failure model to explore the function of APS. GFP-LC3 adenovirus-mediated autophagic vesicle assays, GFP and RFP tandemly tagged LC3 (tfLC3) assays and Western blot analyses were performed to analyze the cell function and cell signaling changes following APS treatment in cardiomyocytes. First, doxorubicin treatment led to C57BL/6J mouse heart failure and increased cardiomyocyte apoptosis, with a disturbed cell autophagic flux. Second, APS restored autophagy in doxorubicin-treated primary neonatal rat ventricular myocytes and in the doxorubicin-induced heart failure mouse model. Third, APS attenuated doxorubicin-induced heart injury by regulating the AMPK/mTOR pathway. The mTOR inhibitor rapamycin significantly abrogated the protective effect of APS. These results suggest that doxorubicin could induce heart failure by disturbing cardiomyocyte autophagic flux, which may cause excessive cell apoptosis. APS could restore normal autophagic flux, ameliorating doxorubicin-induced cardiotoxicity by regulating the AMPK/mTOR pathway. PMID:27902477

  4. Inhibition of mTOR improves the impairment of acidification in autophagic vesicles caused by hepatic steatosis

    SciTech Connect

    Nakadera, Eisuke; Yamashina, Shunhei; Izumi, Kousuke; Inami, Yoshihiro; Sato, Toshifumi; Fukushima, Hirofumi; Kon, Kazuyoshi; Ikejima, Kenichi; Ueno, Takashi; Watanabe, Sumio

    2016-01-22

    Recent investigations revealed that dysfunction of autophagy involved in the progression of chronic liver diseases such as alcoholic and nonalcoholic steatohepatitis and hepatocellular neoplasia. Previously, it was reported that hepatic steatosis disturbs autophagic proteolysis via suppression of both autophagic induction and lysosomal function. Here, we demonstrate that autophagic acidification was altered by a decrease in lysosomal proton pump vacuolar-ATPase (V-ATPase) in steatohepatitis. The number of autophagic vesicles was increased in hepatocytes from obese KKAy mice as compared to control. Similarly, autophagic membrane protein LC3-II and lysosomal protein LAMP-2 expression were enhanced in KKAy mice liver. Nevertheless, both phospho-mTOR and p62 expression were augmented in KKAy mice liver. More than 70% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, the percentage of acidic autolysosomes was decreased in hepatocytes from KKAy mice significantly (40.1 ± 3.48%). Both protein and RNA level of V-ATPase subunits ATP6v1a, ATP6v1b, ATP6v1d in isolated lysosomes were suppressed in KKAy mice as compared to control. Interestingly, incubation with mTOR inhibitor rapamycin increased in the rate of LTR-positive autolysosomes in hepatocytes from KKAy mice and suppressed p62 accumulation in the liver from KKAy mice which correlated to an increase in the V-ATPase subunits expression. These results indicate that down-regulation of V-ATPase due to hepatic steatosis causes autophagic dysfunction via disruption of lysosomal and autophagic acidification. Moreover, activation of mTOR plays a pivotal role on dysregulation of lysosomal and autophagic acidification by modulation of V-ATPase expression and could therefore be a useful therapeutic target to ameliorate dysfunction of autophagy in NAFLD. - Highlights: • Hepatic steatosis causes accumulation of autophagic vesicles in hepatocytes. • Hepatic steatosis disturbs

  5. Mild MPP(+) exposure impairs autophagic degradation through a novel lysosomal acidity-independent mechanism.

    PubMed

    Miyara, Masatsugu; Kotake, Yaichiro; Tokunaga, Wataru; Sanoh, Seigo; Ohta, Shigeru

    2016-10-01

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, but its underlying cause remains unknown. Although recent studies using PD-related neurotoxin MPP(+) suggest autophagy involvement in the pathogenesis of PD, the effect of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of PD, remains largely unclear. We examined the effect of mild MPP(+) exposure (10 and 200 μM for 48 h), which induces a more slowly developing cell death, on autophagic processes and the mechanistic differences with acute MPP(+) toxicity (2.5 and 5 mM for 24 h). In SH-SY5Y cells, mild MPP(+) exposure predominantly inhibited autophagosome degradation, whereas acute MPP(+) exposure inhibited both autophagosome degradation and basal autophagy. Mild MPP(+) exposure reduced lysosomal hydrolase cathepsin D activity without changing lysosomal acidity, whereas acute exposure decreased lysosomal density. Lysosome biogenesis enhancers trehalose and rapamycin partially alleviated mild MPP(+) exposure induced impaired autophagosome degradation and cell death, but did not prevent the pathogenic response to acute MPP(+) exposure, suggesting irreversible lysosomal damage. We demonstrated impaired autophagic degradation by MPP(+) exposure and mechanistic differences between mild and acute MPP(+) toxicities. Mild MPP(+) toxicity impaired autophagosome degradation through novel lysosomal acidity-independent mechanisms. Sustained mild lysosomal damage may contribute to PD. We examined the effects of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of Parkinson's disease, in SH-SY5Y cells. This study demonstrated impaired autophagic degradation through a reduction in lysosomal cathepsin D activity without altering lysosomal acidity by mild MPP(+) exposure. Mechanistic differences between acute and mild MPP(+) toxicity were also observed. Sustained mild damage of lysosome may be an underlying cause

  6. Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice

    PubMed Central

    Wang, Bo; Yang, Qing; Sun, Yuan-yuan; Xing, Yi-fan; Wang, Ying-bin; Lu, Xiao-ting; Bai, Wen-wu; Liu, Xiao-qiong; Zhao, Yu-xia

    2014-01-01

    Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol-induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol-mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long-term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol-induced down-regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H2O2 increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1-dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy. PMID:24889822

  7. Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210.

    PubMed

    Frank, Benjamin; Marcu, Ana; de Oliveira Almeida Petersen, Antonio Luis; Weber, Heike; Stigloher, Christian; Mottram, Jeremy C; Scholz, Claus Juergen; Schurigt, Uta

    2015-07-31

    Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on

  8. The autophagic tumor stroma model of cancer

    PubMed Central

    Pavlides, Stephanos; Tsirigos, Aristotelis; Migneco, Gemma; Whitaker-Menezes, Diana; Chiavarina, Barbara; Flomenberg, Neal; Frank, Philippe G; Casimiro, Mathew C; Wang, Chenguang; Pestell, Richard G; Martinez-Outschoorn, Ubaldo E; Howell, Anthony

    2010-01-01

    A loss of stromal caveolin-1 (Cav-1) in the tumor fibroblast compartment is associated with early tumor recurrence, lymphnode metastasis and tamoxifen-resistance, resulting in poor clinical outcome in breast cancer patients. Here, we have used Cav-1 (−/−) null mice as a pre-clinical model for this “lethal tumor micro-environment”. Metabolic profiling of Cav-1 (−/−) mammary fat pads revealed the upregulation of numerous metabolites (nearly 100), indicative of a major catabolic phenotype. Our results are consistent with the induction of oxidative stress, mitochondrial dysfunction and autophagy/mitophagy. The two most prominent metabolites that emerged from this analysis were ADMA (asymmetric dimethyl arginine) and BHB (beta-hydroxybutyrate; a ketone body), which are markers of oxidative stress and mitochondrial dysfunction, respectively. Transcriptional profiling of Cav-1 (−/−) stromal cells and human tumor stroma from breast cancer patients directly supported an association with oxidative stress, mitochondrial dysfunction and autophagy/mitophagy, as well as ADMA and ketone production. MicroRNA profiling of Cav-1 (−/−) stromal cells revealed the upregulation of two key cancer-related miR's, namely miR-31 and miR-34c. Consistent with our metabolic findings, these miR's are associated with oxidative stress (miR-34c) or activation of the hypoxic response/HIF1α (miR-31), which is sufficient to drive authophagy/mitophagy. Thus, via an unbiased comprehensive analysis of a lethal tumor micro-environment, we have identified a number of candidate biomarkers (ADMA, ketones and miR-31/34c) that could be used to identify high-risk cancer patients at diagnosis, for treatment stratification and/or for evaluating therapeutic efficacy during anti-cancer therapy. We propose that the levels of these key biomarkers (ADMA, ketones/BHB, miR-31 and miR-34c) could be (1) assayed using serum or plasma from cancer patients or (2) performed directly on excised tumor

  9. Agriculture Education. Farm Machinery.

    ERIC Educational Resources Information Center

    Stuttgart Public Schools, AR.

    This curriculum guide is designed for group instruction of secondary agricultural education students enrolled in one or two semester-long courses in farm machinery. The guide presents units of study in the following areas: (1) small gas engines, (2) job opportunities, (3) tractors, (4) engines, (5) hydraulics, (6) electrical system, (7) combine…

  10. Agriculture Power and Machinery.

    ERIC Educational Resources Information Center

    Rogers, Tom

    This guide is intended to assist vocational agriculture teachers who are teaching secondary- or postsecondary-level courses in agricultural power and machinery. The materials presented are based on the Arizona validated occupational competencies and tasks for the following occupations: service manager, shop foreman, service technician, and tractor…

  11. Agriculture Power and Machinery.

    ERIC Educational Resources Information Center

    Rogers, Tom

    This guide is intended to assist vocational agriculture teachers who are teaching secondary- or postsecondary-level courses in agricultural power and machinery. The materials presented are based on the Arizona validated occupational competencies and tasks for the following occupations: service manager, shop foreman, service technician, and tractor…

  12. Structure of yeast Ape1 and its role in autophagic vesicle formation

    PubMed Central

    Su, Ming-Yuan; Peng, Wen-Hsin; Ho, Meng-Ru; Su, Shih-Chieh; Chang, Yuan-Chih; Chen, Guang-Chao; Chang, Chung-I

    2015-01-01

    In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy. PMID:26208681

  13. Brucella abortus Transits through the Autophagic Pathway and Replicates in the Endoplasmic Reticulum of Nonprofessional Phagocytes

    PubMed Central

    Pizarro-Cerdá, Javier; Méresse, Stéphane; Parton, Robert G.; van der Goot, Gisou; Sola-Landa, Alberto; Lopez-Goñi, Ignacio; Moreno, Edgardo; Gorvel, Jean-Pierre

    1998-01-01

    Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At ∼1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61β but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61β- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER. PMID:9826346

  14. Inhibition of UCH-L1 in oligodendroglial cells results in microtubule stabilization and prevents α-synuclein aggregate formation by activating the autophagic pathway: implications for multiple system atrophy

    PubMed Central

    Pukaß, Katharina; Richter-Landsberg, Christiane

    2015-01-01

    α-Synuclein (α-syn) positive glial cytoplasmic inclusions (GCI) originating in oligodendrocytes (ODC) are a characteristic hallmark in multiple system atrophy (MSA). Their occurrence may be linked to a failure of the ubiquitin proteasome system (UPS) or the autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin, and deubiquitinated by deubiquitinating enzymes (DUBs) before proteolytic degradation is performed. The DUB ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a component of the UPS, it is abundantly expressed in neuronal brain cells and has been connected to Parkinson’s disease (PD). It interacts with α-syn and tubulin. The present study was undertaken to investigate whether UCH-L1 is a constituent of ODC, the myelin forming cells of the CNS, and is associated with GCIs in MSA. Furthermore, LDN-57444 (LDN), a specific UCH-L1 inhibitor, was used to analyze its effects on cell morphology, microtubule (MT) organization and the proteolytic degradation system. Towards this an oligodendroglial cell line (OLN cells), stably transfected with α-syn or with α-syn and GFP-LC3, to monitor the autophagic flux, was used. The data show that UCH-L1 is expressed in ODC derived from the brains of newborn rats and colocalizes with α-syn in GCIs of MSA brain sections. LDN treatment had a direct impact on the MT network by affecting tubulin posttranslational modifications, i.e., acetylation and tyrosination. An increase in α-tubulin detyrosination was observed and detyrosinated MT were abundantly recruited to the cellular extensions. Furthermore, small α-syn aggregates, which are constitutively expressed in OLN cells overexpressing α-syn, were abolished, and LDN caused the upregulation of the autophagic pathway. Our data add to the knowledge that the UPS and the autophagy-lysosomal pathway are tightly balanced, and that UCH-L1 and its regulation may play a role in neurodegenerative diseases with oligodendroglia

  15. Bidirectional coupling between ryanodine receptors and Ca2+ release-activated Ca2+ (CRAC) channel machinery sustains store-operated Ca2+ entry in human T lymphocytes.

    PubMed

    Thakur, Pratima; Dadsetan, Sepehr; Fomina, Alla F

    2012-10-26

    The expression and functional significance of ryanodine receptors (RyR) were investigated in resting and activated primary human T cells. RyR1, RyR2, and RyR3 transcripts were detected in human T cells. RyR1/2 transcript levels increased, whereas those of RyR3 decreased after T cell activation. RyR1/2 protein immunoreactivity was detected in activated but not in resting T cells. The RyR agonist caffeine evoked Ca(2+) release from the intracellular store in activated T cells but not in resting T cells, indicating that RyR are functionally up-regulated in activated T cells compared with resting T cells. In the presence of store-operated Ca(2+) entry (SOCE) via plasmalemmal Ca(2+) release-activated Ca(2+) (CRAC) channels, RyR blockers reduced the Ca(2+) leak from the endoplasmic reticulum (ER) and the magnitude of SOCE, suggesting that a positive feedback relationship exists between RyR and CRAC channels. Overexpression of fluorescently tagged RyR2 and stromal interaction molecule 1 (STIM1), an ER Ca(2+) sensor gating CRAC channels, in HEK293 cells revealed that RyR are co-localized with STIM1 in the puncta formed after store depletion. These data indicate that in primary human T cells, the RyR are coupled to CRAC channel machinery such that SOCE activates RyR via a Ca(2+)-induced Ca(2+) release mechanism, which in turn reduces the Ca(2+) concentration within the ER lumen in the vicinity of STIM1, thus facilitating SOCE by reducing store-dependent CRAC channel inactivation. Treatment with RyR blockers suppressed activated T cell expansion, demonstrating the functional importance of RyR in T cells.

  16. Instability in Rotating Machinery

    NASA Technical Reports Server (NTRS)

    1985-01-01

    The proceedings contain 45 papers on a wide range of subjects including flow generated instabilities in fluid flow machines, cracked shaft detection, case histories of instability phenomena in compressors, turbines, and pumps, vibration control in turbomachinery (including antiswirl techniques), and the simulation and estimation of destabilizing forces in rotating machines. The symposium was held to serve as an update on the understanding and control of rotating machinery instability problems.

  17. Improve machinery vibration data

    SciTech Connect

    Haq, I. )

    1993-03-01

    Continuous monitoring and periodic logging of machinery information are the best ways to measure current performance and predict availability. Decisions made on time-based information analysis are only as good as the quality of the data. Methods for data acquisition must conform to standards that ensure data are free of errors and ambiguity. This paper discusses the data acquisition framework; data collection techniques; conventions and documentation; transduces operation; transduces selection and application; transducer noise; and vital features of a diagnostic instrument.

  18. Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD

    PubMed Central

    González-Rodríguez, Á; Mayoral, R; Agra, N; Valdecantos, M P; Pardo, V; Miquilena-Colina, M E; Vargas-Castrillón, J; Lo Iacono, O; Corazzari, M; Fimia, G M; Piacentini, M; Muntané, J; Boscá, L; García-Monzón, C; Martín-Sanz, P; Valverde, Á M

    2014-01-01

    The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to

  19. Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.

    PubMed

    González-Rodríguez, A; Mayoral, R; Agra, N; Valdecantos, M P; Pardo, V; Miquilena-Colina, M E; Vargas-Castrillón, J; Lo Iacono, O; Corazzari, M; Fimia, G M; Piacentini, M; Muntané, J; Boscá, L; García-Monzón, C; Martín-Sanz, P; Valverde, Á M

    2014-04-17

    The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to

  20. Methods for assessing autophagy and autophagic cell death.

    PubMed

    Tasdemir, Ezgi; Galluzzi, Lorenzo; Maiuri, M Chiara; Criollo, Alfredo; Vitale, Ilio; Hangen, Emilie; Modjtahedi, Nazanine; Kroemer, Guido

    2008-01-01

    Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.

  1. Evidence for autophagic gridlock in aging and neurodegeneration.

    PubMed

    Bakhoum, Mathieu F; Bakhoum, Christine Y; Ding, Zhixia; Carlton, Susan M; Campbell, Gerald A; Jackson, George R

    2014-07-01

    Autophagy is essential to neuronal homeostasis, and its impairment is implicated in the development of neurodegenerative pathology. However, the underlying mechanisms and consequences of this phenomenon remain a matter of conjecture. We show that misexpression of human tau in Drosophila induces accumulation of autophagic intermediates with a preponderance of large vacuoles, which we term giant autophagic bodies (GABs), which are reminiscent of dysfunctional autophagic entities. Lowering basal autophagy reduces GABs, whereas increasing autophagy decreases mature autolysosomes. Induction of autophagy is also associated with rescue of the tauopathy phenotype, suggesting that formation of GABs may be a compensatory mechanism rather than a trigger of neurodegeneration. Last, we show that the peculiar Biondi bodies observed in the choroid epithelium of both elderly and Alzheimer's disease human brains express immunoreactive markers similar to those of GABs. Collectively, these data indicate that autophagic gridlock contributes to the development of pathology in aging and neurodegeneration. Published by Mosby, Inc.

  2. Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells.

    PubMed

    Wong, V K W; Li, T; Law, B Y K; Ma, E D L; Yip, N C; Michelangeli, F; Law, C K M; Zhang, M M; Lam, K Y C; Chan, P L; Liu, L

    2013-07-11

    Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase pump, leading to autophagy induction through the activation of the Ca(2+)/calmodulin-dependent kinase kinase-AMP-activated protein kinase-mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.

  3. A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells.

    PubMed

    Pallet, Nicolas; Sirois, Isabelle; Bell, Christina; Hanafi, Laïla-Aïcha; Hamelin, Katia; Dieudé, Mélanie; Rondeau, Christiane; Thibault, Pierre; Desjardins, Michel; Hebert, Marie-Josée

    2013-04-01

    The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase-dependent manner by serum-starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. HTLV-1 Tax deregulates autophagy by recruiting autophagic molecules into lipid raft microdomains.

    PubMed

    Ren, T; Takahashi, Y; Liu, X; Loughran, T P; Sun, S-C; Wang, H-G; Cheng, H

    2015-01-15

    The retroviral oncoprotein Tax from human T-cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T-cell leukemia and lymphoma, has a crucial role in initiating T-lymphocyte transformation by inducing oncogenic signaling activation. We here report that Tax is a determining factor for dysregulation of autophagy in HTLV-1-transformed T cells and Tax-immortalized CD4 memory T cells. Tax facilitated autophagic process by activating inhibitor of κB (IκB) kinase (IKK) complex, which subsequently recruited an autophagy molecular complex containing Beclin1 and Bif-1 to the lipid raft microdomains. Tax engaged a crosstalk between IKK complex and autophagic molecule complex by directly interacting with both complexes, promoting assembly of LC3+ autophagosomes. Moreover, expression of lipid raft-targeted Bif-1 or Beclin1 was sufficient to induce formation of LC3+ autophagosomes, suggesting that Tax recruitment of autophagic molecules to lipid rafts is a dominant strategy to deregulate autophagy in the context of HTLV-1 transformation of T cells. Furthermore, depletion of autophagy molecules such as Beclin1 and PI3 kinase class III resulted in impaired growth of HTLV-1-transformed T cells, indicating a critical role of Tax-deregulated autophagy in promoting survival and transformation of virally infected T cells.

  5. HTLV-1 Tax deregulates autophagy by recruiting autophagic molecules into lipid raft microdomains

    PubMed Central

    Ren, Tong; Takahashi, Yoshinori; Liu, Xin; Loughran, Thomas P.; Sun, Shao-Cong; Wang, Hong-Gang; Cheng, Hua

    2014-01-01

    The retroviral oncoprotein Tax from Human T cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T cell leukemia and lymphoma, plays a crucial role in initiating T lymphocyte transformation by inducing oncogenic signaling activation. We here report that Tax is a determining factor for dysregulation of autophagy in HTLV-1-transformed T cells and Tax-immortalized CD4 memory T cells. Tax facilitated autophagic process by activating IκB kinase complex, which subsequently recruited an autophagy molecular complex containing Beclin1 and Bif-1 to the lipid raft microdomains. Tax engaged a crosstalk between IκB kinase complex and autophagic molecule complex by directly interacting with both complexes, promoting assembly of LC3+ autophagosomes. Moreover, expression of lipid raft-targeted Bif-1 or Beclin1 was sufficient to induce formation of LC3+ autophagosomes, suggesting that Tax recruitment of autophagic molecules to lipid rafts is a dominant strategy to deregulate autophagy in the context of HTLV-1 transformation of T cells. Furthermore, depletion of autophagy molecules such as Beclin1 and PI3 kinase class III resulted in impaired growth of HTLV-1-transformed T cells, indicating a critical role of Tax-deregulated autophagy in promoting survival and transformation of virally infected T cells. PMID:24362528

  6. [D-Lys3]-GHRP-6 exhibits pro-autophagic effects on skeletal muscle.

    PubMed

    Yu, Angus P; Pei, Xiao M; Sin, Thomas K; Yip, Shea P; Yung, Benjamin Y; Chan, Lawrence W; Wong, Cesar S; Siu, Parco M

    2015-02-05

    [D-Lys3]-GHRP-6 is regarded as a highly selective growth-hormone secretagogue receptor (GHSR) antagonist and has been widely used to investigate the dependency of GHSR-1a signalling mediated by acylated ghrelin. However, [D-Lys3]-GHRP-6 has been reported to influence other cellular processes which are unrelated to GHSR-1a. This study aimed to examine the effects of [D-Lys3]-GHRP-6 on autophagic and apoptotic cellular signalling in skeletal muscle. [D-Lys3]-GHRP-6 enhanced the autophagic signalling demonstrated by the increases in protein abundances of beclin-1 and LC3 II-to-LC3 1 ratio in both normal muscle and doxorubicin-injured muscle. [D-Lys3]-GHRP-6 reduced the activation of muscle apoptosis induced by doxorubicin. No histological abnormalities were observed in the [D-Lys3]-GHRP-6-treated muscle. Intriguingly, the doxorubicin-induced increase in centronucleated muscle fibres was not observed in muscle treated with [D-Lys3]-GHRP-6, suggesting the myoprotective effects of [D-Lys3]-GHRP-6 against doxorubicin injury. The [D-Lys3]-GHRP-6-induced activation of autophagy was found to be abolished by the co-treatment of CXCR4 antagonist, suggesting that the pro-autophagic effects of [D-Lys3]-GHRP-6 might be mediated through CXCR4. In conclusion, [D-Lys3]-GHRP-6 exhibits pro-autophagic effects on skeletal muscle under both normal and doxorubicin-injured conditions.

  7. Health State Monitoring of Bladed Machinery with Crack Growth Detection in BFG Power Plant Using an Active Frequency Shift Spectral Correction Method.

    PubMed

    Sun, Weifang; Yao, Bin; He, Yuchao; Chen, Binqiang; Zeng, Nianyin; He, Wangpeng

    2017-08-09

    Power generation using waste-gas is an effective and green way to reduce the emission of the harmful blast furnace gas (BFG) in pig-iron producing industry. Condition monitoring of mechanical structures in the BFG power plant is of vital importance to guarantee their safety and efficient operations. In this paper, we describe the detection of crack growth of bladed machinery in the BFG power plant via vibration measurement combined with an enhanced spectral correction technique. This technique enables high-precision identification of amplitude, frequency, and phase information (the harmonic information) belonging to deterministic harmonic components within the vibration signals. Rather than deriving all harmonic information using neighboring spectral bins in the fast Fourier transform spectrum, this proposed active frequency shift spectral correction method makes use of some interpolated Fourier spectral bins and has a better noise-resisting capacity. We demonstrate that the identified harmonic information via the proposed method is of suppressed numerical error when the same level of noises is presented in the vibration signal, even in comparison with a Hanning-window-based correction method. With the proposed method, we investigated vibration signals collected from a centrifugal compressor. Spectral information of harmonic tones, related to the fundamental working frequency of the centrifugal compressor, is corrected. The extracted spectral information indicates the ongoing development of an impeller blade crack that occurred in the centrifugal compressor. This method proves to be a promising alternative to identify blade cracks at early stages.

  8. Health State Monitoring of Bladed Machinery with Crack Growth Detection in BFG Power Plant Using an Active Frequency Shift Spectral Correction Method

    PubMed Central

    Sun, Weifang; Yao, Bin; He, Yuchao; Zeng, Nianyin; He, Wangpeng

    2017-01-01

    Power generation using waste-gas is an effective and green way to reduce the emission of the harmful blast furnace gas (BFG) in pig-iron producing industry. Condition monitoring of mechanical structures in the BFG power plant is of vital importance to guarantee their safety and efficient operations. In this paper, we describe the detection of crack growth of bladed machinery in the BFG power plant via vibration measurement combined with an enhanced spectral correction technique. This technique enables high-precision identification of amplitude, frequency, and phase information (the harmonic information) belonging to deterministic harmonic components within the vibration signals. Rather than deriving all harmonic information using neighboring spectral bins in the fast Fourier transform spectrum, this proposed active frequency shift spectral correction method makes use of some interpolated Fourier spectral bins and has a better noise-resisting capacity. We demonstrate that the identified harmonic information via the proposed method is of suppressed numerical error when the same level of noises is presented in the vibration signal, even in comparison with a Hanning-window-based correction method. With the proposed method, we investigated vibration signals collected from a centrifugal compressor. Spectral information of harmonic tones, related to the fundamental working frequency of the centrifugal compressor, is corrected. The extracted spectral information indicates the ongoing development of an impeller blade crack that occurred in the centrifugal compressor. This method proves to be a promising alternative to identify blade cracks at early stages. PMID:28792453

  9. Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

    PubMed

    Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

    2014-11-01

    The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.

  10. Targeting the protein ubiquitination machinery in melanoma by the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924).

    PubMed

    Wong, Kit Man; Micel, Lindsey N; Selby, Heather M; Tan, Aik Choon; Pitts, Todd M; Bagby, Stacey M; Spreafico, Anna; Klauck, Peter J; Blakemore, Stephen J; Smith, Peter F; McDonald, Alice; Berger, Allison; Tentler, John J; Eckhardt, S Gail

    2017-02-01

    Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC50 < 0.3 μM) and induced apoptosis in a subset of melanoma cell lines. Sensitive and resistant cell lines exhibited distinct gene expression profiles; sensitive models were enriched for genes involved in DNA repair, replication and cell cycle regulation, while immune response and cell adhesion pathways were upregulated in resistant models. Pevonedistat also reduced tumor growth in melanoma cell line xenografts and PDTX with variable responses. An accumulation of pevonedistat-NEDD8 adduct and CDT1 was observed in sensitive tumors consistent with its mechanism of action. Conclusions This study provided preclinical evidence that NAE inhibition by pevonedistat has anti-tumor activity in melanoma and supports the clinical benefits observed in recent Phase 1 trials of this drug in melanoma patients. Further investigations are warranted to develop rational combinations and determine predictive biomarkers of pevonedistat.

  11. DENSpm overcame Bcl-2 mediated resistance against Paclitaxel treatment in MCF-7 breast cancer cells via activating polyamine catabolic machinery.

    PubMed

    Akyol, Zeynep; Çoker-Gürkan, Ajda; Arisan, Elif Damla; Obakan-Yerlikaya, Pınar; Palavan-Ünsal, Narçin

    2016-12-01

    The Bcl-2 mediated resistance is one of the most critical obstacle in cancer therapy. Conventional chemotherapeutics such as Paclitaxel, a commonly used in the treatment of metastatic breast cancer, is not sufficient to overcome Bcl-2 mediated drug resistance mechanism. Thus, combinational drug regimes are favored by researchers to overcome resistance phenotype against drugs. N1,N11-diethylnorspermine (DENSpm), a polyamine analogue, which is a promising drug candidate induced-cell cycle arrest and apoptosis in various cancer cells such as prostate, melanoma, colon and breast cancer cells via activated polyamine catabolism and reactive oxygen generation. Recent studies indicated the potential therapeutic role of DENSpm in phase I and II trials in breast cancer cases. Although the molecular targets of Paclitaxel in apoptotic cell death mechanism is well documented, the therapeutic effect of DENSpm and Paclitaxel in breast cancer cells has not been investigated yet. In this study, our aim was to determine the time dependent effect of DENSpm and Paclitaxel on apoptotic cell death via determination of polyamine metabolism related targets in wt and Bcl-2 overexpressing MCF-7 breast cancer cells. In our experimental study, Paclitaxel decreased cell viability in dose-dependent manner within 24h. Co-treatment of Paclitaxel (30nM) with DENSpm (20μM) further increased the cytoxicity of Paclitaxel (30nM) compared to alone Paclitaxel (30nM) treatment in MCF-7 Bcl-2+ breast cancer cells. In addition, we determined that resistance against Paclitaxel-induced apoptotic cell death in Bcl-2 overexpressed MCF-7 cells was overcome due to activation of polyamine catabolic pathway, which caused depletion of polyamines. DENSpm combinational treatment might increase the effect of low cytotoxic paclitaxel in drug-resistant breast cancer cases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity*♦

    PubMed Central

    Chen, Robert H. C.; Wislet-Gendebien, Sabine; Samuel, Filsy; Visanji, Naomi P.; Zhang, Gang; Marsilio, Diana; Langman, Tammy; Fraser, Paul E.; Tandon, Anurag

    2013-01-01

    α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity. PMID:23344955

  13. Effects of Epigallocatechin-3-Gallate on Autophagic Lipolysis in Adipocytes

    PubMed Central

    Kim, Sang-Nam; Kwon, Hyun-Jung; Akindehin, Seun; Jeong, Hyun Woo

    2017-01-01

    Previous studies demonstrated effects of green tea on weight loss; however, green tea-induced modulation of adipocyte function is not fully understood. Here, we investigated effects of the major green tea phytochemical, epigallocatechin-3-gallate (EGCG) on triglyceride contents, lipolysis, mitochondrial function, and autophagy, in adipocytes differentiated from C3H10T1/2 cells and immortalized pre-adipocytes in vitro. EGCG reduced the triglycerol content significantly in adipocytes by 25%, comparable to the nutrient starvation state. EGCG did not affect protein kinase A signaling or brown adipocyte marker expression in adipocytes; however, EGCG increased autophagy, as measured by autophagy flux analysis and immunoblot analysis of LC3B, ATG7, and Beclin1. EGCG treatment reduced mitochondrial membrane potential by 56.8% and intracellular ATP levels by 49.1% compared to controls. Although mammalian target of rapamycin signaling was not upregulated by EGCG treatment, EGCG treatment induced AMP-activated protein kinase phosphorylation, indicating an energy-depleted state. In addition, EGCG increased the association between RAB7 and lipid droplets, suggesting that lipophagy was activated. Finally, knockdown of Rab7 attenuated the EGCG-dependent reduction in lipid contents. Collectively, these results indicated that EGCG upregulated autophagic lipolysis in adipocytes, supporting the therapeutic potential of EGCG as a caloric restriction mimetic to prevent obesity and obesity-related metabolic diseases. PMID:28665330

  14. Effects of Epigallocatechin-3-Gallate on Autophagic Lipolysis in Adipocytes.

    PubMed

    Kim, Sang-Nam; Kwon, Hyun-Jung; Akindehin, Seun; Jeong, Hyun Woo; Lee, Yun-Hee

    2017-06-30

    Previous studies demonstrated effects of green tea on weight loss; however, green tea-induced modulation of adipocyte function is not fully understood. Here, we investigated effects of the major green tea phytochemical, epigallocatechin-3-gallate (EGCG) on triglyceride contents, lipolysis, mitochondrial function, and autophagy, in adipocytes differentiated from C₃H10T1/2 cells and immortalized pre-adipocytes in vitro. EGCG reduced the triglycerol content significantly in adipocytes by 25%, comparable to the nutrient starvation state. EGCG did not affect protein kinase A signaling or brown adipocyte marker expression in adipocytes; however, EGCG increased autophagy, as measured by autophagy flux analysis and immunoblot analysis of LC3B, ATG7, and Beclin1. EGCG treatment reduced mitochondrial membrane potential by 56.8% and intracellular ATP levels by 49.1% compared to controls. Although mammalian target of rapamycin signaling was not upregulated by EGCG treatment, EGCG treatment induced AMP-activated protein kinase phosphorylation, indicating an energy-depleted state. In addition, EGCG increased the association between RAB7 and lipid droplets, suggesting that lipophagy was activated. Finally, knockdown of Rab7 attenuated the EGCG-dependent reduction in lipid contents. Collectively, these results indicated that EGCG upregulated autophagic lipolysis in adipocytes, supporting the therapeutic potential of EGCG as a caloric restriction mimetic to prevent obesity and obesity-related metabolic diseases.

  15. Bortezomib induces autophagic death in proliferating human endothelial cells

    SciTech Connect

    Belloni, Daniela; Veschini, Lorenzo; Foglieni, Chiara; Dell'Antonio, Giacomo; Caligaris-Cappio, Federico; Ferrarini, Marina; Ferrero, Elisabetta

    2010-04-01

    The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.

  16. Overexpression of Smooth Muscle Myosin Heavy Chain Leads to Activation of the Unfolded Protein Response and Autophagic Turnover of Thick Filament-associated Proteins in Vascular Smooth Muscle Cells*

    PubMed Central

    Kwartler, Callie S.; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V.; Walker, Lori; Hill, Joseph A.; Epstein, Henry F.; Taegtmeyer, Heinrich; Milewicz, Dianna M.

    2014-01-01

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. PMID:24711452

  17. Overexpression of smooth muscle myosin heavy chain leads to activation of the unfolded protein response and autophagic turnover of thick filament-associated proteins in vascular smooth muscle cells.

    PubMed

    Kwartler, Callie S; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V; Walker, Lori; Hill, Joseph A; Epstein, Henry F; Taegtmeyer, Heinrich; Milewicz, Dianna M

    2014-05-16

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. MP Resulting in Autophagic Cell Death of Microglia through Zinc Changes against Spinal Cord Injury

    PubMed Central

    Li, Dingding; Wang, Guannan; Han, Donghe; Bi, Jing; Li, Chenyuan; Wang, Hongyu; Liu, Zhiyuan; Gao, Wei; Gao, Kai; Yao, Tianchen; Wan, Zhanghui; Li, Haihong; Mei, Xifan

    2016-01-01

    Methylprednisolone pulse therapy (MPPT), as a public recognized therapy of spinal cord injury (SCI), is doubted recently, and the exact mechanism of MP on SCI is unclear. This study sought to investigate the exact effect of MP on SCI. We examined the effect of MP in a model of SCI in vivo and an LPS induced model in vitro. We found that administration of MP produced an increase in the Basso, Beattie, and Bresnahan scores and motor neurons counts of injured rats. Besides the number of activated microglia was apparently reduced by MP in vivo, and Beclin-1 dependent autophagic cell death of microglia was induced by MP in LPS induced model. At the same time, MP increases cellular zinc concentration and level of ZIP8, and TPEN could revert effect of MP on autophagic cell death of microglia. Finally, we have found that MP could inhibit NF-κβ in LPS induced model. These results show that the MP could result in autophagic cell death of microglia, which mainly depends on increasing cellular labile zinc, and may be associated with inhibition of NF-κβ, and that MP can produce neuroprotective effect in SCI. PMID:27057544

  19. Altered autophagic flux enhances inflammatory responses during inflammation-induced preterm labor

    PubMed Central

    Agrawal, Varkha; Jaiswal, Mukesh K.; Mallers, Timothy; Katara, Gajendra K.; Gilman-Sachs, Alice; Beaman, Kenneth D.; Hirsch, Emmet

    2015-01-01

    Cellular organelles and proteins are degraded and recycled through autophagy, a process during which vesicles known as autophagosomes fuse with lysosomes. Altered autophagy occurs in various diseases, but its role in preterm labor (PTL) is unknown. We investigated the role of autophagic flux in two mouse models of PTL compared to controls: 1) inflammation-induced PTL (IPTL), induced by toll-like receptor agonists; and 2) non-inflammation (hormonally)-induced PTL (NIPTL). We demonstrate that the autophagy related genes Atg4c and Atg7 (involved in the lipidation of microtubule-associated protein 1 light chain 3 (LC3) B-I to the autophagosome-associated form, LC3B-II) decrease significantly in uterus and placenta during IPTL but not NIPTL. Autophagic flux is altered in IPTL, as shown by the accumulation of LC3B paralogues and diminishment of lysosome associated membrane protein (LAMP)-1, LAMP-2 and the a2 isoform of V-ATPase (a2V, an enzyme involved in lysosome acidification). These alterations in autophagy are associated with increased activation of NF-κB and proinflammatory cytokines/chemokines in both uterus and placenta. Similar changes are seen in macrophages exposed to TLR ligands and are enhanced with blockade of a2V. These novel findings represent the first evidence of an association between altered autophagic flux and hyper-inflammation and labor in IPTL. PMID:25797357

  20. Autophagic flux promotes cisplatin resistance in human ovarian carcinoma cells through ATP-mediated lysosomal function.

    PubMed

    Ma, Liwei; Xu, Ye; Su, Jing; Yu, Huimei; Kang, Jinsong; Li, Hongyan; Li, Xiaoning; Xie, Qi; Yu, Chunyan; Sun, Liankun; Li, Yang

    2015-11-01

    Lysosomes are involved in promoting resistance of cancer cells to chemotherapeutic agents. However, the mechanisms underlying lysosomal influence of cisplatin resistance in ovarian cancer remain incompletely understood. We report that, compared with cisplatin-sensitive SKOV3 cells, autophagy increases in cisplatin-resistant SKOV3/DDP cells treated with cisplatin. Inhibition of early-stage autophagy enhanced cisplatin-mediated cytotoxicity in SKOV3/DDP cells, but autophagy inhibition at a later stage by disturbing autophagosome-lysosome fusion is more effective. Notably, SKOV3/DDP cells contained more lysosomes than cisplatin-sensitive SKOV3 cells. Abundant lysosomes and lysosomal cathepsin D activity were required for continued autolysosomal degradation and maintenance of autophagic flux in SKOV3/DDP cells. Furthermore, SKOV3/DDP cells contain abundant lysosomal ATP required for lysosomal function, and inhibition of lysosomal ATP accumulation impaired lysosomal function and blocked autophagic flux. Therefore, our findings suggest that lysosomes at least partially contribute to cisplatin resistance in ovarian cancer cells through their role in cisplatin-induced autophagic processes, and provide insight into the mechanism of cisplatin resistance in tumors.

  1. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells.

    PubMed

    Fazi, Barbara; Bursch, Wilfried; Fimia, Gian Maria; Nardacci, Roberta; Piacentini, Mauro; Di Sano, Federica; Piredda, Lucia

    2008-05-01

    The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displaying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumor cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment.

  2. Sulfasalazine induces autophagic cell death in oral cancer cells via Akt and ERK pathways.

    PubMed

    Han, Hye-Yeon; Kim, Hyungwoo; Jeong, Sung-Hee; Lim, Do-Seon; Ryu, Mi Heon

    2014-01-01

    Sulfasalazine (SSZ) is an anti-inflammatory drug that has been used to treat inflammatory bowel disease and rheumatoid arthritis for decades. Recently, some reports have suggested that SSZ also has anti-cancer properties against human tumors. However, little is known about the effects of SSZ on oral cancer. The aim of this study was to investigate the anti-cancer effects of SSZ in oral squamous cell carcinoma (OSCC) cells and to elucidate the mechanisms involved. The authors investigated the anti-proliferative effect of SSZ using the MTT method in HSC-4 cells (an OSCC cell line). Cell cycle analysis, acidic vesicular organelle (AVO) staining, monodansylcadaverine (MDC) staining and Western blotting were also conducted to investigate the cytotoxic mechanism of SSZ. SSZ significantly inhibited the proliferation of HSC-4 cells in a dose-dependent manner. In addition, SSZ induced autophagic cell death, increased microtubule-associated protein 1 light chain (MAP1- LC; also known as LC) 3-II levels, as well as induced punctate AVO and MDC staining, resulted in autophagic cell death. Furthermore, these observations were accompanied by the inhibition of the Akt pathway and the activation of ERK pathway. These results suggest that SSZ promotes autophagic cell death via Akt and ERK pathways and has chemotherapeutic potential for the treatment of oral cancer.

  3. Altered autophagic flux enhances inflammatory responses during inflammation-induced preterm labor.

    PubMed

    Agrawal, Varkha; Jaiswal, Mukesh K; Mallers, Timothy; Katara, Gajendra K; Gilman-Sachs, Alice; Beaman, Kenneth D; Hirsch, Emmet

    2015-03-23

    Cellular organelles and proteins are degraded and recycled through autophagy, a process during which vesicles known as autophagosomes fuse with lysosomes. Altered autophagy occurs in various diseases, but its role in preterm labor (PTL) is unknown. We investigated the role of autophagic flux in two mouse models of PTL compared to controls: 1) inflammation-induced PTL (IPTL), induced by toll-like receptor agonists; and 2) non-inflammation (hormonally)-induced PTL (NIPTL). We demonstrate that the autophagy related genes Atg4c and Atg7 (involved in the lipidation of microtubule-associated protein 1 light chain 3 (LC3) B-I to the autophagosome-associated form, LC3B-II) decrease significantly in uterus and placenta during IPTL but not NIPTL. Autophagic flux is altered in IPTL, as shown by the accumulation of LC3B paralogues and diminishment of lysosome associated membrane protein (LAMP)-1, LAMP-2 and the a2 isoform of V-ATPase (a2V, an enzyme involved in lysosome acidification). These alterations in autophagy are associated with increased activation of NF-κB and proinflammatory cytokines/chemokines in both uterus and placenta. Similar changes are seen in macrophages exposed to TLR ligands and are enhanced with blockade of a2V. These novel findings represent the first evidence of an association between altered autophagic flux and hyper-inflammation and labor in IPTL.

  4. Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy

    PubMed Central

    Karagounis, Ilias V; Kalamida, Dimitra; Mitrakas, Achilleas; Pouliliou, Stamatia; Liousia, Maria V; Giatromanolaki, Alexandra; Koukourakis, Michael I

    2016-01-01

    Background: The cellular autophagic response to radiation is complex. Various cells and tissues respond differentially to radiation, depending on both the dose of exposure and the time post irradiation. In the current study, we determined the autophagosomal and lysosomal response to radiation in lung cancer cell lines by evaluating the expression of the associated proteins, as well as the effect of relevant gene silencing in radio and chemosensitisation. Furthermore, tumour sensitisation was evaluated in in vivo autophagic gene silencing model after irradiation. Methods: A549 and H1299 cell lines were utilised as in vitro cancer models. Both cell lines were transfected with various small-interfering RNAs, silencing auto-lysosomal genes, and irradiated with 4 Gy. Cell growth response was evaluated with AlamarBlue assay. Western blot and confocal microscopy were utilised for the characterisation of the auto-lysosomal flux. Also, the H1299 cell line was stable transfected with small-hairpin RNA of the MAP1LC3A gene, and the tumour radiosensitisation in Athymic Nude-Foxn1nu was evaluated. Results: Following exposure to 4 Gy of radiation, A549 cells exhibited a significant induction of the autophagic flux, which was not supported by transcriptional activation of auto-lysosomal genes (LC3A, LC3B, p62, TFEB and LAMP2a), resulting in aggresome accumulation. Recovery of transcriptional activity and autophagy efficacy occurred 7 days post irradiation. Alternatively, H1299 cells, a relatively radio-resistant cell line, sharply responded with an early (at 2 days) transcriptional activation of auto-lysosomal genes that sustained an effective autophagosomal flux, resulting in adequate aggresome clearance. Subsequently, we tested the silencing of four genes (LC3A, LC3B, TFEB and LAMP2a), confirming a significant radiosensitisation and chemosensitisation to various chemotherapeutic agents, including cisplatin and taxanes. In mouse xenografts, exposure to radiation

  5. 49. Machinery rooms on north tower. Facing north. Machinery rooms ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    49. Machinery rooms on north tower. Facing north. Machinery rooms contain all motors, motor controllers, and gears for operating one span, in this case, the north span. Note bell with continuous operating clapper for use as fog signals. - Henry Ford Bridge, Spanning Cerritos Channel, Los Angeles-Long Beach Harbor, Los Angeles, Los Angeles County, CA

  6. RAB24 facilitates clearance of autophagic compartments during basal conditions

    PubMed Central

    Ylä-Anttila, Päivi; Mikkonen, Elisa; Happonen, Kaisa E; Holland, Petter; Ueno, Takashi; Simonsen, Anne; Eskelinen, Eeva-Liisa

    2015-01-01

    RAB24 belongs to a family of small GTPases and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy shows an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicate that this is due to a hindrance in their clearance. Formation of autophagosomes is shown to be unaffected by RAB24-silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 is also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions. PMID:26325487

  7. Muscle atrophy, ubiquitin-proteasome, and autophagic pathways in dysferlinopathy.

    PubMed

    Fanin, Marina; Nascimbeni, Anna C; Angelini, Corrado

    2014-09-01

    Muscle fiber atrophy and the molecular pathways underlying this process have not been investigated in dysferlinopathy patients. In 22 muscles from dysferlinopathy patients we investigated fiber atrophy by morphometry and ubiquitin-proteasome and autophagic pathways using protein and/or transcriptional analysis of atrophy- and autophagy-related genes (MuRF1, atrogin1, LC3, p62, Bnip3). Dysferlinopathy showed significant fiber atrophy and higher MuRF-1 protein and mRNA levels, which correlated with fiber size, suggesting activation of the atrophy program by proteasome induction. Some of the MuRF-1 upregulation and proteasome induction may be attributed to the prominent regeneration found. A potential role of impaired autophagy was suggested by p62-positive protein aggregates in atrophic fibers and significantly higher levels of LC3-II and p62 proteins and overexpression of p62 and Bnip3 mRNA. Damaged muscle fibers and prominent inflammatory changes may also enhance autophagy due to the insufficient level of proteasomal degradation of mutant dysferlin. Copyright © 2014 Wiley Periodicals, Inc.

  8. Nanoparticles modulate autophagic effect in a dispersity-dependent manner

    NASA Astrophysics Data System (ADS)

    Huang, Dengtong; Zhou, Hualu; Gao, Jinhao

    2015-09-01

    Autophagy plays a key role in human health and disease, especially in cancer and neurodegeneration. Many autophagy regulators are developed for therapy. Diverse nanomaterials have been reported to induce autophagy. However, the underlying mechanisms and universal rules remain unclear. Here, for the first time, we show a reliable and general mechanism by which nanoparticles induce autophagy and then successfully modulate autophagy via tuning their dispersity. Various well-designed univariate experiments demonstrate that nanomaterials induce autophagy in a dispersity-dependent manner. Aggregated nanoparticles induce significant autophagic effect in comparison with well-dispersed nanoparticles. As the highly stable nanoparticles may block autophagic degradation in autolysosomes, endocytosis and intracellular accumulation of nanoparticles can be responsible for this interesting phenomenon. Our results suggest dispersity-dependent autophagic effect as a common cellular response to nanoparticles, reveal the relationship between properties of nanoparticles and autophagy, and offer a new alternative way to modulate autophagy.

  9. Thioredoxin-interacting protein induced α-synuclein accumulation via inhibition of autophagic flux: Implications for Parkinson's disease.

    PubMed

    Su, Cun-Jin; Feng, Yu; Liu, Teng-Teng; Liu, Xu; Bao, Jun-Jie; Shi, Ai-Ming; Hu, Duan-Min; Liu, Tong; Yu, Yun-Li

    2017-09-01

    Thioredoxin-interacting protein (TXNIP) is associated with activation of oxidative stress through inhibition of thioredoxin (Trx). However, some evidences point out that TXNIP acts as a scaffolding protein in signaling complex independent of cellular redox regulation. The autophagy-lysosomal pathway plays important roles in the clearance of misfolded proteins and dysfunctional organelles. Lysosomal dysfunction has been involved in several neurodegenerative disorders including Parkinson's disease (PD). Although researchers have reported that TXNIP inhibited autophagic flux, the specific mechanism is rarely studied. In this study, we investigated the effects of TXNIP on autophagic flux and α-synuclein accumulation by Western blot in HEK293 cells transfected with TXNIP plasmid. Further, we explored the influence of TXNIP on DA neuron survival in substantia nigra by IHC. We found that TXNIP induced LC3-II expression, but failed to degrade p62, a substrate of autophagy. Also, TXNIP aggravated α-synuclein accumulation. We also found that TXNIP inhibited the expression of ATP13A2, a lysosomal membrane protein. Moreover, we found that overexpression of ATP13A2 attenuated the impairment of autophagic flux and α-synuclein accumulation induced by TXNIP. Furthermore, overexpression of TXNIP in substantia nigra resulted in loss of DA neuron. Our data suggested that TXNIP blocked autophagic flux and induced α-synuclein accumulation through inhibition of ATP13A2, indicating TXNIP was a disease-causing protein in PD. © 2017 John Wiley & Sons Ltd.

  10. Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

    PubMed Central

    Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

    2015-01-01

    To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

  11. Dynamic organization of the mitochondrial protein import machinery.

    PubMed

    Straub, Sebastian P; Stiller, Sebastian B; Wiedemann, Nils; Pfanner, Nikolaus

    2016-11-01

    Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

  12. MACHINERY RESONANCE AND DRILLING

    SciTech Connect

    Leishear, R.; Fowley, M.

    2010-01-23

    New developments in vibration analysis better explain machinery resonance, through an example of drill bit chattering during machining of rusted steel. The vibration of an operating drill motor was measured, the natural frequency of an attached spring was measured, and the two frequencies were compared to show that the system was resonant. For resonance to occur, one of the natural frequencies of a structural component must be excited by a cyclic force of the same frequency. In this case, the frequency of drill bit chattering due to motor rotation equaled the spring frequency (cycles per second), and the system was unstable. A soft rust coating on the steel to be drilled permitted chattering to start at the drill bit tip, and the bit oscillated on and off of the surface, which increased the wear rate of the drill bit. This resonant condition is typically referred to as a motor critical speed. The analysis presented here quantifies the vibration associated with this particular critical speed problem, using novel techniques to describe resonance.

  13. Direct molecular interactions between Beclin 1 and the canonical NFκB activation pathway.

    PubMed

    Niso-Santano, Mireia; Criollo, Alfredo; Malik, Shoaib Ahmad; Michaud, Michael; Morselli, Eugenia; Mariño, Guillermo; Lachkar, Sylvie; Galluzzi, Lorenzo; Maiuri, Maria Chaira; Kroemer, Guido

    2012-02-01

    General (macro)autophagy and the activation of NFκB constitute prominent responses to a large array of intracellular and extracellular stress conditions. The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase (IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB activation pathway, limits autophagy induction by physiological or pharmacological triggers, while constitutive active IKK subunits suffice to stimulate autophagy. The activation of IKK usually relies on TGFβ-activated kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy in multiple settings. TAK1 interacts with two structurally similar co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in resting conditions both TAB2 and TAB3 bind the essential autophagic factor Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3 dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The inhibitory interaction between TABs and Beclin 1 is mediated by their coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point to the existence of a direct molecular crosstalk between the canonical NFκB activation pathway and the autophagic core machinery that guarantees the coordinated induction of these processes in response to stress.

  14. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... distribution; (iii) Machinery space fire detection, alarm, and extinguishing systems; and (iv) Machinery space... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... spaces. (c) Fire detection and alarms. An approved automatic fire detection and alarm system must be...

  15. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... distribution; (iii) Machinery space fire detection, alarm, and extinguishing systems; and (iv) Machinery space... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... spaces. (c) Fire detection and alarms. An approved automatic fire detection and alarm system must be...

  16. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... distribution; (iii) Machinery space fire detection, alarm, and extinguishing systems; and (iv) Machinery space... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... spaces. (c) Fire detection and alarms. An approved automatic fire detection and alarm system must be...

  17. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... distribution; (iii) Machinery space fire detection, alarm, and extinguishing systems; and (iv) Machinery space... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... spaces. (c) Fire detection and alarms. An approved automatic fire detection and alarm system must be...

  18. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... distribution; (iii) Machinery space fire detection, alarm, and extinguishing systems; and (iv) Machinery space... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... spaces. (c) Fire detection and alarms. An approved automatic fire detection and alarm system must be...

  19. Inhibition of ERα/ERK/P62 cascades induces “autophagic switch” in the estrogen receptor-positive breast cancer cells exposed to gemcitabine

    PubMed Central

    He, Mengye; Chen, Luoquan; Song, Yinjing; Xiao, Peng; Wan, Xiaopeng; Dai, Feng; Pan, Ting; Wang, Qingqing

    2016-01-01

    Several clinical trials revealed that estrogen receptor (ER) status had relevance to the response of mammary malignancy to chemotherapy. Autophagy has emerged as an important cellular mechanism of tumor cells in response to anticancer therapy. The aim of this study is to investigate whether gemcitabine induces autophagy, and more importantly, whether such autophagy is functional relevant to the therapeutic effects of gemcitabine in breast cancer cells in relation to the ER status. In our study, autophagy was induced both in ER+ MCF-7 and ER− MDA-MB-231 cells by gemcitabine markedly, while the autophagy plays distinct roles – cytoprotective in ER− MDA-MB-231 and cytotoxic in ER+ MCF-7 cells. Gemcitabine treatment leads to the activation of ERα-ERK-P62 signal pathway in MCF-7 cells which may augment the autophagic degradation, thus results in the excessive activation of autophagy and irreversible autophagic cell death eventually. Inhibition of ERα-ERK-P62 cascades in MCF-7 cells by small interfering RNA or PD98059 impairs the autophagic degradation, and leads to “autophagic switch” – from cytotoxic autophagy to cytoprotection. Moreover, stable overexpression of ERα in the ER− BCap37 breast cancer cell line enhances the gemcitabine-induced autophagy flux and switches the autophagic cytoprotection in ER− BCap37 to cytotoxicity effect in ER+ BCap37 cells. Our study firstly demonstrated that ER status influences gemcitabine efficacy via modulating the autophagy in breast cancer cells. PMID:27384485

  20. Autophagic degradation of peroxisomes in mammals.

    PubMed

    Zientara-Rytter, Katarzyna; Subramani, Suresh

    2016-04-15

    Peroxisomes are essential organelles required for proper cell function in all eukaryotic organisms. They participate in a wide range of cellular processes including the metabolism of lipids and generation, as well as detoxification, of hydrogen peroxide (H2O2). Therefore, peroxisome homoeostasis, manifested by the precise and efficient control of peroxisome number and functionality, must be tightly regulated in response to environmental changes. Due to the existence of many physiological disorders and diseases associated with peroxisome homoeostasis imbalance, the dynamics of peroxisomes have been widely examined. The increasing volume of reports demonstrating significant involvement of the autophagy machinery in peroxisome removal leads us to summarize current knowledge of peroxisome degradation in mammalian cells. In this review we present current models of peroxisome degradation. We particularly focus on pexophagy-the selective clearance of peroxisomes through autophagy. We also critically discuss concepts of peroxisome recognition for pexophagy, including signalling and selectivity factors. Finally, we present examples of the pathological effects of pexophagy dysfunction and suggest promising future directions. © 2016 Authors; published by Portland Press Limited.

  1. Autophagic degradation of peroxisomes in mammals

    PubMed Central

    Katarzyna, Zientara-Rytter; Suresh, Subramani

    2016-01-01

    Peroxisomes are essential organelles required for proper cell function in all eukaryotic organisms. They participate in a wide range of cellular processes including the metabolism of lipids and generation, as well as detoxification, of hydrogen peroxide. Therefore, peroxisome homeostasis, manifested by the precise and efficient control of peroxisome number and functionality, must be tightly regulated in response to environmental changes. Due to the existence of many physiological disorders and diseases associated with peroxisome homeostasis imbalance, the dynamics of peroxisomes have been widely examined. The increasing volume of reports demonstrating significant involvement of the autophagy machinery in peroxisome removal leads us to summarize current knowledge of peroxisome degradation in mammalian cells. In this review we present current models of peroxisome degradation. We particularly focus on pexophagy - the selective clearance of peroxisomes through autophagy. We also critically discuss concepts of peroxisome recognition for pexophagy, including signaling and selectivity factors. Finally, we present examples of the pathological effects of pexophagy dysfunction and suggest promising future directions. PMID:27068951

  2. The autophagic paradox in cancer therapy.

    PubMed

    Wu, W K K; Coffelt, S B; Cho, C H; Wang, X J; Lee, C W; Chan, F K L; Yu, J; Sung, J J Y

    2012-02-23

    Autophagy, hallmarked by the formation of double-membrane bound organelles known as autophagosomes, is a lysosome-dependent pathway for protein degradation. The role of autophagy in carcinogenesis is context dependent. As a tumor-suppressing mechanism in early-stage carcinogenesis, autophagy inhibits inflammation and promotes genomic stability. Moreover, disruption of autophagy-related genes accelerates tumorigenesis in animals. However, autophagy may also act as a pro-survival mechanism to protect cancer cells from various forms of cellular stress. In cancer therapy, adaptive autophagy in cancer cells sustains tumor growth and survival in face of the toxicity of cancer therapy. To this end, inhibition of autophagy may sensitize cancer cells to chemotherapeutic agents and ionizing radiation. Nevertheless, in certain circumstances, autophagy mediates the therapeutic effects of some anticancer agents. Data from recent studies are beginning to unveil the apparently paradoxical nature of autophagy as a cell-fate decision machinery. Taken together, modulation of autophagy is a novel approach for enhancing the efficacy of existing cancer therapy, but its Janus-faced nature may complicate the clinical development of autophagy modulators as anticancer therapeutics.

  3. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis

    DOE PAGES

    Marshall, Richard S.; Li, Faqiang; Gemperline, David C.; ...

    2015-05-21

    Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. In this paper, we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuingmore » proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Finally and collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.« less

  4. Rotenone Inhibits Autophagic Flux Prior to Inducing Cell Death

    PubMed Central

    2012-01-01

    Rotenone, which selectively inhibits mitochondrial complex I, induces oxidative stress, α-synuclein accumulation, and dopaminergic neuron death, principal pathological features of Parkinson's disease. The autophagy–lysosome pathway degrades damaged proteins and organelles for the intracellular maintenance of nutrient and energy balance. While it is known that rotenone causes autophagic vacuole accumulation, the mechanism by which this effect occurs has not been thoroughly investigated. Treatment of differentiated SH-SY5Y cells with rotenone (10 μM) induced the accumulation of autophagic vacuoles at 6 h and 24 h as indicated by Western blot analysis for microtubule associated protein-light chain 3-II (MAP-LC3-II). Assessment of autophagic flux at these time points indicated that autophagic vacuole accumulation resulted from a decrease in their effective lysosomal degradation, which was substantiated by increased levels of autophagy substrates p62 and α-synuclein. Inhibition of lysosomal degradation may be explained by the observed decrease in cellular ATP levels, which in turn may have caused the observed concomitant increase in acidic vesicle pH. The early (6 h) effects of rotenone on cellular energetics and autophagy–lysosome pathway function preceded the induction of cell death and apoptosis. These findings indicate that the classical mitochondrial toxin rotenone has a pronounced effect on macroautophagy completion that may contribute to its neurotoxic potential. PMID:23259041

  5. The autophagic- lysosomal pathway determines the fate of glial cells under manganese- induced oxidative stress conditions.

    PubMed

    Gorojod, R M; Alaimo, A; Porte Alcon, S; Pomilio, C; Saravia, F; Kotler, M L

    2015-10-01

    Manganese (Mn) overexposure is frequently associated with the development of a neurodegenerative disorder known as Manganism. The Mn-mediated generation of reactive oxygen species (ROS) promotes cellular damage, finally leading to apoptotic cell death in rat astrocytoma C6 cells. In this scenario, the autophagic pathway could play an important role in preventing cytotoxicity. In the present study, we found that Mn induced an increase in the amount and total volume of acidic vesicular organelles (AVOs), a process usually related to the activation of the autophagic pathway. Particularly, the generation of enlarged AVOs was a ROS- dependent event. In this report we demonstrated for the first time that Mn induces autophagy in glial cells. This conclusion emerged from the results obtained employing a battery of autophagy markers: a) the increase in LC3-II expression levels, b) the formation of autophagic vesicles labeled with monodansylcadaverine (MDC) or LC3 and, c) the increase in Beclin 1/ Bcl-2 and Beclin 1/ Bcl-X(L) ratio. Autophagy inhibition employing 3-MA and mAtg5(K130R) resulted in decreased cell viability indicating that this event plays a protective role in Mn- induced cell death. In addition, mitophagy was demonstrated by an increase in LC3 and TOM-20 colocalization. On the other hand, we proposed the occurrence of lysosomal membrane permeabilization (LMP) based in the fact that cathepsins B and D activities are essential for cell death. Both cathepsin B inhibitor (Ca-074 Me) or cathepsin D inhibitor (Pepstatin A) completely prevented Mn- induced cytotoxicity. In addition, low dose of Bafilomycin A1 showed a similar effect, a finding that adds evidence about the lysosomal role in Mn cytotoxicity. Finally, in vivo experiments demonstrated that Mn induces injury and alters LC3 expression levels in rat striatal astrocytes. In summary, our results demonstrated that autophagy is activated to counteract the harmful effect caused by Mn. These data is valuable to

  6. The Research of Computer Aided Farm Machinery Designing Method Based on Ergonomics

    NASA Astrophysics Data System (ADS)

    Gao, Xiyin; Li, Xinling; Song, Qiang; Zheng, Ying

    Along with agricultural economy development, the farm machinery product type Increases gradually, the ergonomics question is also getting more and more prominent. The widespread application of computer aided machinery design makes it possible that farm machinery design is intuitive, flexible and convenient. At present, because the developed computer aided ergonomics software has not suitable human body database, which is needed in view of farm machinery design in China, the farm machinery design have deviation in ergonomics analysis. This article puts forward that using the open database interface procedure in CATIA to establish human body database which aims at the farm machinery design, and reading the human body data to ergonomics module of CATIA can product practical application virtual body, using human posture analysis and human activity analysis module to analysis the ergonomics in farm machinery, thus computer aided farm machinery designing method based on engineering can be realized.

  7. Rotenone impairs autophagic flux and lysosomal functions in Parkinson's disease.

    PubMed

    Wu, F; Xu, H-D; Guan, J-J; Hou, Y-S; Gu, J-H; Zhen, X-C; Qin, Z-H

    2015-01-22

    Rotenone is an environmental neurotoxin that induces accumulation of α-synuclein and degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc), but the molecular mechanisms are not fully understood. We investigated whether rotenone induced impairment of autophagic flux and lysosomal functions. Autophagy flux, accumulation of α-synuclein, lysosomal membrane integrity and neurodegeneration were assessed in the rotenone-treated rat model and PC12 cells, and the effects of the autophagy inducer trehalose on rotenone's cytotoxicity were also studied. Rotenone administration significantly reduced motor activity and caused a loss of tyrosine hydroxylase in SNpc of Lewis rats. The degeneration of nigral dopaminergic neurons was accompanied by the deposition of α-synuclein aggregates, autophagosomes and redistribution of cathepsin D from lysosomes to the cytosol. In cultured PC12 cells, rotenone also induced increases in protein levels of α-synuclein, microtubule-associated protein 1 light chain 3-II, Beclin 1, and p62. Rotenone increased lysosomal membrane permeability as evidenced by leakage of N-acetyl-beta-d-glucosaminidase and cathepsin D, the effects were blocked by reactive oxygen species scavenger tiron. Autophagy inducer trehalose enhanced the nuclear translocation of transcription factor EB, accelerated the clearance of autophagosomes and α-synuclein and attenuated rotenone-induced cell death of PC12 cells. Meanwhile, administration of trehalose to rats in drinking water (2%) decreased rotenone-induced dopaminergic neurons loss in SNpc. These studies indicate that the lysosomal dysfunction contributes to rotenone's neurotoxicity and restoration of lysosomal function could be a new therapeutic strategy for Parkinson's disease. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  8. Metformin represses glucose starvation induced autophagic response in microvascular endothelial cells and promotes cell death.

    PubMed

    Samuel, Samson Mathews; Ghosh, Suparna; Majeed, Yasser; Arunachalam, Gnanapragasam; Emara, Mohamed M; Ding, Hong; Triggle, Chris R

    2017-05-15

    Metformin, the most frequently administered drug for the treatment of type 2 diabetes, is being investigated for its potential in the treatment of various types of cancer; however, the cellular basis for this putative anti-cancer action remains controversial. In the current study we examined the effect of metformin on endoplasmic reticulum (ER) stress and autophagy in glucose-starved micro-vascular endothelial cells (MECs). The rationale for our experimental protocol is that in a growing tumor MECs are subjected to hypoxia and nutrient/glucose starvation that results from the reduced supply and relatively high consumption of glucose. Mouse MECs (MMECs) were glucose-starved for up to 48h in the absence or presence of metformin (50μM and 2mM) and the status of ER stress, autophagic, cell survival and apoptotic markers were assessed. Activation of ER stress and autophagy was observed in glucose starved MECs as evidenced by the significant increase in the levels of ER stress and autophagic markers while accumulation of LC3B stained punctae in the MECs confirmed autophagic activation. Treatment with 2mM metformin, independent of AMPK, significantly reversed glucose starvation induced ER stress and autophagy in MECs, but, at 24h, did not decrease cell viability; however, at 48h, persistent ER stress and metformin associated inhibition of autophagy decreased cell viability, caused cell cycle arrest in G2/M and increased the number of cells in the sub-G0/G1 phase of cell cycle. Treatment with metformin reduced phosphorylation of Akt and mTOR and inhibited downstream targets of mTOR. Our findings support the argument that treatment with metformin when used in combination with glycolytic inhibitors will inhibit pro-survival autophagy and promote cell death and potentially prove to be the basis for an effective anti-cancer strategy. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Disturbed Flow Induces Autophagy, but Impairs Autophagic Flux to Perturb Mitochondrial Homeostasis

    PubMed Central

    Li, Rongsong; Jen, Nelson; Wu, Lan; Lee, Juhyun; Fang, Karen; Quigley, Katherine; Lee, Katherine; Wang, Sky; Zhou, Bill; Vergnes, Laurent; Chen, Yun-Ru; Li, Zhaoping; Reue, Karen; Ann, David K.

    2015-01-01

    Abstract Aim: Temporal and spatial variations in shear stress are intimately linked with vascular metabolic effects. Autophagy is tightly regulated in intracellular bulk degradation/recycling system for maintaining cellular homeostasis. We postulated that disturbed flow modulates autophagy with an implication in mitochondrial superoxide (mtO2•−) production. Results: In the disturbed flow or oscillatory shear stress (OSS)-exposed aortic arch, we observed prominent staining of p62, a reverse marker of autophagic flux, whereas in the pulsatile shear stress (PSS)-exposed descending aorta, p62 was attenuated. OSS significantly increased (i) microtubule-associated protein light chain 3 (LC3) II to I ratios in human aortic endothelial cells, (ii) autophagosome formation as quantified by green fluorescent protein (GFP)-LC3 dots per cell, and (iii) p62 protein levels, whereas manganese superoxide dismutase (MnSOD) overexpression by recombinant adenovirus, N-acetyl cysteine treatment, or c-Jun N-terminal kinase (JNK) inhibition reduced OSS-mediated LC3-II/LC3-I ratios and mitochondrial DNA damage. Introducing bafilomycin to Earle's balanced salt solution or to OSS condition incrementally increased both LC3-II/LC3-I ratios and p62 levels, implicating impaired autophagic flux. In the OSS-exposed aortic arch, both anti-phospho-JNK and anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) staining for DNA damage were prominent, whereas in the PSS-exposed descending aorta, the staining was nearly absent. Knockdown of ATG5 with siRNA increased OSS-mediated mtO2•−, whereas starvation or rapamycin-induced autophagy reduced OSS-mediated mtO2•−, mitochondrial respiration, and complex II activity. Innovation: Disturbed flow-mediated oxidative stress and JNK activation induce autophagy. Conclusion: OSS impairs autophagic flux to interfere with mitochondrial homeostasis. Antioxid. Redox Signal. 23, 1207–1219. PMID:26120766

  10. Involvement of ROS in Curcumin-induced Autophagic Cell Death.

    PubMed

    Lee, Youn Ju; Kim, Nam-Yi; Suh, Young-Ah; Lee, Chuhee

    2011-02-01

    Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell

  11. Involvement of ROS in Curcumin-induced Autophagic Cell Death

    PubMed Central

    Lee, Youn Ju; Kim, Nam-Yi; Suh, Young-Ah

    2011-01-01

    Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell

  12. Low concentration of mercury induces autophagic cell death in rat hepatocytes.

    PubMed

    Chatterjee, Sarmishtha; Ray, Atish; Mukherjee, Sandip; Agarwal, Soumik; Kundu, Rakesh; Bhattacharya, Shelley

    2014-08-01

    In the present study, we attempted to elucidate the induction of autophagy in rat hepatocytes by a low concentration of mercury. Hepatocytes treated with different doses of mercuric chloride (HgCl2; 1, 2.5, 5 and 10 µM) and at different time intervals (0 min, 30 min, 1 h, 2 h and 4 h) show autophagic cell death only at 5 µM HgCl2 within 30 min of incubation. At 1 and 2.5 µM HgCl2, no cell death is recorded, while apoptosis is found at 10 µM HgCl2, as evidenced by the activation of caspase 3. Autophagic cell death is confirmed by the presence of monodansylcadaverine (MDC) positive hepatocytes which is found to be highest at 1 h. Atg5-Atg12 covalent-conjugation triggers the autophagic pathway within 30 min of 5 µM HgCl2 treatment and continues till 4 h of incubation. In addition, damage-regulated autophagy modulator (DRAM) expression gradually increases from 30 min to 4 h of treatment with mercury and a corresponding linear decrease in p53 has been observed. It is concluded that a low concentration (5 µM HgCl2) of mercury induces autophagy or nonapoptotic programmed cell death following an Atg5-Atg12 covalent-conjugation pathway, which is modulated by DRAM in a p53-dependent manner.

  13. A Molecular Reporter for Monitoring Autophagic Flux in Nervous System In Vivo.

    PubMed

    Castillo, K; Valenzuela, V; Oñate, M; Hetz, C

    2017-01-01

    The relevance of autophagy in neuronal health has been extensively reported in a plethora of conditions affecting the nervous system, such as neurodegenerative diseases, cancer, diabetes, and tissue injury, where altered autophagic activity may contribute to the pathological process. Autophagy is a dynamic pathway involving the formation of a membrane surrounding and enclosing cargoes that are delivered to lysosomal compartments for degradation. Cargoes can include large protein aggregates, organelles, or even pathogens. Traditionally, autophagy assessment relies on the measurement of LC3-II protein levels or the visualization of LC3-positive puncta. However, these approaches represent a static measurement of autophagy markers, making difficult the dissection of the actual changes in the autophagy process (activation, inhibition, or no effects), due to the dynamic regulation of LC3 viral levels. To circumvent this limitation, we previously developed an adeno-associated vector (AAV) to deliver a molecular autophagy sensor to the neuronal compartment in vivo. Here, we describe the detailed design and methods to use an engineered AAV harboring the monomeric tandem mCherry-GFP-LC3 to determine autophagic fluxes in the nervous system. Key methodological details to succeed in the use of this reporter are provided. © 2017 Elsevier Inc. All rights reserved.

  14. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    SciTech Connect

    Inami, Yoshihiro; Yamashina, Shunhei; Izumi, Kousuke; Ueno, Takashi; Tanida, Isei; Ikejima, Kenichi; Watanabe, Sumio

    2011-09-09

    Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  15. A triazole derivative elicits autophagic clearance of polyglutamine aggregation in neuronal cells

    PubMed Central

    Hsieh, Chang Heng; Lee, Li-Ching; Leong, Wai-Yin; Yang, Tsai-Chen; Yao, Ching-Fa; Fang, Kang

    2016-01-01

    Trinucleotide CAG repeat expansion in the coding region of genes has a propensity to form polyglutamine (polyQ) aggregates that contribute to neuronal disorders. Strategies in elevating autophagy to disintegrate the insoluble aggregates without injuring cells have become a major goal for therapy. In this work, a triazole derivative, OC-13, was found accelerating autophagic clearance of polyQ aggregation in human neuroblastoma cells following induction of the enhanced green fluorescence-conjugated chimeric protein that enclosed 79 polyQ repeats (Q79-EGFP). OC-13 accelerated autophagy development and removed nuclear Q79-EGFP aggregates. The increase of Beclin-1, turnover of LC3-I to LC3-II and degradation of p62 supported autophagy activation. Pretreatment of autophagy inhibitor, bafilomycin A1, not only suppressed autophagolysome fusion, but also impeded aggregate eradication. The study also showed that c-Jun N-terminal kinase/Beclin-1 pathway was activated during OC-13 treatment and c-Jun N-terminal kinase inhibitor impaired autophagy and final breakdown. Autophagic clearance of the insoluble aggregates demonstrated the feasibility of OC-13 in alleviating neuronal disorders because of expanded glutamine stretches. PMID:27695292

  16. Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity.

    PubMed

    Vanrell, María C; Cueto, Juan A; Barclay, Jeremías J; Carrillo, Carolina; Colombo, María I; Gottlieb, Roberta A; Romano, Patricia S

    2013-07-01

    Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.

  17. Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

    PubMed Central

    Vanrell, María C.; Cueto, Juan A.; Barclay, Jeremías J.; Carrillo, Carolina; Colombo, María I.; Gottlieb, Roberta A.; Romano, Patricia S.

    2013-01-01

    Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings. PMID:23697944

  18. Primary cilia and autophagic dysfunction in Huntington's disease

    PubMed Central

    Kaliszewski, M; Knott, A B; Bossy-Wetzel, E

    2015-01-01

    Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by a single-gene mutation: a CAG expansion in the huntingtin (HTT) gene that results in production of a mutated protein, mutant HTT, with a polyglutamine tail (polyQ-HTT). Although the molecular pathways of polyQ-HTT toxicity are not fully understood, because protein misfolding and aggregation are central features of HD, it has long been suspected that cellular housekeeping processes such as autophagy might be important to disease pathology. Indeed, multiple lines of research have identified abnormal autophagy in HD, characterized generally by increased autophagic induction and inefficient clearance of substrates. To date, the origin of autophagic dysfunction in HD remains unclear and the search for actors involved continues. To that end, recent studies have suggested a bidirectional relationship between autophagy and primary cilia, signaling organelles of most mammalian cells. Interestingly, primary cilia structure is defective in HD, suggesting a potential link between autophagic dysfunction, primary cilia and HD pathogenesis. In addition, because polyQ-HTT also accumulates in primary cilia, the possibility exists that primary cilia might play additional roles in HD: perhaps by disrupting signaling pathways or acting as a reservoir for secretion and propagation of toxic, misfolded polyQ-HTT fragments. Here, we review recent research suggesting potential links between autophagy, primary cilia and HD and speculate on possible pathogenic mechanisms and future directions for the field. PMID:26160070

  19. Metaxins 1 and 2, two proteins of the mitochondrial protein sorting and assembly machinery, are essential for Bak activation during TNF alpha triggered apoptosis.

    PubMed

    Cartron, Pierre-François; Petit, Elise; Bellot, Grégory; Oliver, Lisa; Vallette, François M

    2014-09-01

    The proteins Bax and Bak are central in the execution phase of apoptosis; however, little is known about the partners involved in the control of this complex process. Here, we show that mitochondrial Bak is incorporated into a VDAC2/Mtx1/Mtx2 multi-protein complex in both resting and dying cells. VDAC2 is a porin that has previously been described as a partner of Bak while Mtx1 and Mtx2 are two proteins of the mitochondrial sorting and assembly machinery (SAM) that have been implicated in TNF-induced apoptosis. We show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to interact with Mtx1. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

    PubMed Central

    Chiu, Chang-Fang; Weng, Jing-Ru; Jadhav, Appaso; Wu, Chia-Yung; Sargeant, Aaron M.; Bai, Li-Yuan

    2016-01-01

    T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy. PMID:27537872

  1. Practical machinery management for process plants. Volume 3: Machinery component maintenance and repair

    SciTech Connect

    Bloch, H.P.; Geitner, F.K.

    1985-01-01

    This work is a reference for machinery engineers concerned with machinery and component installation, maintenance, and repair. This third volume covers maintenance organization, machinery turnaround management, turbomachinery overhauls, machinery foundations and grouting, process machinery piping, alignment and balancing of machinery components, ball bearing maintenance and replacement, mechanical seals, welded repairs to pump shafts and other rotating equipment components, centrifugal compressor rotor repair, selection and application of O-rings, and more.

  2. The Endocrine Machinery.

    ERIC Educational Resources Information Center

    Fillman, David

    1987-01-01

    Promotes a reductionist approach to teaching about the endocrine system in high school biology and anatomy courses. Encourages the study of how hormones travel to the cells and affect them. Provides suggestions for activities and discussion questions, along with sample diagrams and flow charts. (TW)

  3. The Endocrine Machinery.

    ERIC Educational Resources Information Center

    Fillman, David

    1987-01-01

    Promotes a reductionist approach to teaching about the endocrine system in high school biology and anatomy courses. Encourages the study of how hormones travel to the cells and affect them. Provides suggestions for activities and discussion questions, along with sample diagrams and flow charts. (TW)

  4. Phellinus linteus Mycelium Alleviates Myocardial Ischemia-Reperfusion Injury through Autophagic Regulation.

    PubMed

    Su, Hsing-Hui; Chu, Ya-Chun; Liao, Jiuan-Miaw; Wang, Yi-Hsin; Jan, Ming-Shiou; Lin, Chia-Wei; Wu, Chiu-Yeh; Tseng, Chin-Yin; Yen, Jiin-Cherng; Huang, Shiang-Suo

    2017-01-01

    The incidence of myocardial ischemia-reperfusion (IR) injury is rapidly increasing around the world and this disease is a major contributor to global morbidity and mortality. It is known that regulation of programmed cell death including apoptosis and autophagy reduces the impact of myocardial IR injury. In this study, the cardioprotective effects and underlying mechanisms of Phellinus linteus (Berk. and Curt.) Teng, Hymenochaetaceae (PL), a type of medicinal mushroom, were examined in rats subjected to myocardial IR injury. The left main coronary artery of rats was ligated for 1 h and reperfused for 3 h. The arrhythmia levels were monitored during the entire process and the infarct size was evaluated after myocardial IR injury. Furthermore, the expression levels of proteins in apoptotic and autophagic pathways were observed. Pretreatment with PL mycelium (PLM) significantly reduced ventricular arrhythmia and mortality due to myocardial IR injury. PLM also significantly decreased myocardial infarct size and plasma lactate dehydrogenase level after myocardial IR injury. Moreover, PLM administration resulted in decreased caspase 3 and caspase 9 activation and increased Bcl-2/Bax ratio. Phosphorylation level of AMPK was elevated while mTOR level was reduced. Becline-1 and p62 levels decreased. These findings suggest that PLM is effective in protecting the myocardium against IR injury. The mechanism involves mediation through suppressed pro-apoptotic signaling and regulation of autophagic signaling, including stimulation of AMPK-dependent pathway and inhibition of beclin-1-dependent pathway, resulting in enhancement of protective autophagy and inhibition of excessive autophagy.

  5. Phellinus linteus Mycelium Alleviates Myocardial Ischemia-Reperfusion Injury through Autophagic Regulation

    PubMed Central

    Su, Hsing-Hui; Chu, Ya-Chun; Liao, Jiuan-Miaw; Wang, Yi-Hsin; Jan, Ming-Shiou; Lin, Chia-Wei; Wu, Chiu-Yeh; Tseng, Chin-Yin; Yen, Jiin-Cherng; Huang, Shiang-Suo

    2017-01-01

    The incidence of myocardial ischemia-reperfusion (IR) injury is rapidly increasing around the world and this disease is a major contributor to global morbidity and mortality. It is known that regulation of programmed cell death including apoptosis and autophagy reduces the impact of myocardial IR injury. In this study, the cardioprotective effects and underlying mechanisms of Phellinus linteus (Berk. and Curt.) Teng, Hymenochaetaceae (PL), a type of medicinal mushroom, were examined in rats subjected to myocardial IR injury. The left main coronary artery of rats was ligated for 1 h and reperfused for 3 h. The arrhythmia levels were monitored during the entire process and the infarct size was evaluated after myocardial IR injury. Furthermore, the expression levels of proteins in apoptotic and autophagic pathways were observed. Pretreatment with PL mycelium (PLM) significantly reduced ventricular arrhythmia and mortality due to myocardial IR injury. PLM also significantly decreased myocardial infarct size and plasma lactate dehydrogenase level after myocardial IR injury. Moreover, PLM administration resulted in decreased caspase 3 and caspase 9 activation and increased Bcl-2/Bax ratio. Phosphorylation level of AMPK was elevated while mTOR level was reduced. Becline-1 and p62 levels decreased. These findings suggest that PLM is effective in protecting the myocardium against IR injury. The mechanism involves mediation through suppressed pro-apoptotic signaling and regulation of autophagic signaling, including stimulation of AMPK-dependent pathway and inhibition of beclin-1-dependent pathway, resulting in enhancement of protective autophagy and inhibition of excessive autophagy. PMID:28420993

  6. Mitigated NSAID-induced apoptotic and autophagic cell death with Smad7 overexpression

    PubMed Central

    Lee, Ho-Jae; Park, Jong Min; Hahm, Ki Baik

    2017-01-01

    Non-steroidal anti-inflammatory drugs damaged gastrointestinal mucosa in cyclooxygenase-dependent and -independent pathway, among which apopototic or autophagic cell death in gastrointestinal cells might be one of key cytotoxic mechanisms responsible for NSAID-induced damages. Therefore, alleviating this cell death after NSAIDs can be a rescuing strategy. In this study, we explored the role of Smad7 on NSAID-induced cytotoxicity in gastric epithelial cells. Using RGM1 cells, we have compared biological changes between mock-transfected and Smad7-overexpressed cells. As results, significantly decreased cytotoxicity accompanied with decreased levels of cleaved caspase-3 and poly (ADP-ribose) polymerase, Bax, and autophagic vesicles concurrent with decreased expressions of autophagy protein 5 and microtubule-associated protein light chain 3B-II were noted in Smad7-overexpressed cells with indomethacin administration compared to mock-transfected cells. Contrast to mitigated apoptotic execution, anti-apoptotic Bcl-2 and Beclin-1 were significantly increased in Smad7-overexpressed cells compared to mock-transfected cells. Smad7 siRNA significantly reversed these protective actions of Smad7 against indomethacin, in which p38 mitogen-activated protein kinase was significantly intervened. Furthermore, indomethacin-induced Smad7 degradation through ubiquitin-proteasome pathway was relevant to increased cytotoxicity, while chloroquine as autophagy inhibitor significantly attenuated indomethacin-induced cytotoxicity through Smad7 preservation via repressed ubiquitination. Conclusively, either genetic overexpression or pharmacological induction of Smad7 significantly attenuated indomethacin-induced gastric cell damages. PMID:28163383

  7. Tractor & Machinery Safety. 1984 Revision.

    ERIC Educational Resources Information Center

    Montana State Office of Public Instruction, Helena. Dept. of Vocational Education Services.

    This curriculum guide is intended for use in teaching an instructional unit in tractor and machinery safety that is geared toward college freshmen. Addressed in the individual lessons of the unit are the following topics: understanding the importance of safe and efficient tractor operation, understanding the characteristics of tractors, preparing…

  8. Combined aerobic exercise and enzyme replacement therapy rejuvenates the mitochondrial-lysosomal axis and alleviates autophagic blockage in Pompe disease.

    PubMed

    Nilsson, M I; MacNeil, L G; Kitaoka, Y; Suri, R; Young, S P; Kaczor, J J; Nates, N J; Ansari, M U; Wong, T; Ahktar, M; Brandt, L; Hettinga, B P; Tarnopolsky, M A

    2015-10-01

    A unifying feature in the pathogenesis of aging, neurodegenerative disease, and lysosomal storage disorders is the progressive deposition of macromolecular debris impervious to enzyme catalysis by cellular waste disposal mechanisms (e.g., lipofuscin). Aerobic exercise training (AET) has pleiotropic effects and stimulates mitochondrial biogenesis, antioxidant defense systems, and autophagic flux in multiple organs and tissues. Our aim was to explore the therapeutic potential of AET as an ancillary therapy to mitigate autophagic buildup and oxidative damage and rejuvenate the mitochondrial-lysosomal axis in Pompe disease (GSD II/PD). Fourteen weeks of combined recombinant acid α-glucosidase (rhGAA) and AET polytherapy attenuated mitochondrial swelling, fortified antioxidant defense systems, reduced oxidative damage, and augmented glycogen clearance and removal of autophagic debris/lipofuscin in fast-twitch skeletal muscle of GAA-KO mice. Ancillary AET potently augmented the pool of PI4KA transcripts and exerted a mild restorative effect on Syt VII and VAMP-5/myobrevin, collectively suggesting improved endosomal transport and Ca(2+)- mediated lysosomal exocytosis. Compared with traditional rhGAA monotherapy, AET and rhGAA polytherapy effectively mitigated buildup of protein carbonyls, autophagic debris/lipofuscin, and P62/SQSTM1, while enhancing MnSOD expression, nuclear translocation of Nrf-2, muscle mass, and motor function in GAA-KO mice. Combined AET and rhGAA therapy reactivates cellular clearance pathways, mitigates mitochondrial senescence, and strengthens antioxidant defense systems in GSD II/PD. Aerobic exercise training (or pharmacologic targeting of contractile-activity-induced pathways) may have therapeutic potential for mitochondrial-lysosomal axis rejuvenation in lysosomal storage disorders and related conditions (e.g., aging and neurodegenerative disease).

  9. Rotenone Induces the Formation of 4-Hydroxynonenal Aggresomes. Role of ROS-Mediated Tubulin Hyperacetylation and Autophagic Flux Disruption.

    PubMed

    Bonet-Ponce, Luis; Saez-Atienzar, Sara; da Casa, Carmen; Sancho-Pelluz, Javier; Barcia, Jorge M; Martinez-Gil, Natalia; Nava, Eduardo; Jordan, Joaquín; Romero, Francisco J; Galindo, Maria F

    2016-11-01

    Oxidative stress causes cellular damage by (i) altering protein stability, (ii) impairing organelle function, or (iii) triggering the formation of 4-HNE protein aggregates. The catabolic process known as autophagy is an antioxidant cellular response aimed to counteract these stressful conditions. Therefore, autophagy might act as a cytoprotective response by removing impaired organelles and aggregated proteins. In the present study, we sought to understand the role of autophagy in the clearance of 4-HNE protein aggregates in ARPE-19 cells under rotenone exposure. Rotenone induced an overproduction of reactive oxygen species (ROS), which led to an accumulation of 4-HNE inclusions, and an increase in the number of autophagosomes. The latter resulted from a disturbed autophagic flux rather than an activation of the autophagic synthesis pathway. In compliance with this, rotenone treatment induced an increase in LC3-II while upstream autophagy markers such as Beclin- 1, Vsp34 or Atg5-Atg12, were decreased. Rotenone reduced the autophagosome-to-lysosome fusion step by increasing tubulin acetylation levels through a ROS-mediated pathway. Proof of this is the finding that the free radical scavenger, N-acetylcysteine, restored autophagy flux and reduced rotenone-induced tubulin hyperacetylation. Indeed, this dysfunctional autophagic response exacerbates cell death triggered by rotenone, since 3-methyladenine, an autophagy inhibitor, reduced cell mortality, while rapamycin, an inductor of autophagy, caused opposite effects. In summary, we shed new light on the mechanisms involved in the autophagic responses disrupted by oxidative stress, which take place in neurodegenerative diseases such as Huntington or Parkinson diseases, and age-related macular degeneration.

  10. Regulation of Glycolytic Metabolism by Autophagy in Liver Cancer Involves Selective Autophagic Degradation of HK2 (hexokinase 2).

    PubMed

    Jiao, Lin; Zhang, Hai-Liang; Li, Dan-Dan; Yang, Ke-Li; Tang, Jun; Li, Xuan; Ji, Jiao; Yu, Yan; Wu, Rui-Yan; Ravichandran, Senthilkumar; Liu, Jian-Jun; Feng, Gong-Kan; Chen, Min-Shan; Zeng, Yi-Xin; Deng, Rong; Zhu, Xiao-Feng

    2017-10-05

    Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p<0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.

  11. The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

    PubMed Central

    Jiang, Yanxialei; Lee, Jeeyoung; Lee, Jung Hoon; Lee, Joon Won; Kim, Ji Hyeon; Choi, Won Hoon; Yoo, Young Dong; Cha-Molstad, Hyunjoo; Kim, Bo Yeon; Kwon, Yong Tae; Noh, Sue Ah; Kim, Kwang Pyo; Lee, Min Jae

    2016-01-01

    ABSTRACT The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux. PMID:27560450

  12. Apoptotic and autophagic cell death induced by glucolaxogenin in cervical cancer cells.

    PubMed

    Sánchez-Sánchez, L; Escobar, M L; Sandoval-Ramírez, J; López-Muñoz, H; Fernández-Herrera, M A; Hernández-Vázquez, J M V; Hilario-Martínez, C; Zenteno, E

    2015-12-01

    The antiproliferative and cytotoxic activity of glucolaxogenin and its ability to induce apoptosis and autophagy in cervical cancer cells are reported. We ascertained that glucolaxogenin exerts an inhibitory effect on the proliferation of HeLa, CaSki and ViBo cells in a dose-dependent manner. Analysis of DNA distribution in the cell-cycle phase of tumor cells treated with glucolaxogenin suggests that the anti-proliferative activity of this steroid is not always dependent on the cell cycle. Cytotoxic activity was evaluated by detection of the lactate dehydrogenase enzyme in supernatants from tumor cell cultures treated with the steroid. Glucolaxogenin exhibited null cytotoxic activity. With respect to the apoptotic activity, the generation of apoptotic bodies, the presence of active caspase-3 and annexin-V, as well as the DNA fragmentation observed in all tumor lines after treatment with glucolaxogenin suggests that this compound does indeed induce cell death by apoptosis. Also, a significantly increased presence of the LC3-II, LC3 and Lamp-1 proteins was evidenced with the ultrastructural existence of autophagic vacuoles in cells treated with this steroidal glycoside, indicating that glucolaxogenin also induces autophagic cell death. It is important to note that this compound showed no cytotoxic effect and did not affect the proliferative capacity of mononuclear cells obtained from normal human peripheral blood activated by phytohaemagglutinin. Thus, glucolaxogenin is a compound with anti-proliferative properties that induces programmed cell death in cancer cell lines, though it is selective with respect to normal lymphocytic cells. These findings indicate that this glycoside could have a selective action on tumor cells and, therefore, be worthy of consideration as a therapeutic candidate with anti-tumor potential.

  13. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms....

  14. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms. (a...

  15. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms. (a...

  16. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms. (a...

  17. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms. (a...

  18. 30 CFR 57.14204 - Machinery lubrication.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery lubrication. 57.14204 Section 57.14204 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL... Equipment Safety Practices and Operational Procedures § 57.14204 Machinery lubrication. Machinery...

  19. 30 CFR 56.14204 - Machinery lubrication.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery lubrication. 56.14204 Section 56.14204 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL... Equipment Safety Practices and Operational Procedures § 56.14204 Machinery lubrication. Machinery...

  20. Selenomethionine Mitigates Cognitive Decline by Targeting Both Tau Hyperphosphorylation and Autophagic Clearance in an Alzheimer's Disease Mouse Model.

    PubMed

    Zhang, Zhong-Hao; Wu, Qiu-Yan; Zheng, Rui; Chen, Chen; Chen, Yao; Liu, Qiong; Hoffmann, Peter R; Ni, Jia-Zuan; Song, Guo-Li

    2017-03-01

    Tau pathology was recently identified as a key driver of disease progression and an attractive therapeutic target in Alzheimer's disease (AD). Selenomethionine (Se-Met), a major bioactive form of selenium (Se) in organisms with significant antioxidant capacity, reduced the levels of total tau and hyperphosphorylated tau and ameliorated cognitive deficits in younger triple transgenic AD (3xTg-AD) mice. Whether Se-Met has a similar effect on tau pathology and the specific mechanism of action in older 3xTg-AD mice remains unknown. Autophagy is a major self-degradative process to maintain cellular homeostasis and function. Autophagic dysfunction has been implicated in the pathogenesis of multiple age-dependent diseases, including AD. Modulation of autophagy has been shown to retard the accumulation of misfolded and aggregated proteins and to delay the progression of AD. Here, we found that 3xTg-AD mice showed significant improvement in cognitive ability after a 3-month treatment with Se-Met beginning at 8 months of age. In addition to attenuating the hyperphosphorylation of tau by modulating the activity of Akt/glycogen synthase kinase-3β and protein phosphatase 2A, Se-Met-induced reduction of tau was also mediated by an autophagy-based pathway. Specifically, Se-Met improved the initiation of autophagy via the AMP-activated protein kinase-mTOR (mammalian target of rapamycin) signaling pathway and enhanced autophagic flux to promote the clearance of tau in 3xTg-AD mice and primary 3xTg neurons. Thus, our results demonstrate for the first time that Se-Met mitigates cognitive decline by targeting both the hyperphosphorylation of tau and the autophagic clearance of tau in AD mice. These data strongly support Se-Met as a potent nutraceutical for AD therapy.SIGNIFICANCE STATEMENT Selenium has been widely recognized as a vital trace element abundant in the brain with effects of antioxidant, anticancer, and anti-inflammation. In this study, we report that selenomethionine

  1. The Ubiquitination Machinery of the Ubiquitin System

    PubMed Central

    Callis, Judy

    2014-01-01

    The protein ubiquitin is a covalent modifier of proteins, including itself. The ubiquitin system encompasses the enzymes required for catalysing attachment of ubiquitin to substrates as well as proteins that bind to ubiquitinated proteins leading them to their final fate. Also included are activities that remove ubiquitin independent of, or in concert with, proteolysis of the substrate, either by the proteasome or proteases in the vacuole. In addition to ubiquitin encoded by a family of fusion proteins, there are proteins with ubiquitin-like domains, likely forming ubiquitin's β-grasp fold, but incapable of covalent modification. However, they serve as protein-protein interaction platforms within the ubiquitin system. Multi-gene families encode all of these types of activities. Within the ubiquitination machinery “half” of the ubiquitin system are redundant, partially redundant, and unique components affecting diverse developmental and environmental responses in plants. Notably, multiple aspects of biotic and abiotic stress responses require, or are modulated by, ubiquitination. Finally, aspects of the ubiquitin system have broad utility: as components to enhance gene expression or to regulate protein abundance. This review focuses on the ubiquitination machinery: ubiquitin, unique aspects about the synthesis of ubiquitin and organization of its gene family, ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases, or E3s. Given the large number of E3s in Arabidopsis this review covers the U box, HECT and RING type E3s, with the exception of the cullin-based E3s. PMID:25320573

  2. Interference in autophagosome fusion by rare earth nanoparticles disrupts autophagic flux and regulation of an interleukin-1β producing inflammasome.

    PubMed

    Li, Ruibin; Ji, Zhaoxia; Qin, Hongqiang; Kang, Xuedong; Sun, Bingbing; Wang, Meiying; Chang, Chong Hyun; Wang, Xiang; Zhang, Haiyuan; Zou, Hanfa; Nel, Andre E; Xia, Tian

    2014-10-28

    Engineered nanomaterials (ENMs) including multiwall carbon nanotubes (MWCNTs) and rare earth oxide (REO) nanoparticles, which are capable of activating the NLRP3 inflammasome and inducing IL-1β production, have the potential to cause chronic lung toxicity. Although it is known that lysosome damage is an upstream trigger in initiating this pro-inflammatory response, the same organelle is also an important homeostatic regulator of activated NLRP3 inflammasome complexes, which are engulfed by autophagosomes and then destroyed in lysosomes after fusion. Although a number of ENMs have been shown to induce autophagy, no definitive research has been done on the homeostatic regulation of the NLRP3 inflammasome during autophagic flux. We used a myeloid cell line (THP-1) and bone marrow derived macrophages (BMDM) to compare the role of autophagy in regulating inflammasome activation and IL-1β production by MWCNTs and REO nanoparticles. THP-1 cells express a constitutively active autophagy pathway and are also known to mimic NLRP3 activation in pulmonary macrophages. We demonstrate that, while activated NLRP3 complexes could be effectively removed by autophagosome fusion in cells exposed to MWCNTs, REO nanoparticles interfered in autophagosome fusion with lysosomes. This leads to the accumulation of the REO-activated inflammasomes, resulting in robust and sustained IL-1β production. The mechanism of REO nanoparticle interference in autophagic flux was clarified by showing that they disrupt lysosomal phosphoprotein function and interfere in the acidification that is necessary for lysosome fusion with autophagosomes. Binding of LaPO4 to the REO nanoparticle surfaces leads to urchin-shaped nanoparticles collecting in the lysosomes. All considered, these data demonstrate that in contradistinction to autophagy induction by some ENMs, specific materials such as REOs interfere in autophagic flux, thereby disrupting homeostatic regulation of activated NLRP3 complexes.

  3. Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery.

    PubMed

    Liao, Sumei; Klein, Marlise I; Heim, Kyle P; Fan, Yuwei; Bitoun, Jacob P; Ahn, San-Joon; Burne, Robert A; Koo, Hyun; Brady, L Jeannine; Wen, Zezhang T

    2014-07-01

    Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

  4. Autophagic-lysosomal pathway functions in the masseter and tongue muscles in the klotho mouse, a mouse model for aging.

    PubMed

    Iida, Ryo-hei; Kanko, Syuhei; Suga, Takeo; Morito, Mitsuhiko; Yamane, Akira

    2011-02-01

    Klotho mutant (kl/kl) mice, a type of short-lived mouse models, display several aging-related phenotypes. To investigate whether the atrophy of skeletal muscles is induced in these mice via activation of the ubiquitin-proteasomal pathway and/or the autophagic-lysosomal pathway through an alteration of insulin/IGF-I signaling, we analyzed the activity of the two pathways for protein degradation and components of the insulin/IGF signaling pathway in their skeletal muscles. The masseter, tongue, and gastrocnemius muscles in kl/kl showed marked reductions in muscle weight and in myofiber diameter compared with +/+. The autophagic-lysosomal pathway in kl/kl was activated in the masseter and tongue, but not in the gastrocnemius, compared with that in +/+, whereas the ubiquitin-proteasomal pathway in these three muscles of kl/kl was not altered. No marked difference in the phosphorylation levels of insulin/IGF-I signaling components, such as insulin/IGF-I receptor, Akt, and FoxO in three muscles studied were found between kl/kl and +/+, but the phosphorylation levels of signaling component at the downstream of mTOR such as 4E-BP1 and p70 S6K were suppressed in the masseter and tongue of kl/kl compared with +/+. Deficiency of essential amino acids is reported to activate the autophagy-lysosomal pathway through the down-regulation of mTOR, not through IGF-Akt-FoxO. The masseter and tongue seem to be more actively moved than limb muscles in kl/kl, because they are essential for survival activities such as mastication, swallowing, and respiration. Thus, the deficiency of amino acid by the active movement of the masseter and tongue seems to stimulate the autophagic-lysosomal pathway via the down-regulation of mTOR signalling pathway.

  5. ESCRT-0 dysfunction compromises autophagic degradation of protein aggregates and facilitates ER stress-mediated neurodegeneration via apoptotic and necroptotic pathways.

    PubMed

    Oshima, Ryuji; Hasegawa, Takafumi; Tamai, Keiichi; Sugeno, Naoto; Yoshida, Shun; Kobayashi, Junpei; Kikuchi, Akio; Baba, Toru; Futatsugi, Akira; Sato, Ikuro; Satoh, Kennichi; Takeda, Atsushi; Aoki, Masashi; Tanaka, Nobuyuki

    2016-04-26

    Endosomal sorting required for transport (ESCRT) complexes orchestrate endo-lysosomal sorting of ubiquitinated proteins, multivesicular body formation and autophagic degradation. Defects in the ESCRT pathway have been implicated in many neurodegenerative diseases, but the underlying molecular mechanisms that link them to neurodegeneration remain unknown. In this study, we showed that forebrain-specific ablation of ESCRT-0/Hrs induced marked hippocampal neuronal cell loss accompanied by the accumulation of ubiquitinated proteins, including α-synuclein, TDP-43 and huntingtin as well as the autophagic substrate SQSTM1/p62. Consistent with this, silencing of Hrs in cultured cells not only led to α-synuclein and TDP-43 accumulation in addition to impaired autophagic flux but also suppressed cell viability through the induction of ER stress followed by the activation of JNK and RIPK1, a key regulator of necroptosis. Moreover, necrostatin-1, a specific inhibitor of RIPK1, and pan-caspase inhibitors partially reduced the neurotoxicity in the Hrs-silenced cells. Altogether, these findings suggest that the disruption of ESCRT-0/Hrs in the nervous system compromises autophagic/lysosomal degradation of neurodegenerative disease-related proteins, which thereby triggers ER stress-mediated apoptotic and necroptotic cell death.

  6. Essentials of Proteolytic Machineries in Chloroplasts.

    PubMed

    Nishimura, Kenji; Kato, Yusuke; Sakamoto, Wataru

    2017-01-09

    Plastids are unique organelles that can alter their structure and function in response to environmental and developmental stimuli. Chloroplasts are one type of plastid and are the sites for various metabolic processes, including photosynthesis. For optimal photosynthetic activity, the chloroplast proteome must be properly shaped and maintained through regulated proteolysis and protein quality control mechanisms. Enzymatic functions and activities are conferred by protein maturation processes involving consecutive proteolytic reactions. Protein abundances are optimized by the balanced protein synthesis and degradation, which is depending on the metabolic status. Malfunctioning proteins are promptly degraded. Twenty chloroplast proteolytic machineries have been characterized to date. Specifically, processing peptidases and energy-driven processive proteases are the major players in chloroplast proteome biogenesis, remodeling, and maintenance. Recently identified putative proteases are potential regulators of photosynthetic functions. Here we provide an updated, comprehensive overview of chloroplast protein degradation machineries and discuss their importance for photosynthesis. Wherever possible, we also provide structural insights into chloroplast proteases that implement regulated proteolysis of substrate proteins/peptides. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  7. Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent.

    PubMed

    Torrado, Belén; Graña, Martín; Badano, José L; Irigoín, Florencia

    2016-01-01

    Gli2 is the primary transcriptional activator of Hedgehog signalling in mammals. Upon stimulation of the pathway, Gli2 moves into the cilium before reaching the nucleus. However, the mechanisms underlying its entry into the cilium are not completely understood. Since several similarities have been reported between nuclear and ciliary import, we investigated if the nuclear import machinery participates in Gli2 ciliary entry. Here we show that while two conserved classical nuclear localization signals mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import. However, blocking Imp-mediated transport through overexpression of GTP-locked Ran reduced the percentage of Gli2 positive cilia, an effect that was not explained by increased CRM1-dependent export of Gli2 from the cilium. We explored the participation of Imp-β2 in Gli2 ciliary traffic and observed that this transporter is involved in moving Gli2 into the cilium, as has been described for other ciliary proteins. In addition, our data indicate that Imp-β2 might also collaborate in Gli2 nuclear entry. How does Imp-β2 determine the final destination of a protein that can localize to two distinct subcellular compartments remains an open question. Therefore, our data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediating its ciliary and nuclear translocation.

  8. Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

    PubMed Central

    Torrado, Belén; Graña, Martín; Badano, José L.; Irigoín, Florencia

    2016-01-01

    Gli2 is the primary transcriptional activator of Hedgehog signalling in mammals. Upon stimulation of the pathway, Gli2 moves into the cilium before reaching the nucleus. However, the mechanisms underlying its entry into the cilium are not completely understood. Since several similarities have been reported between nuclear and ciliary import, we investigated if the nuclear import machinery participates in Gli2 ciliary entry. Here we show that while two conserved classical nuclear localization signals mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import. However, blocking Imp-mediated transport through overexpression of GTP-locked Ran reduced the percentage of Gli2 positive cilia, an effect that was not explained by increased CRM1-dependent export of Gli2 from the cilium. We explored the participation of Imp-β2 in Gli2 ciliary traffic and observed that this transporter is involved in moving Gli2 into the cilium, as has been described for other ciliary proteins. In addition, our data indicate that Imp-β2 might also collaborate in Gli2 nuclear entry. How does Imp-β2 determine the final destination of a protein that can localize to two distinct subcellular compartments remains an open question. Therefore, our data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediating its ciliary and nuclear translocation. PMID:27579771

  9. Punicalagin induces apoptotic and autophagic cell death in human U87MG glioma cells

    PubMed Central

    Wang, Shyang-guang; Huang, Ming-hung; Li, Jui-hsiang; Lai, Fu-i; Lee, Horng-mo; Hsu, Yuan-nian

    2013-01-01

    Aim: To investigate the effects of punicalagin, a polyphenol isolated from Punica granatum, on human U87MG glioma cells in vitro. Methods: The viability of human U87MG glioma cells was evaluated using MTT assay. Cell cycle was detected with flow cytometry analysis. The levels of Bcl-2, cleaved caspase-9, cleaved poly(ADP-ribose) polymerase (PARP), phosphor-AMPK and phosphor-p27 at Thr198 were measured using immunoblot analyses. Caspase-3 activity was determined with spectrophotometer. To determine autophagy, LC3 cleavage and punctate patterns were examined. Results: Punicalagin (1-30 μg/mL) dose-dependently inhibited the cell viability in association with increased cyclin E level and decreased cyclin B and cyclin A levels. The treatment also induced apoptosis as shown by the cleavage of PARP, activation of caspase-9, and increase of caspase-3 activity in the cells. However, pretreatment of the cells with the pan-caspase inhibitor z-DEVD-fmk (50 μmol/L) did not completely prevent the cell death. On the other hand, punicalagin treatment increased LC3-II cleavage and caused GFP-LC3-II-stained punctate pattern in the cells. Suppressing autophagy of cells with chloroquine (1-10 μmol/L) dose-dependently alleviated the cell death caused by punicalagin. Punicalagin (1-30 μg/mL) also increased the levels phosphor-AMPK and phosphor-p27 at Thr198 in the cells, which were correlated with the induction of autophagic cell death. Conclusion: Punicalagin induces human U87MG glioma cell death through both apoptotic and autophagic pathways. PMID:24077634

  10. Autophagic Recycling Plays a Central Role in Maize Nitrogen Remobilization

    PubMed Central

    Chung, Taijoon; Pennington, Janice G.; Federico, Maria L.; Kaeppler, Heidi F.; Kaeppler, Shawn M.

    2015-01-01

    Autophagy is a primary route for nutrient recycling in plants by which superfluous or damaged cytoplasmic material and organelles are encapsulated and delivered to the vacuole for breakdown. Central to autophagy is a conjugation pathway that attaches AUTOPHAGY-RELATED8 (ATG8) to phosphatidylethanolamine, which then coats emerging autophagic membranes and helps with cargo recruitment, vesicle enclosure, and subsequent vesicle docking with the tonoplast. A key component in ATG8 function is ATG12, which promotes lipidation upon its attachment to ATG5. Here, we fully defined the maize (Zea mays) ATG system transcriptionally and characterized it genetically through atg12 mutants that block ATG8 modification. atg12 plants have compromised autophagic transport as determined by localization of a YFP-ATG8 reporter and its vacuolar cleavage during nitrogen or fixed-carbon starvation. Phenotypic analyses showed that atg12 plants are phenotypically normal and fertile when grown under nutrient-rich conditions. However, when nitrogen-starved, seedling growth is severely arrested, and as the plants mature, they show enhanced leaf senescence and stunted ear development. Nitrogen partitioning studies revealed that remobilization is impaired in atg12 plants, which significantly decreases seed yield and nitrogen-harvest index. Together, our studies demonstrate that autophagy, while nonessential, becomes critical during nitrogen stress and severely impacts maize productivity under suboptimal field conditions. PMID:25944100

  11. Listeria phospholipases subvert host autophagic defenses by stalling pre-autophagosomal structures

    PubMed Central

    Tattoli, Ivan; Sorbara, Matthew T; Yang, Chloe; Tooze, Sharon A; Philpott, Dana J; Girardin, Stephen E

    2013-01-01

    Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)-dependent cytosolic escape of Listeria triggered a transient amino-acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro-autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3-phosphate (PI3P) levels, causing pre-autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co-infection experiments, wild-type Listeria protected PlcA/B-deficient bacteria from autophagy-mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense. PMID:24162724

  12. Bioactive natural products against prostate cancer: mechanism of action and autophagic/apoptotic molecular pathways.

    PubMed

    Gioti, Katerina; Tenta, Roxane

    2015-05-01

    Prostate cancer is one of the leading causes of death worldwide for men. There is increasing evidence that diet and lifestyle play a crucial role in prostate cancer biology and tumorigenesis. Due to the fact that conventional chemotherapy is not adequately effective against prostate cancer and has severe side effects, numerous in vitro studies have been conducted in order to identify the potent cytotoxic or chemopreventive activity of naturally occurring compounds and their respective molecular mechanisms of action. In this context, many natural compounds isolated from plants have been found to inhibit cancer growth and to induce cell cycle arrest, suppress angiogenesis, and promote apoptotic or autophagic cell death. Therefore, in this article, the most promising bioactive natural products and their respective mechanisms of action for the prevention or/and treatment of prostate cancer are presented.

  13. The natural compounds piperovatine and piperlonguminine induce autophagic cell death on Trypanosoma cruzi.

    PubMed

    Veiga-Santos, Phercyles; Desoti, Vânia Cristina; Miranda, Nathielle; Ueda-Nakamura, Tânia; Dias-Filho, Benedito Prado; Silva, Sueli Oliveira; Cortez, Diogenes Aparício Garcia; de Mello, João Carlos Palazzo; Nakamura, Celso Vataru

    2013-03-01

    The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Our group has been attempting to find alternative drugs isolated from natural products as a potential source of pharmacological agents against Trypanosoma cruzi. Here, we demonstrate the antitrypanosomal activity of the amides piperovatine and piperlonguminine isolated from Piper ovatum against epimastigotes and intracellular amastigotes. We also investigated the mechanisms of action of these compounds on extracellular amastigote and epimastigote forms of T. cruzi. These amides showed low toxicity to LLCMK(2) mammalian cells. By using transmission and scanning electron microscopy, we observed that the compounds caused severe alterations in T. cruzi. These alterations were mainly located in plasma membrane and mitochondria. Furthermore, the study of treated parasites labeled with Rh123, PI and MDC corroborate with our TEM data. These mitochondrial dysfunctions induced by the amides might trigger biochemical alterations that lead to cell death. Altogether, our data evidence a possible autophagic process.

  14. Inhibition of autophagic flux by salinomycin results in anti-cancer effect in hepatocellular carcinoma cells.

    PubMed

    Klose, Johannes; Stankov, Metodi V; Kleine, Moritz; Ramackers, Wolf; Panayotova-Dimitrova, Diana; Jäger, Mark D; Klempnauer, Jürgen; Winkler, Michael; Bektas, Hüseyin; Behrens, Georg M N; Vondran, Florian W R

    2014-01-01

    Salinomycin raised hope to be effective in anti-cancer therapies due to its capability to overcome apoptosis-resistance in several types of cancer cells. Recently, its effectiveness against human hepatocellular carcinoma (HCC) cells both in vitro and in vivo was demonstrated. However, the mechanism of action remained unclear. Latest studies implicated interference with the degradation pathway of autophagy. This study aimed to determine the impact of Salinomycin on HCC-autophagy and whether primary human hepatocytes (PHH) likewise are affected. Following exposure of HCC cell lines HepG2 and Huh7 to varying concentrations of Salinomycin (0-10 µM), comprehensive analysis of autophagic activity using western-blotting and flow-cytometry was performed. Drug effects were analyzed in the settings of autophagy stimulation by starvation or PP242-treatment and correlated with cell viability, proliferation, apoptosis induction, mitochondrial mass accumulation and reactive oxygen species (ROS) formation. Impact on apoptosis induction and cell function of PHH was analyzed. Constitutive and stimulated autophagic activities both were effectively suppressed in HCC by Salinomycin. This inhibition was associated with dysfunctional mitochondria accumulation, increased apoptosis and decreased proliferation and cell viability. Effects of Salinomycin were dose and time dependent and could readily be replicated by pharmacological and genetic inhibition of HCC-autophagy alone. Salinomycin exposure to PHH resulted in transient impairment of synthesis function and cell viability without apoptosis induction. In conclusion, our data suggest that Salinomycin suppresses late stages of HCC-autophagy, leading to impaired recycling and accumulation of dysfunctional mitochondria with increased ROS-production all of which are associated with induction of apoptosis.

  15. The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death.

    PubMed

    Qiao, Shuxi; Tao, Shasha; Rojo de la Vega, Montserrat; Park, Sophia L; Vonderfecht, Amanda A; Jacobs, Suesan L; Zhang, Donna D; Wondrak, Georg T

    2013-12-01

    Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A, E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells.

  16. microRNA-20a Inhibits Autophagic Process by Targeting ATG7 and ATG16L1 and Favors Mycobacterial Survival in Macrophage Cells

    PubMed Central

    Guo, Le; Zhao, Jin; Qu, Yuliang; Yin, Runting; Gao, Qian; Ding, Shuqin; Zhang, Ying; Wei, Jun; Xu, Guangxian

    2016-01-01

    Autophagy plays important roles in the host immune response against mycobacterial infection. Mycobacterium tuberculosis (M. tuberculosis) can live in macrophages owing to its ability to evade attacks by regulating autophagic response. MicroRNAs (miRNAs) are small noncoding, endogenously encoded RNA which plays critical roles in precise regulation of macrophage functions. Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that BCG infection of macrophages resulted in enhanced expression of miRNA-20a, which inhibits autophagic process by targeting ATG7 and ATG16L1 and promotes BCG survival in macrophages. Forced overexpression of miR-20a decreased the expression levels of LC3-II and the number of LC3 puncta in macrophages, and promoted BCG survival in macrophages, while transfection with miR-20a inhibitor had the opposite effect. Moreover, the inhibitory effect of miR-20a on autophagy was further confirmed by transmission electron microscopy (TEM) analysis. Quantification of autophagosomes per cellular cross-section revealed a significant reduction upon transfection with miR-20a mimic, but transfection with miR-20a inhibitor increased the number of autophagosomes per cellular cross-section. Moreover, silencing of ATG7 significantly inhibited autophagic response, and transfection with ATG7 siRNA plus miR-20a mimic could further decrease autophagic response. Collectively, our data reveal that miR-20a inhibits autophagic response and promotes BCG survival in macrophages by targeting ATG7 and ATG16L1, which may have implications for a better understanding of pathogenesis of M. tuberculosis infection. PMID:27803889

  17. Sulindac sulfide induces autophagic death in gastric epithelial cells via survivin down-regulation: a mechanism of NSAIDs-induced gastric injury.

    PubMed

    Chiou, Shiun-Kwei; Hoa, Neil; Hodges, Amy

    2011-06-01

    Sulindac sulfide, a nonsteroidal anti-inflammatory drug (NSAID), has anti-tumorigenic and anti-inflammatory activities, but causes gastric mucosal damage. NSAIDs cause gastric injury in part by down-regulation of Survivin, an apoptosis inhibitor, resulting in apoptosis induction. Autophagy is a process that promotes cellular health by destroying unwanted cellular materials. Excessive autophagy induction could lead to a non-apoptotic cell death (autophagic cell death). The present study showed that sulindac sulfide at a physiological concentration also induces autophagic death in human gastric epithelial AGS and rat gastric epithelial RGM-1 cells, and that Survivin down-regulation is a mechanism involved: Sulindac sulfide treatment increased LC3b-II and APG7 levels and cytosolic vacuole formation, indications of autophagy induction, in AGS and RGM-1 cells. Sulindac sulfide treatment induced AGS and RGM-1 cell death, which was significantly reduced by pretreatment with the autophagy inhibitors 3-methyladenine and chloroquine, indicating that sulindac sulfide induced autophagic cell death. Stable overexpression of Survivin in RGM-1 cells did not inhibit the induction of LC3b-II levels or vacuole formation by sulindac sulfide, but significantly reduced the resulting cell death, suggesting that Survivin may inhibit autophagic cell death downstream of LC3b-II induction and vacuole formation. Indeed, siRNA depletion of LC3b in AGS cells inhibited the down-regulation of Survivin levels and the induction of cell death by sulindac sulfide, confirming that down-regulation of Survivin occurs in the autophagy pathway downstream of LC3b-II induction by sulindac sulfide. Induction of Survivin-dependent autophagic cell death is a novel mechanism by which sulindac sulfide induces gastric mucosal injury. Published by Elsevier Inc.

  18. Effect and proposed mechanism of vitamin C modulating amino acid regulation of autophagic proteolysis.

    PubMed

    Karim, Md Razaul; Kadowaki, Motoni

    2017-08-10

    Autophagy is an intracellular bulk degradation process, induced under nutrient starvation. Failure of autophagy has been recognized as a contributor to aging and multiple age related neurodegenerative diseases. Improving autophagy is a beneficial anti-aging strategy, however very few physiological regulators have been identified. Here, we demonstrate that vitamin C is a nutritional stimulator of autophagy. Supplementation of fresh hepatocytes with vitamin C increased autophagic proteolysis significantly in the presence of amino acids in a dose- and time-dependent manner, although no effect was observed in the absence of amino acids. In addition, inhibitor studies with 3-methyladenine, chloroquine, leupeptin and β-lactone confirmed that vitamin C is active through the lysosomal autophagy and not the proteasome pathway. Furthermore, the autophagy marker LC3 protein was significantly increased by vitamin C, suggesting its possible site of action is at the formation step. Both the reduced (ascorbic acid, AsA) and oxidized form (dehydroascorbic acid, DHA) of vitamin C exhibited equal enhancing effect, indicating that the effect does not depend on the anti-oxidation functionality of vitamin C. To understand the mechanism, we established that the effective dose (50 μM) was 15× lower than the intracellular content suggesting these would be only a minor influx from the extracellular pool. Moreover, transporter inhibitor studies in an AsA deficient ODS model rat revealed more accurately that the enhancing effect on autophagic proteolysis still existed, even though the intracellular influx of AsA was blocked. Taken together, these results provide evidence that vitamin C can potentially act through extracellular signaling. Copyright © 2017. Published by Elsevier B.V.

  19. Changes in Respiratory Mitochondrial Machinery and Cytochrome and Alternative Pathway Activities in Response to Energy Demand Underlie the Acclimation of Respiration to Elevated CO2 in the Invasive Opuntia ficus-indica1[OA

    PubMed Central

    Gomez-Casanovas, Nuria; Blanc-Betes, Elena; Gonzalez-Meler, Miquel A.; Azcon-Bieto, Joaquim

    2007-01-01

    Studies on long-term effects of plants grown at elevated CO2 are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO2, the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO2 concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO2 during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO2 also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO2, the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO2. Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO2, the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO2 suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO2. However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO2, total mitochondrial ATP production was decreased by plant growth at elevated CO2 when compared to ambient-grown plants. Because plant growth at elevated CO2 increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O2 consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO2 results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested. PMID:17660349

  20. Changes in respiratory mitochondrial machinery and cytochrome and alternative pathway activities in response to energy demand underlie the acclimation of respiration to elevated CO2 in the invasive Opuntia ficus-indica.

    PubMed

    Gomez-Casanovas, Nuria; Blanc-Betes, Elena; Gonzalez-Meler, Miquel A; Azcon-Bieto, Joaquim

    2007-09-01

    Studies on long-term effects of plants grown at elevated CO(2) are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO(2), the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO(2) concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO(2) during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO(2) also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO(2), the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO(2). Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO(2), the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO(2) suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO(2). However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO(2), total mitochondrial ATP production was decreased by plant growth at elevated CO(2) when compared to ambient-grown plants. Because plant growth at elevated CO(2) increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O(2) consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO(2) results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested.

  1. Cellular metabolic and autophagic pathways: traffic control by redox signaling.

    PubMed

    Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

    2013-10-01

    It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy-lysosomal pathway functions to maintain mitochondrial quality and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Cellular Metabolic and Autophagic Pathways: Traffic Control by Redox Signaling

    PubMed Central

    Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

    2013-01-01

    It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy-lysosomal pathway functions to maintain mitochondrial quality, and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function. PMID:23702245

  3. The novel pterostilbene derivative ANK-199 induces autophagic cell death through regulating PI3 kinase class III/beclin 1/Atg‑related proteins in cisplatin‑resistant CAR human oral cancer cells.

    PubMed

    Hsieh, Min-Tsang; Chen, Hao-Ping; Lu, Chi-Cheng; Chiang, Jo-Hua; Wu, Tian-Shung; Kuo, Daih-Huang; Huang, Li-Jiau; Kuo, Sheng-Chu; Yang, Jai-Sing

    2014-08-01

    Pterostilbene is an effective chemopreventive agent against multiple types of cancer cells. A novel pterostilbene derivative, ANK-199, was designed and synthesized by our group. Its antitumor activity and mechanism in cisplatin-resistant CAR human oral cancer cells were investigated in this study. Our results show that ANK-199 has an extremely low toxicity in normal oral cell lines. The formation of autophagic vacuoles and acidic vesicular organelles (AVOs) was observed in the ANK-199-treated CAR cells by monodansylcadaverine (MDC) and acridine orange (AO) staining, suggesting that ANK-199 is able to induce autophagic cell death in CAR cells. Neither DNA fragmentation nor DNA condensation was observed, which means that ANK-199-induced cell death is not triggered by apoptosis. In accordance with morphological observation, 3-MA, a specific inhibitor of PI3K kinase class III, can inhibit the autophagic vesicle formation induced by ANK-199. In addition, ANK-199 is also able to enhance the protein levels of autophagic proteins, Atg complex, beclin 1, PI3K class III and LC3-II, and mRNA expression of autophagic genes Atg7, Atg12, beclin 1 and LC3-II in the ANK-199-treated CAR cells. A molecular signaling pathway induced by ANK-199 was therefore summarized. Results presented in this study show that ANK-199 may become a novel therapeutic reagent for the treatment of oral cancer in the near future (patent pending).

  4. Membrane manipulations by the ESCRT machinery.

    PubMed

    Odorizzi, Greg

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRTs) collectively comprise a machinery that was first known for its function in the degradation of transmembrane proteins in the endocytic pathway of eukaryotic cells. Since their discovery, however, ESCRTs have been recognized as playing important roles at the plasma membrane, which appears to be the original site of function for the ESCRT machinery. This article reviews some of the major research findings that have shaped our current understanding of how the ESCRT machinery controls membrane dynamics and considers new roles for the ESCRT machinery that might be driven by these mechanisms.

  5. 46 CFR 176.804 - Machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... control mechanisms including primary and alternate means of starting machinery; (c) Inspection of all... remote operating positions; (g) Operational test of all overboard discharge and intake valves and...

  6. Cathepsin S-mediated autophagic flux in tumor-associated macrophages accelerate tumor development by promoting M2 polarization

    PubMed Central

    2014-01-01

    Background Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues, but relatively little is known about the key molecular in these cells that contribute to malignant phenotypes. Autophagic activity is a critical factor in tumor development that contributes to enhancing cellular fitness and survival in the hostile tumor microenvironment. However, the molecular basis and relations between autophagy and TAMs polarization remain unclear. Methods Cathepsin S (Cat S) expression was analyzed in human colon carcinoma and normal colon tissues. In vivo effects were evaluated using PancO2 subcutaneous tumor model and SL4 hepatic metastasis model. Immunofluorescence staining, flow cytometry and real-time PCR were done to examine TAMs polarization. Western blotting assay, transmission electron microscopy, mCherry-GFP-LC3 transfection and DQ-BSA degradation assays were carried out to determine its role in regulating autophagy. Results In the present study, we showed that the enhanced expression of Cat S correlated with the severity of histologic grade as well as clinical stage, metastasis, and recurrence, which are known indicators of a relatively poor prognosis of human colon carcinoma. Cat S knockout led to decreased tumor growth and metastasis. Moreover, Cat S knockout inhibited M2 macrophage polarization during tumor development. We further demonstrated that Cat S was required for not only autophagic flux but also the fusion processes of autophagosomes and lysosomes in TAMs. Importantly, we found that Cat S contributed to tumor development by regulating the M2 phenotype of TAMs through the activation of autophagy. Conclusions These results indicated that Cat S-mediated autophagic flux is an important mechanism for inducing M2-type polarization of TAMs, which leads to tumor development. These data provide strong evidence for a

  7. Carbazole alkaloids from Murraya koenigii trigger apoptosis and autophagic flux inhibition in human oral squamous cell carcinoma cells.

    PubMed

    Utaipan, Tanyarath; Athipornchai, Anan; Suksamrarn, Apichart; Jirachotikoon, Canussanun; Yuan, Xiaohong; Lertcanawanichakul, Monthon; Chunglok, Warangkana

    2017-01-01

    Carbazole alkaloids, a major constituent of Murraya koenigii (L.) Sprengel (Rutaceae), exhibit biological effects such as anticancer activity via the induction of apoptosis, and they represent candidate chemotherapeutic agents. Oral squamous cell carcinoma (OSCC) is the most prevalent cancer of the oral cavity and a growing and serious health problem worldwide. In this study, we investigated the anticancer properties and mechanisms of action of two carbazole alkaloids derived from M. koenigii leaves, mahanine and isomahanine, in the OSCC cell line CLS-354. At 15 μM, mahanine and isomahanine were cytotoxic to CLS-354 cells, triggering apoptosis via caspase-dependent and -independent mechanisms. Autophagosomes, visualised using monodansylcadaverine (MDC) labelling, were numerous in carbazole alkaloid-treated cells. Mahanine and isomahanine markedly induced the expression of the autophagosome marker microtubule-associated protein 1 light chain 3, type II (LC3B-II). Genetic and chemical inhibition of autophagy via silencing of the Autophagy protein 5 gene and exposure to bafilomycin A1 (BafA1), respectively, did not arrest carbazole alkaloid-induced apoptosis, indicating that it occurs independently of autophagic activation. Surprisingly, both carbazole alkaloids caused increased accumulation of p62/sequestosome1 (p62/SQSTM1), with coordinated expression of LC3B-II and cleaved caspase-3, suggesting inhibition of autophagic flux. Our results suggest that inhibition of autophagic flux is associated with carbazole alkaloid-induced apoptosis. Our findings provide evidence of a novel cytotoxic action of natural carbazole alkaloids and support their use as candidate chemotherapeutic agents for the treatment of OSCC.

  8. Modeling of the MAPK machinery activation in response to various abiotic and biotic stresses in plants by a system biology approach.

    PubMed

    Pathak, Rajesh Kumar; Taj, Gohar; Pandey, Dinesh; Arora, Sandeep; Kumar, Anil

    2013-01-01

    Mitogen-Activated Protein Kinases (MAPKs) cascade plays an important role in regulating plant growth and development, generating cellular responses to the extracellular stimuli. MAPKs cascade mainly consist of three sub-families i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK) and mitogen activated protein kinase (MAPK), several cascades of which are activated by various abiotic and biotic stresses. In this work we have modeled the holistic molecular mechanisms essential to MAPKs activation in response to several abiotic and biotic stresses through a system biology approach and performed its simulation studies. As extent of abiotic and biotic stresses goes on increasing, the process of cell division, cell growth and cell differentiation slow down in time dependent manner. The models developed depict the combinatorial and multicomponent signaling triggered in response to several abiotic and biotic factors. These models can be used to predict behavior of cells in event of various stresses depending on their time and exposure through activation of complex signaling cascades.

  9. 5. FIRST FLOOR INTERIOR, NITROGEN MACHINERY, MACHINERY ROOM (SEE N4) ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. FIRST FLOOR INTERIOR, NITROGEN MACHINERY, MACHINERY ROOM (SEE N-4) FROM EASTERN ENTRANCE, LOOKING EAST. - Oakland Naval Supply Center, Cold Storage Warehouse, South of C Street between First & Second Street, Oakland, Alameda County, CA

  10. On the mechanochemical machinery underlying chromatin remodeling

    NASA Astrophysics Data System (ADS)

    Yusufaly, Tahir I.

    This dissertation discuss two recent efforts, via a unique combination of structural bioinformatics and density functional theory, to unravel some of the details concerning how molecular machinery within the eukaryotic cell nucleus controls chromatin architecture. The first, a study of the 5-methylation of cytosine in 5'-CG-3' : 5'-CG-3' base-pair steps, reveals that the methyl groups roughen the local elastic energy landscape of the DNA. This enhances the probability of the canonical B-DNA structure transitioning into the undertwisted A-like and overtwisted C-like forms seen in nucleosomes, or looped segments of DNA bound to histones. The second part focuses on the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. The arginine residues are ob- served to apply a tunable mechanical load to the backbone, enabling precision-controlled activation of DNA deformations.

  11. The HSP90 chaperone machinery.

    PubMed

    Schopf, Florian H; Biebl, Maximilian M; Buchner, Johannes

    2017-06-01

    The heat shock protein 90 (HSP90) chaperone machinery is a key regulator of proteostasis under both physiological and stress conditions in eukaryotic cells. As HSP90 has several hundred protein substrates (or 'clients'), it is involved in many cellular processes beyond protein folding, which include DNA repair, development, the immune response and neurodegenerative disease. A large number of co-chaperones interact with HSP90 and regulate the ATPase-associated conformational changes of the HSP90 dimer that occur during the processing of clients. Recent progress has allowed the interactions of clients with HSP90 and its co-chaperones to be defined. Owing to the importance of HSP90 in the regulation of many cellular proteins, it has become a promising drug target for the treatment of several diseases, which include cancer and diseases associated with protein misfolding.

  12. Role of hypoxia‑inducible factor‑1α in autophagic cell death in microglial cells induced by hypoxia.

    PubMed

    Wang, Xintao; Ma, Jun; Fu, Qiang; Zhu, Lei; Zhang, Zhiling; Zhang, Fan; Lu, Nan; Chen, Aimin

    2017-03-01

    Microglial cells are phagocytic cells of the central nervous system (CNS) and have been proposed to be a primary component of the innate immune response and maintain efficient CNS homeostasis. Microglial cells are activated during various phases of tissue repair and participate in various pathological conditions in the CNS. Following spinal cord injury (SCI), anoxemia is a key problem that results in tissue destruction. Hypoxia‑inducible factor 1‑α (HIF‑1α) may protect hypoxic cells from apoptosis or necrosis under ischemic and anoxic conditions. However, numerous studies have revealed that hypoxia upregulates HIF‑1α expression leading to the death of microglial cells. The present study investigated the alterations in HIF‑1α expression levels and the mechanism of autophagic cell death mediated by HIF‑1α in microglial cells induced by hypoxia. Hypoxia was demonstrated to induce HIF‑1α expression and autophagic cell death in microglial cells. Enhanced autophagy reduced cell death during the initial stages by restraining the functions of autophagy‑associated genes (microtubule‑associated protein 1A/1B‑light chain 3 phosphatidylethanolamine conjugate and Beclin‑1) and modulating the expression of inflammatory cytokines (tumor necrosis factor‑α and interleukin‑1β). Target value was determined by Cell Counting Kit 8 and cell death by flow cytometry. Transmission electron microscopy, immunohistochemical staining, reverse transcription‑quantitative polymerase chain reaction, western blotting, and ELISA were used for further analysis. However, increased expression of HIF‑1α induced cell death and autophagic cell death in microglial cells. Furthermore, the effects of the HIF‑1α inhibitor 2‑methoxyestradiol and HIF‑1α small interfering RNA on the death and autophagy of microglial cells in vitro were investigated. These investigations revealed the suppression of autophagy, the decrease of cell viability and the increase of

  13. Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma

    PubMed Central

    Zhang, L; Zhang, W; Wang, Y-F; Liu, B; Zhang, W-F; Zhao, Y-F; Kulkarni, A B; Sun, Z-J

    2015-01-01

    Survivin is ubiquitously expressed in patients with head neck squamous cell carcinoma (HNSCC) and is associated with poor survival and chemotherapy resistance. Sepantronium bromide (YM155) is a selective survivin suppressant that exhibits potent antitumor activities by inducing apoptosis and autophagy in various types of cancer. However, the curative effects and underlying mechanisms of YM155 in HNSCC remain unclear. This study showed that survivin overexpression positively correlated with p-S6, p-Rb and LAMP2 but negatively correlated with the autophagic marker LC3 in human HNSCC tissues. In vitro studies revealed that YM155 triggered apoptosis of HNSCC cells in mitochondria and death receptor-dependent manner. The treatment also significantly enhanced autophagy by upregulating Beclin1, which led to cell death. YM155 not only downregulated the expression of survivin but also remarkably suppressed the activation of the mTOR signaling pathway in vitro and in vivo. YM155 displayed potent antitumor activities in both CAL27 xenograft and transgenic HNSCC mice models by delaying tumor onset and suppressing tumor growth. Furthermore, YM155 combined with docetaxel promoted tumor regression better than either treatment alone without causing considerable body weight loss in the HNSCC xenograft models. Overall, targeting survivin by YM155 can benefit HNSCC therapy by increasing apoptotic and autophagic cell death, and suppressing prosurvival pathways. PMID:26018732

  14. 46 CFR 115.804 - Machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...) Operational test of the means provided for pumping bilges; and (i) Test of machinery alarms including bilge... managing operator shall be prepared to conduct tests and have the vessel ready for inspections of machinery... ahead and astern; (b) Operational test and inspection of engine control mechanisms including primary...

  15. 46 CFR 115.804 - Machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) Operational test of the means provided for pumping bilges; and (i) Test of machinery alarms including bilge... managing operator shall be prepared to conduct tests and have the vessel ready for inspections of machinery... ahead and astern; (b) Operational test and inspection of engine control mechanisms including primary...

  16. 46 CFR 115.804 - Machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...) Operational test of the means provided for pumping bilges; and (i) Test of machinery alarms including bilge... managing operator shall be prepared to conduct tests and have the vessel ready for inspections of machinery... ahead and astern; (b) Operational test and inspection of engine control mechanisms including primary...

  17. 46 CFR 115.804 - Machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...) Operational test of the means provided for pumping bilges; and (i) Test of machinery alarms including bilge... managing operator shall be prepared to conduct tests and have the vessel ready for inspections of machinery... ahead and astern; (b) Operational test and inspection of engine control mechanisms including primary...

  18. 46 CFR 115.804 - Machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) Operational test of the means provided for pumping bilges; and (i) Test of machinery alarms including bilge... managing operator shall be prepared to conduct tests and have the vessel ready for inspections of machinery... ahead and astern; (b) Operational test and inspection of engine control mechanisms including primary...

  19. Testosterone regulates the autophagic clearance of androgen binding protein in rat Sertoli cells

    PubMed Central

    Ma, Yi; Yang, Hao-Zheng; Xu, Long-Mei; Huang, Yi-Ran; Dai, Hui-Li; Kang, Xiao-Nan

    2015-01-01

    Dysregulation of androgen-binding protein (ABP) is associated with a number of endocrine and andrology diseases. However, the ABP metabolism in Sertoli cells is largely unknown. We report that autophagy degrades ABP in rat Sertoli cells, and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. Further studies identified that the autophagic clearance of ABP might be selectively regulated by testosterone, independent of stress (hypoxia)-induced autophagic degradation. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. We report a novel finding with respect to the mechanisms by which ABP is cleared, and by which the process is regulated in Sertoli cells. PMID:25745956

  20. Synergistic induction of apoptosis and caspase-independent autophagic cell death by a combination of nitroxide Tempo and heat shock in human leukemia U937 cells.

    PubMed

    Zhao, Qing-Li; Fujiwara, Yoshisada; Kondo, Takashi

    2010-10-01

    We have shown that heat stress or a superoxide dismutase mimic nitroxide, Tempo, induces apoptosis, while their combination causes nonapoptotic cell death; however, the underlying mechanism for this switch remains unclear. Here we identified for the first time that 10 mM Tempo present during heating at 44°C for 30 min rapidly induced autophagy in U937 leukemic cells in spite of Bax activation and mitochondrial outer membrane (MOM) permeabilization. This co-treatment inhibited the processing of heat-activated procaspases-2, -8, -9 and -3 into active small subunits, leading to the inhibition of caspase-dependent apoptosis, and instead caused the induction of autophagy. The inactivation of caspases, a key event, could result from oxidation of active-site-CysSH of all caspases by a prooxidant oxo-ammonium cation, an intermediate derived Tempo during dismutation of heat-induced superoxide anion. In addition, the co-treatment caused mitochondrial calcium overloads, the mitochondrial inner membrane permeabilization, profound mitochondrial dysfunction, and liberation of Beclin 1 from the Bcl-2/Beclin 1 complex, all of which contributed to induction of autophagy. These autophagic cells underwent propidium iodide-positive necrosis in a delayed fashion, leading to the complete proliferative inhibition. Remarkably, ruthenium red and BAPTA, which interfere with mitochondrial calcium uptake, facilitated autophagic necrotic death. Cyclosporin A, which binds to cyclophilin D, had a similar necrotic effect. 3-Methyladenine facilitated the necrosis of autophagic cells. In contrast, 5 mM Tempo-44°C/10 min or 44°C/30 min induced Bax-mediated MOM permeabilization and caspase-dependent apoptosis more potently than Tempo alone. Thus, Tempo is a unique thermosensitizer to synergistically induce apoptosis and autophagic cell death.

  1. Morin hydrate augments phagocytosis mechanism and inhibits LPS induced autophagic signaling in murine macrophage.

    PubMed

    Jakhar, Rekha; Paul, Souren; Chauhan, Anil Kumar; Kang, Sun Chul

    2014-10-01

    Morin, a natural flavonoid that is the primary bioactive constituent of the family Moraceae, has been found to be associated with many therapeutic properties. In this study, we evaluated the immunomodulatory activities of increasing concentration of morin hydrate in vitro. Three different concentrations of morin hydrate (5, 10, and 15μM) were used to evaluate their effect on splenocyte proliferation, phagocytic activity of macrophages, cytokine secretion and complement inhibition. We also evaluated the role of morin hydrate on lipopolysaccharide (LPS) induced autophagy. Our study demonstrated that morin hydrate elicited a significant increase in splenocyte proliferation, phagocytic capacity and suppressed the production of cytokines and nitric oxide in activated macrophages. Humoral immunity measured by anti-complement activity showed an increase in inhibition of the complement system after the addition of morin hydrate, where morin hydrate at 15μM concentration induced a significant inhibition. Depending on our results, we can also conclude that morin hydrate protects macrophages from LPS induced autophagic cell death. Our findings suggest that morin hydrate represents a structurally diverse class of flavonoid and this structural variability can profoundly affect its cell-type specificity and its biological activities. Supplementation of immune cells with morin hydrate has an upregulating and immunoprotective effect that shows potential as a countermeasure to the immune dysfunction and suggests an interesting use in inflammation related diseases.

  2. 46 CFR 58.01-45 - Machinery space, ventilation.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Machinery space, ventilation. 58.01-45 Section 58.01-45... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-45 Machinery space, ventilation. Each machinery space must be ventilated to ensure that, when machinery or boilers are operating at full power in all...

  3. 46 CFR 58.01-45 - Machinery space, ventilation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Machinery space, ventilation. 58.01-45 Section 58.01-45... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-45 Machinery space, ventilation. Each machinery space must be ventilated to ensure that, when machinery or boilers are operating at full power in all...

  4. 46 CFR 58.01-45 - Machinery space, ventilation.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Machinery space, ventilation. 58.01-45 Section 58.01-45... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-45 Machinery space, ventilation. Each machinery space must be ventilated to ensure that, when machinery or boilers are operating at full power in all...

  5. 46 CFR 58.01-45 - Machinery space, ventilation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Machinery space, ventilation. 58.01-45 Section 58.01-45... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-45 Machinery space, ventilation. Each machinery space must be ventilated to ensure that, when machinery or boilers are operating at full power in...

  6. 46 CFR 58.01-45 - Machinery space, ventilation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery space, ventilation. 58.01-45 Section 58.01-45... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-45 Machinery space, ventilation. Each machinery space must be ventilated to ensure that, when machinery or boilers are operating at full power in...

  7. Reduced cathepsins B and D cause impaired autophagic degradation that can be almost completely restored by overexpression of these two proteases in Sap C-deficient fibroblasts.

    PubMed

    Tatti, Massimo; Motta, Marialetizia; Di Bartolomeo, Sabrina; Scarpa, Susanna; Cianfanelli, Valentina; Cecconi, Francesco; Salvioli, Rosa

    2012-12-01

    Saposin (Sap) C deficiency, a rare variant form of Gaucher disease, is due to mutations in the Sap C coding region of the prosaposin (PSAP) gene. Sap C is required as an activator of the lysosomal enzyme glucosylceramidase (GCase), which catalyzes glucosylceramide (GC) degradation. Deficit of either GCase or Sap C leads to the accumulation of undegraded GC and other lipids in lysosomes of monocyte/macrophage lineage. Recently, we reported that Sap C mutations affecting a cysteine residue result in increased autophagy. Here, we characterized the basis for the autophagic dysfunction. We analyzed Sap C-deficient and GCase-deficient fibroblasts and observed that autophagic disturbance was only associated with lack of Sap C. By a combined fluorescence microscopy and biochemical studies, we demonstrated that the accumulation of autophagosomes in Sap C-deficient fibroblasts is not due to enhanced autophagosome formation but to delayed degradation of autolysosomes caused, in part, to decreased amount and reduced enzymatic activity of cathepsins B and D. On the contrary, in GCase-deficient fibroblasts, the protein level and enzymatic activity of cathepsin D were comparable with control fibroblasts, whereas those of cathepsin B were almost doubled. Moreover, the enhanced expression of both these lysosomal proteases in Sap C-deficient fibroblasts resulted in close to functional autophagic degradation. Our data provide a novel example of altered autophagy as secondary event resulting from insufficient lysosomal function.

  8. TRPV1 participates in the activation of clock molecular machinery in the brown adipose tissue in response to light-dark cycle.

    PubMed

    Moraes, Maria Nathalia; Mezzalira, Nathana; de Assis, Leonardo Vinicius Monteiro; Menaker, Michael; Guler, Ali; Castrucci, Ana Maria L

    2017-02-01

    Transient receptor potential (TRPs) channels are involved in thermogenesis, and temperature and energy balance control. Mice lacking TrpV1 become more obese and develop insulin resistance when fed with high fat diet; however, a relationship between metabolic disorders, TRP channels, and clock genes is still unknown. Based on this, we hypothesized that TRPV1 channels would be involved in the synchronization of clock genes in the peripheral tissues. To address this question, we used wild type (WT) and TrpV1 knockout (KO) mice kept in constant darkness (DD) or in light-dark cycle (LD). In WT mouse brown adipose tissue (BAT), TrpV1 oscillated with higher expression at scotophase, Per1 and Per2 showed the same profile, and Bmal1 transcript only oscillated in DD. Interestingly, the oscillatory profile of these clock genes was abolished in TrpV1 KO mice. WT mouse Ucp1 was upregulated in LD as compared to DD, showing no temporal variation; mice lacking TrpV1 showed Ucp1 oscillation with a peak at the photophase. Remarkably, TrpV1 KO mice displayed less total activity than WT only when submitted to LD. We provide evidence that TRPV1 is an important modulator of BAT clock gene oscillations. Therefore, temperature and/or light-dependent regulation of TRPV1 activity might provide novel pharmacological approaches to treat metabolic disorders.

  9. The vector-related influences of autophagic microRNA delivery by Lipofectamine 2000 and polyethylenimine 25K on mouse embryonic fibroblast cells.

    PubMed

    Lin, Chia-Wei; Jan, Ming-Shiou; Kuo, Jung-Hua Steven

    2017-04-01

    Despite the greater potential for clinical applications of autophagic microRNA (miRNA) delivery, the vector-related effects of such delivery on cells have not been fully explored. In this study, autophagic mmu-miR-494-3p (miR-494) in mouse embryonic fibroblast (MEF) cells was selected as a cargo miRNA, and two commonly used non-viral carriers (Lipofectamine 2000 (Lipo) and polyethylenimine 25K (PEI)), were used as delivery vectors to mechanistically elucidate its vector-related effects. The cellular uptake, nuclear localization, and quantitative miR-494 levels of the complexes of miR-494 with Lipo (miR-494 lipoplexes) were lower than those of the complexes of miR-494 with PEI (miR-494 polyplexes) in MEF cells. The indicator of autophagic activity (LC3 (microtubule-associated protein 1 light chain 3)-II/LC3-I ratio) in cells treated with miR-494 lipoplexes was higher than that in cells treated with miR-494 polyplexes. Lipo alone and PEI alone induced slight increases in the quantitative levels of miR-494 in cells, but Lipo resulted in higher gene and protein expressions of target Igf1, higher LC3-II/LC3-I ratios, and higher autophagosome formation than PEI. We also demonstrated that the delivery of miR-494 by Lipo was more involved in apoptotic caspase-3 pathways than such delivery by PEI. By applying knock-out atg5 gene in MEF cells, we found that autophagy played a protective role in cell survival and also affected cellular uptake, the quantitative level of miR-494, and target gene Igf1 regulation of delivery systems. Taken together, these results indicate that there are different degrees of responses in MEF cells for autophagic miR-494 delivery through the use of Lipo or PEI vectors that also induce autophagy in cells. Therefore, Lipo and PEI vectors cannot be treated as inert molecules, and their effects must be known and evaluated when they are used in autophagic miRNA delivery systems. Most importantly, understanding these vector-related effects on cells will

  10. Silencing Livin induces apoptotic and autophagic cell death, increasing chemotherapeutic sensitivity to cisplatin of renal carcinoma cells.

    PubMed

    Wang, Zhiyang; Liu, Shuai; Ding, Kejia; Ding, Sentai; Li, Chensheng; Lu, Jiaju; Gao, Dexuan; Zhang, Tong; Bi, Dongbin

    2016-11-01

    Renal cell carcinoma (RCC) accounts for 3 % of all adult malignancies and is the most lethal urological cancer. Livin is a member of the inhibitor of apoptosis protein (IAP) family, which is associated with tumor resistance to radiotherapy and chemotherapy. Clinical data also showed that patients with high tumor grades and stages have higher expression levels of Livin in RCC cells. Autophagy is a survival mechanism activated in response to nutrient deprivation. A possible role of Livin in the autophagy of RCC cells has not been investigated; therefore, this pioneer study was carried out. Livin was silenced in RCC cells (slow virus infection [SVI]-shLivin cells) by lentiviral transfection. Then, mRNA and protein expression levels in the transfected cells were assessed by quantitative fluorescence PCR and Western blotting, respectively. In addition, acridine orange staining and electron microscopy were used to assess autophagy in SVI-shLivin cells. The cisplatin IC50 values for RCC cells were measured by the CCK8 assay. Potent antitumor activities were observed in xenograft mouse models generated with Livin-silenced RCC cells in terms of delayed tumor onset and suppressed tumor growth. These results suggested that Livin silencing could increase the chemotherapeutic sensitivity of RCC cells to cisplatin and induce autophagic cell death. A possible mechanism of Bcl-2 and Akt pathway involvement was discussed specifically in this study. Overall, Livin silencing induces apoptotic and autophagic cell death and increases chemotherapeutic sensitivity of RCC cells to cisplatin.

  11. iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic defects and impaired calcium homeostasis.

    PubMed

    Schöndorf, David C; Aureli, Massimo; McAllister, Fiona E; Hindley, Christopher J; Mayer, Florian; Schmid, Benjamin; Sardi, S Pablo; Valsecchi, Manuela; Hoffmann, Susanna; Schwarz, Lukas Kristoffer; Hedrich, Ulrike; Berg, Daniela; Shihabuddin, Lamya S; Hu, Jing; Pruszak, Jan; Gygi, Steven P; Sonnino, Sandro; Gasser, Thomas; Deleidi, Michela

    2014-06-06

    Mutations in the acid β-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gaucher's disease (GD), are the strongest genetic risk factor for Parkinson's disease (PD) known to date. Here we generate induced pluripotent stem cells from subjects with GD and PD harbouring GBA1 mutations, and differentiate them into midbrain dopaminergic neurons followed by enrichment using fluorescence-activated cell sorting. Neurons show a reduction in glucocerebrosidase activity and protein levels, increase in glucosylceramide and α-synuclein levels as well as autophagic and lysosomal defects. Quantitative proteomic profiling reveals an increase of the neuronal calcium-binding protein 2 (NECAB2) in diseased neurons. Mutant neurons show a dysregulation of calcium homeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium. Importantly, correction of the mutations rescues such pathological phenotypes. These findings provide evidence for a link between GBA1 mutations and complex changes in the autophagic/lysosomal system and intracellular calcium homeostasis, which underlie vulnerability to neurodegeneration.

  12. Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

    PubMed Central

    Nemazanyy, Ivan; Blaauw, Bert; Paolini, Cecilia; Caillaud, Catherine; Protasi, Feliciano; Mueller, Amelie; Proikas-Cezanne, Tassula; Russell, Ryan C; Guan, Kun-Liang; Nishino, Ichizo; Sandri, Marco; Pende, Mario; Panasyuk, Ganna

    2013-01-01

    The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3-kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15-deficient mouse tissues are competent for LC3-positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7−/− mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15−/− mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases. PMID:23630012

  13. HDLs inhibit endoplasmic reticulum stress and autophagic response induced by oxidized LDLs

    PubMed Central

    Muller, C; Salvayre, R; Nègre-Salvayre, A; Vindis, C

    2011-01-01

    The apoptotic effect of oxidized LDLs (oxLDLs) is mediated through a complex sequence of signaling events involving a deregulation of the cytosolic Ca2+ homeostasis. OxLDLs also trigger ER stress that may lead to cellular dysfunction and apoptosis, through the activation of the IRE1α/c-Jun N-terminal kinase pathway. Moreover, ER stress and oxidized lipids have been shown to trigger autophagy. The antiatherogenic high-density lipoproteins (HDLs) display protective effects against oxLDLs toxicity. To more deeply investigate the mechanisms mediating the protective effects of HDLs, we examined whether ER stress and autophagy were implicated in oxLDLs-induced apoptosis and whether HDLs prevented these stress processes. We report that, in human endothelial cells, HDLs prevent the oxLDL-induced activation of the ER stress sensors IRE1α, eIF2α and ATF6 and subsequent activation of the proapoptotic mediators JNK and CHOP. OxLDLs also trigger the activation of autophagy, as assessed by LC3 processing and Beclin-1 expression. The autophagic process is independent of the proapoptotic arms of ER stress, but Beclin-1 contributes to PS exposure and subsequent phagocytosis of oxLDLs exposed cells. Induction of autophagy and PS exposure by oxLDLs is prevented by HDLs. Finally, the cytosolic Ca2+ deregulation triggered by oxLDLs is a common signaling pathway that mediates ER stress-induced cell death and autophagy, all these events being blocked by HDLs. PMID:21113143

  14. Eclalbasaponin II induces autophagic and apoptotic cell death in human ovarian cancer cells.

    PubMed

    Cho, Yoon Jin; Woo, Jeong-Hwa; Lee, Jae-Seung; Jang, Dae Sik; Lee, Kyung-Tae; Choi, Jung-Hye

    2016-09-01

    Triterpenoids echinocystic acid and its glycosides, isolated from several Eclipta prostrata, have been reported to possess various biological activities such as anti-inflammatory, anti-bacterial, and anti-diabetic activity. However, the cytotoxicity of the triterpenoids in human cancer cells and their molecular mechanism of action are poorly understood. In the present study, we found that eclalbasaponin II with one glucose moiety has potent cytotoxicity in three ovarian cancer cells and two endometrial cancer cells compared to an aglycone echinocystic acid and eclalbasaponin I with two glucose moiety. Eclalbasaponin II treatment dose-dependently increased sub G1 population. Annexin V staining revealed that eclalbasaponin II induced apoptosis in SKOV3 and A2780 ovarian cancer cells. In addition, eclalbasaponin II-induced cell death was associated with characteristics of autophagy; an increase in acidic vesicular organelle content and elevation of the levels of LC3-II. Interestingly, autophagy inhibitor BaF1 suppressed the eclalbasaponin II-induced apoptosis. Moreover, eclalbasaponin II activated JNK and p38 signaling and inhibited the mTOR signaling. We further demonstrated that pre-treatment with a JNK and p38 inhibitor and mTOR activator attenuated the eclalbasaponin II-induced autophagy. This suggests that eclalbasaponin II induces apoptotic and autophagic cell death through the regulation of JNK, p38, and mTOR signaling in human ovarian cancer cells. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  15. p-Cresol mediates autophagic cell death in renal proximal tubular cells.

    PubMed

    Lin, Hsin-Hung; Huang, Chiu-Ching; Lin, Tze-Yi; Lin, Ching-Yuang

    2015-04-02

    Higher serum level of p-cresol (PC) in chronic kidney disease (CKD) patients has been linked with CKD progression. The toxic effect of PC on diverse cells has been reported by prior studies, except for renal tubular cells. Both autophagy and apoptosis contribute to renal tubular cell death, yet evidence of its response to PC is limited and their crosstalk is still unclear. Autophagy is an important cellular process involved in toxin-induced cell death. Renal tubular cell death in tubular injury is thought to be one of the key events causing the progression of CKD. Thus, we treated rat (NRK-52E) and human (HRPTEC) renal proximal tubular cells (RPTC) with PC and found the cell proliferation was significantly decreased. Cell apoptosis was significantly increased and accompanied with the activation of autophagy as evidenced by increases in LC3-II, beclin 1 and Atg 4. We also found an increase of p62 by c-Jun activation. p62 accumulation could mediate the activation of caspase 8-dependent cell apoptosis. Conversely, knockdown of p62 by siRNA of p62 had the opposite effect by arresting LC3-II accumulation and promoting increasing cell viability. We conclude that PC triggered autophagic RPTC death via JNK-mediated p62 accumulation and then activated caspase 8-dependent cell death pathway. PC can be considered as one of the key events causing progression of CKD, which might affect drug disposition in CKD cases.

  16. Zanthoxylum fruit extract from Japanese pepper promotes autophagic cell death in cancer cells

    PubMed Central

    Nozaki, Reo; Kono, Toru; Bochimoto, Hiroki; Watanabe, Tsuyoshi; Oketani, Kaori; Sakamaki, Yuichi; Okubo, Naoto; Nakagawa, Koji; Takeda, Hiroshi

    2016-01-01

    Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) have multiple physiological activities (e.g., antiviral activity). However, the potential anticancer activity of ZFE has not been fully examined. In this study, we investigated the ability of ZFE to induce autophagic cell death (ACD). ZFE caused remarkable autophagy-like cytoplasmic vacuolization, inhibited cell proliferation, and ultimately induced cell death in the human cancer cell lines DLD-1, HepG2, and Caco-2, but not in A549, MCF-7, or WiDr cells. ZFE increased the level of LC3-II protein, a marker of autophagy. Knockdown of ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, in cancer cells that could be induced to undergo cell death by ZFE, the extract increased the phosphorylation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 attenuated both vacuolization and cell death. Based on morphology and expression of marker proteins, ZFE-induced cell death was neither apoptosis nor necrosis. Normal intestinal cells were not affected by ZFE. Taken together, our findings show that ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development. PMID:27626481

  17. Inhibiting autophagy potentiates the anticancer activity of IFN1@/IFNα in chronic myeloid leukemia cells.

    PubMed

    Zhu, Shan; Cao, Lizhi; Yu, Yan; Yang, Liangchun; Yang, Minghua; Liu, Ke; Huang, Jun; Kang, Rui; Livesey, Kristen M; Tang, Daolin

    2013-03-01

    IFN1@ (interferon, type 1, cluster, also called IFNα) has been extensively studied as a treatment for patients with chronic myeloid leukemia (CML). The mechanism of anticancer activity of IFN1@ is complex and not well understood. Here, we demonstrate that autophagy, a mechanism of cellular homeostasis for the removal of dysfunctional organelles and proteins, regulates IFN1@-mediated cell death. IFN1@ activated the cellular autophagic machinery in immortalized or primary CML cells. Activation of JAK1-STAT1 and RELA signaling were required for IFN1@-induced expression of BECN1, a key regulator of autophagy. Moreover, pharmacological and genetic inhibition of autophagy enhanced IFN1@-induced apoptosis by activation of the CASP8-BID pathway. Taken together, these findings provide evidence for an important mechanism that links autophagy to immunotherapy in leukemia.

  18. Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

    PubMed

    Siebenkäs, Cornelia; Chiappinelli, Katherine B; Guzzetta, Angela A; Sharma, Anup; Jeschke, Jana; Vatapalli, Rajita; Baylin, Stephen B; Ahuja, Nita

    2017-01-01

    Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

  19. Genome-scale modeling of the protein secretory machinery in yeast.

    PubMed

    Feizi, Amir; Österlund, Tobias; Petranovic, Dina; Bordel, Sergio; Nielsen, Jens

    2013-01-01

    The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking. Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm was developed which mimics secretory machinery and assigns each secretory protein to a particular secretory class that determines the set of PTMs and transport steps specific to each protein. Protein abundances were integrated with the model in order to gain system level estimation of the metabolic demands associated with the processing of each specific protein as well as a quantitative estimation of the activity of each component of the secretory machinery.

  20. Autophagonizer, a novel synthetic small molecule, induces autophagic cell death

    SciTech Connect

    Choi, In-Kwon; Cho, Yoon Sun; Jung, Hye Jin; Kwon, Ho Jeong

    2010-03-19

    Autophagy is an apoptosis-independent mechanism of cell death that protects the cell from environmental imbalances and infection by pathogens. We identified a novel small molecule, 2-(3-Benzyl-4-oxo-3,4,5,6,7,8-hexahydro-benzo[4,5]thieno[2,3-d] pyrimidin-2-ylsulfanylmethyl)-oxazole-4-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (referred as autophagonizer), using high-content cell-based screening and the autophagosome marker EGFP-LC3. Autophagonizer inhibited growth and induced cell death in the human tumor cell lines MCF7, HeLa, HCT116, A549, AGS, and HT1080 via a caspase-independent pathway. Conversion of cytosolic LC3-I to autophagosome-associated LC3-II was greatly enhanced by autophagonizer treatment. Transmission electron microscopy and acridine orange staining revealed increased autophagy in the cytoplasm of autophagonizer-treated cells. In conclusion, autophagonizer is a novel autophagy inducer with unique structure, which induces autophagic cell death in the human tumor cell lines.

  1. Beclin-1: autophagic regulator and therapeutic target in cancer.

    PubMed

    Fu, Lei-lei; Cheng, Yan; Liu, Bo

    2013-05-01

    Beclin-1 (the mammalian ortholog of yeast ATG6) has been well-characterized to play a pivotal role in autophagy that is a major catabolic pathway in which the cell degrades macromolecules and damaged organelles. Beclin-1 structure has been identified to contain three identifiable domains, including a short Bcl-2-homology-3 (BH3) motif, a central coiled-coil domain (CCD) and a C-terminal half encompassing the evolutionarily conserved domain (ECD). Recent data indicate that Beclin-1 may interact with some co-factors such as Class III phosphatidylinositol 3-kinase (PI3KCIII)/Vps34, Vps15, ATG14L/Barkor, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, Survivin, Akt and Bcl-2/Bcl-XL to positively or negatively orchestrate the Beclin-1 interactome, thereby co-regulating the autophagy process. Here, we summarize that Beclin-1 serves not only as a key autophagic regulator with its specific interactors, but as a potential therapeutic target in cancer.

  2. Artocarpus communis Induces Autophagic Instead of Apoptotic Cell Death in Human Hepatocellular Carcinoma Cells.

    PubMed

    Tzeng, Cheng-Wei; Tzeng, Wen-Sheng; Lin, Liang-Tzung; Lee, Chiang-Wen; Yen, Ming-Hong; Yen, Feng-Lin; Lin, Chun-Ching

    2015-01-01

    For centuries, natural plant extracts have played an important role in traditional medicine for curing and preventing diseases. Studies have revealed that Artocarpus communis possess various bioactivities, such as anti-inflammation, anti-oxidant, and anticancer activities. A. communis offers economic value as a source of edible fruit, yields timber, and is widely used in folk medicines. However, little is known about its molecular mechanisms of anticancer activity. Here, we demonstrate the antiproliferative activity of A. communis methanol extract (AM) and its dichloromethane fraction (AD) in two human hepatocellular carcinoma (HCC) cell lines, HepG2 and PLC/PRF/5. Colony assay showed the long-term inhibitory effect of both extracts on cell growth. DNA laddering and immunoblotting analyses revealed that both extracts did not induce apoptosis in the hepatoma cell lines. AM and AD-treated cells demonstrated different cell cycle distribution compared to UV-treated cells, which presented apoptotic cell death with high sub-G1 ratio. Instead, acridine orange staining revealed that AM and AD triggered autophagosome accumulation. Immunoblotting showed a significant expression of autophagy-related proteins, which indicated the autophagic cell death (ACD) of hepatoma cell lines. This study therefore demonstrates that A. communis AM and its dichloromethane fraction can induce ACD in HCC cells and elucidates the potential of A. communis extracts for development as anti tumor therapeutic agents that utilize autophagy as mechanism in mediating cancer cell death.

  3. Prazosin induces p53-mediated autophagic cell death in H9C2 cells.

    PubMed

    Yang, Yi-Fan; Wu, Chau-Chung; Chen, Wen-Pin; Chen, Yuh-Lien; Su, Ming-Jai

    2011-08-01

    Prazosin, a quinazoline-based α(1)-adrenoceptor antagonist, is known to induce cell death, and this effect is independent of its α-blockade activity. However, the detailed molecular mechanisms involved are still not fully understood. In this study, we found that prazosin, but not doxazosin, could induce patterns of autophagy in H9C2 cells, including intracellular vacuole formation, microtubule-associated protein 1 light chain 3 (LC3) conversion, and acidic vesicular organelle (AVO) augmentation. Western blot analysis of phosphorylated proteins showed that exposure to prazosin increased the levels of phospho-p53 and phospho-adenosine monophosphate-activated protein kinase (AMPK) but dramatically decreased the levels of phospho-mammalian target of rapamycin (mTOR), phospho-protein kinase B (Akt), and phospho-ribosomal protein S6 kinase (p70S6K). Furthermore, although pretreatments with the pharmacological autophagy inhibitor 3-methyladenine and the p53 inhibitor pifithrin-α suppressed prazosin-induced AVO formation, they did not reverse prazosin-induced decline in cell viability but enhanced prazosin-induced caspase-3 activation. From these results we suggest that prazosin induces autophagic cell death via a p53-mediated mechanism. When the autophagy pathway was inhibited, prazosin still induced programmed cell death, at least in part through apoptotic caspase-3 cascade enhancement. Thus, our results indicate a potential new target in prazosin-induced cell death.

  4. Dysregulation of Endoplasmic Reticulum Stress and Autophagic Responses by the Antiretroviral Drug Efavirenz

    PubMed Central

    Bertrand, Luc

    2015-01-01

    Increasing evidence demonstrates that the antiretroviral drugs (ARVds) used for human immunodeficiency virus (HIV) treatment have toxic effects that result in various cellular and tissue pathologies; however, their impact on the cells composing the blood-brain barrier is poorly understood. The current study focused on ARVds, used either in combination or alone, on the induction of endoplasmic reticulum (ER) stress responses in human brain endothelial cells. Among studied drugs (efavirenz, tenofovir, emtricitabine, lamivudine, and indinavir), only efavirenz increased ER stress via upregulation and activation of protein kinase-like ER kinase PERK and inositol requiring kinase 1α (IRE1α). At the same time, efavirenz diminished autophagic activity, a surprising result because typically the induction of ER stress is linked to enhanced autophagy. These results were confirmed in microvessels of HIV transgenic mice chronically administered with efavirenz. In a series of further experiments, we identified that efavirenz dysregulated ER stress and autophagy by blocking the activity of the Beclin-1/Atg14/PI3KIII complex in regard to synthesis of phosphatidylinositol 3-phosphate, a process that is linked to the formation of autophagosomes. Because autophagy is a protective mechanism involved in the removal of dysfunctional proteins and organelles, its inhibition can contribute to the toxicity of efavirenz or the development of neurodegenerative disease in HIV patients treated with this drug. PMID:25987489

  5. Autophagic degradation of tau in primary neurons and its enhancement by trehalose.

    PubMed

    Krüger, Ulrike; Wang, Yipeng; Kumar, Satish; Mandelkow, Eva-Maria

    2012-10-01

    Modulating the tau level may represent a therapeutic target for Alzheimer's disease (AD), as accumulating evidence shows that Abeta-induced neurodegeneration is mediated by tau. It is therefore important to understand the expression and degradation of tau in neurons. Recently we showed that overexpressed mutant tau and tau aggregates are degraded via the autophagic pathway in an N2a cell model. Here we investigated whether autophagy is involved in the degradation of endogenous tau in cultured primary neurons. We activated this pathway in primary neurons with trehalose, an enhancer of autophagy. This resulted in the reduction of endogenous tau protein. Tau phosphorylation at several sites elevated in AD pathology had little influence on its degradation by autophagy. Furthermore, by using a neuronal cell model of tauopathy, we showed that activation of autophagy suppresses tau aggregation and eliminates cytotoxicity. Notably, apart from activating autophagy, trehalose also inhibits tau aggregation directly. Thus, trehalose may be a good candidate for developing therapeutic strategies for AD and other tauopathies.

  6. Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells.

    PubMed

    Chung, Kyung Min; Jeong, Eun-Ji; Park, Hyunhee; An, Hyun-Kyu; Yu, Seong-Woon

    2016-01-01

    Cytoplasmic Ca(2+) actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca(2+) levels is observed in various neuropathological states including Alzheimer's and Parkinson's diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca(2+) release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca(2+)-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs-especially RyR3-were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca(2+) regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca(2+) in neural stem cell biology.

  7. Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells.

    PubMed

    Canu, Nadia; Tufi, Roberta; Serafino, Anna Lucia; Amadoro, Giuseppina; Ciotti, Maria Teresa; Calissano, Pietro

    2005-03-01

    Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstrated that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.

  8. Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

    PubMed Central

    Chung, Kyung Min; Jeong, Eun-Ji; Park, Hyunhee; An, Hyun-Kyu; Yu, Seong-Woon

    2016-01-01

    Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca2+ release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs—especially RyR3—were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology. PMID:27199668

  9. Acupuncture promotes mTOR-independent autophagic clearance of aggregation-prone proteins in mouse brain

    PubMed Central

    Tian, Tian; Sun, Yanhong; Wu, Huangan; Pei, Jian; Zhang, Jing; Zhang, Yi; Wang, Lu; Li, Bin; Wang, Lihua; Shi, Jiye; Hu, Jun; Fan, Chunhai

    2016-01-01

    Acupuncture has historically been practiced to treat medical disorders by mechanically stimulating specific acupoints with fine needles. Despite its well-documented efficacy, its biological basis remains largely elusive. In this study, we found that mechanical stimulation at the acupoint of Yanglingquan (GB34) promoted the autophagic clearance of α-synuclein (α-syn), a well known aggregation-prone protein closely related to Parkinson’s disease (PD), in the substantia nigra par compacta (SNpc) of the brain in a PD mouse model. We found the protein clearance arose from the activation of the autophagy-lysosome pathway (ALP) in a mammalian target of rapamycin (mTOR)-independent approach. Further, we observed the recovery in the activity of dopaminergic neurons in SNpc, and improvement in the motor function at the behavior level of PD mice. Whereas acupuncture and rapamycin, a chemical mTOR inhibitor, show comparable α-syn clearance and therapeutic effects in the PD mouse model, the latter adopts a distinctly different, mTOR-dependent, autophagy induction process. Due to this fundamental difference, acupuncture may circumvent adverse effects of the rapamycin treatment. The newly discovered connection between acupuncture and autophagy not only provides a new route to understanding the molecular mechanism of acupuncture but also sheds new light on cost-effective and safe therapy of neurodegenerative diseases. PMID:26792101

  10. Acupuncture promotes mTOR-independent autophagic clearance of aggregation-prone proteins in mouse brain.

    PubMed

    Tian, Tian; Sun, Yanhong; Wu, Huangan; Pei, Jian; Zhang, Jing; Zhang, Yi; Wang, Lu; Li, Bin; Wang, Lihua; Shi, Jiye; Hu, Jun; Fan, Chunhai

    2016-01-21

    Acupuncture has historically been practiced to treat medical disorders by mechanically stimulating specific acupoints with fine needles. Despite its well-documented efficacy, its biological basis remains largely elusive. In this study, we found that mechanical stimulation at the acupoint of Yanglingquan (GB34) promoted the autophagic clearance of α-synuclein (α-syn), a well known aggregation-prone protein closely related to Parkinson's disease (PD), in the substantia nigra par compacta (SNpc) of the brain in a PD mouse model. We found the protein clearance arose from the activation of the autophagy-lysosome pathway (ALP) in a mammalian target of rapamycin (mTOR)-independent approach. Further, we observed the recovery in the activity of dopaminergic neurons in SNpc, and improvement in the motor function at the behavior level of PD mice. Whereas acupuncture and rapamycin, a chemical mTOR inhibitor, show comparable α-syn clearance and therapeutic effects in the PD mouse model, the latter adopts a distinctly different, mTOR-dependent, autophagy induction process. Due to this fundamental difference, acupuncture may circumvent adverse effects of the rapamycin treatment. The newly discovered connection between acupuncture and autophagy not only provides a new route to understanding the molecular mechanism of acupuncture but also sheds new light on cost-effective and safe therapy of neurodegenerative diseases.

  11. Dealcoholated red wine induces autophagic and apoptotic cell death in an osteosarcoma cell line.

    PubMed

    Tedesco, I; Russo, M; Bilotto, S; Spagnuolo, C; Scognamiglio, A; Palumbo, R; Nappo, A; Iacomino, G; Moio, L; Russo, G L

    2013-10-01

    Until recently, the supposed preventive effects of red wine against cardiovascular diseases, the so-called "French Paradox", has been associated to its antioxidant properties. The interest in the anticancer capacity of polyphenols present in red wine strongly increased consequently to the enormous number of studies on resveratrol. In this study, using lyophilized red wine, we present evidence that its anticancer effect in a cellular model is mediated by apoptotic and autophagic cell death. Using a human osteosarcoma cell line, U2Os, we found that the lyophilized red wine was cytotoxic in a dose-dependent manner with a maximum effect in the range of 100-200 μg/ml equivalents of gallic acid. A mixed phenotype of types I/II cell death was evidenced by means of specific assays following treatment of U2Os with lyophilized red wine, e.g., autophagy and apoptosis. We found that cell death induced by lyophilized red wine proceeded through a mechanism independent from its anti-oxidant activity and involving the inhibition of PI3K/Akt kinase signaling. Considering the relative low concentration of each single bioactive compound in lyophilized red wine, our study suggests the activation of synergistic mechanism able to inhibit growth in malignant cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 human leukemia cells.

    PubMed

    Siedlecka-Kroplewska, K; Jozwik, A; Boguslawski, W; Wozniak, M; Zauszkiewicz-Pawlak, A; Spodnik, J H; Rychlowski, M; Kmiec, Z

    2013-10-01

    Pterostilbene, a naturally occurring structural analog of resveratrol, has been reported to exert antiproliferative and proapoptotic effects in various cancer types. Recently, it has been demonstrated to induce both autophagy and apoptosis in human bladder and breast cancer cell lines. The aim of this study was to evaluate the effects of pterostilbene on HL60 human leukemia cells. Cell morphology was examined using confocal and electron microscopy. Cell viability was determined by MTT, neutral red uptake and trypan blue exclusion assays. LC3 processing was studied based on Western blotting and immunofluorescence analyses. Flow cytometry was used to study cell cycle distribution, phosphatidylserine externalization, caspase activation, disruption of mitochondrial membrane potential and intracellular production of reactive oxygen species. DNA degradation was examined by gel electrophoresis. We found that treatment of HL60 cells with pterostilbene at the IC90 concentration resulted in the G0/G1 cell cycle arrest. Pterostilbene induced conversion of cytosolic LC3-I to membrane-bound LC3-II and accumulation of large LC3-positive vacuolar structures. Pterostilbene also led to phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase activation and disruption of mitochondrial membrane potential. Moreover, it did not induce oxidative stress. Our results suggest that pterostilbene induces accumulation of autophagic vacuoles followed by cell death in HL60 cells.

  13. Productivity Growth Average in Farm Machinery Manufacturing.

    ERIC Educational Resources Information Center

    Herman, Arthur S.; Ferris, John W.

    1982-01-01

    Productivity in farm machinery manufacturing is examined. The authors discuss how the national economy affects productivity, how the growth of agriculture and technology has changed the industry, and how future trends may cause change in the industry. (CT)

  14. Asparagine slows down the breakdown of storage lipid and degradation of autophagic bodies in sugar-starved embryo axes of germinating lupin seeds.

    PubMed

    Borek, Sławomir; Paluch-Lubawa, Ewelina; Pukacka, Stanisława; Pietrowska-Borek, Małgorzata; Ratajczak, Lech

    2017-02-01

    The research was conducted on embryo axes of yellow lupin (Lupinus luteus L.), white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet), which were isolated from imbibed seeds and cultured for 96h in vitro under different conditions of carbon and nitrogen nutrition. Isolated embryo axes were fed with 60mM sucrose or were sugar-starved. The effect of 35mM asparagine (a central amino acid in the metabolism of germinating lupin seeds) and 35mM nitrate (used as an inorganic kind of nitrogen) on growth, storage lipid breakdown and autophagy was investigated. The sugar-starved isolated embryo axes contained more total lipid than axes fed with sucrose, and the content of this storage compound was even higher in sugar-starved isolated embryo axes fed with asparagine. Ultrastructural observations showed that asparagine significantly slowed down decomposition of autophagic bodies, and this allowed detailed analysis of their content. We found peroxisomes inside autophagic bodies in cells of sugar-starved Andean lupin embryo axes fed with asparagine, which led us to conclude that peroxisomes may be degraded during autophagy in sugar-starved isolated lupin embryo axes. One reason for the slower degradation of autophagic bodies was the markedly lower lipolytic activity in axes fed with asparagine. Copyright © 2016 The Author(s). Published by Elsevier GmbH.. All rights reserved.

  15. Use laser-optics for machinery alignment

    SciTech Connect

    Bloch, H.P.

    1987-10-01

    Many sources attribute most bearing overload and destructive vibration in industrial machinery to shaft misalignment. There is considerable disagreement as to the alignment quality required. There is also little agreement on suitable calculation methods and achievable accuracy for anticipated thermal growth of machinery (necessary if running alignment is to remain acceptable). This article examines existing alignment quality guidelines for relevance and consistency, and reviews the application of laser-optic alignment systems based on three years of field experience.

  16. Impaired retrograde transport of axonal autophagosomes contributes to autophagic stress in Alzheimer’s disease neurons

    PubMed Central

    Tammineni, Prasad; Ye, Xuan; Feng, Tuancheng; Aikal, Daniyal; Cai, Qian

    2017-01-01

    Neurons face unique challenges of transporting nascent autophagic vacuoles (AVs) from distal axons toward the soma, where mature lysosomes are mainly located. Autophagy defects have been linked to Alzheimer’s disease (AD). However, the mechanisms underlying altered autophagy remain unknown. Here, we demonstrate that defective retrograde transport contributes to autophagic stress in AD axons. Amphisomes predominantly accumulate at axonal terminals of mutant hAPP mice and AD patient brains. Amyloid-β (Aβ) oligomers associate with AVs in AD axons and interact with dynein motors. This interaction impairs dynein recruitment to amphisomes through competitive interruption of dynein-Snapin motor-adaptor coupling, thus immobilizing them in distal axons. Consistently, deletion of Snapin in mice causes AD-like axonal autophagic stress, whereas overexpressing Snapin in hAPP neurons reduces autophagic accumulation at presynaptic terminals by enhancing AV retrograde transport. Altogether, our study provides new mechanistic insight into AD-associated autophagic stress, thus establishing a foundation for ameliorating axonal pathology in AD. DOI: http://dx.doi.org/10.7554/eLife.21776.001 PMID:28085665

  17. The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

    SciTech Connect

    Lyu, Qing; Tou, Fangfang; Su, Hong; Wu, Xiaoyong; Chen, Xinyi; Zheng, Zhi

    2015-06-19

    Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary, our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. - Highlights: • Peiminine induces autophagy and upregulates autophagic flux. • Peiminine represses colorectal carcinoma tumor growth. • Peiminine induces autophagic cell death. • Peiminine represses mTOR phosphorylation by influencing PI3K/Akt and AMPK pathway.

  18. Autophagic lysosomal reformation depends on mTOR reactivation in H2O2-induced autophagy.

    PubMed

    Zhang, Jiqian; Zhou, Wei; Lin, Jun; Wei, Pengfei; Zhang, Yunjiao; Jin, Peipei; Chen, Ming; Man, Na; Wen, Longping

    2016-01-01

    Autophagic lysosomal reformation, a key cellular process for maintaining lysosome homeostasis in elevated autophagy, so far has only been reported for cells under certain forms of starvation. For this reason, it is controversial that whether this phenomenon is starvation-specific and its importance in lysosomal regeneration at the late stage of autophagy is often challenged. Here we show that exogenous hydrogen peroxide (H2O2) induced lysosome depletion and recovery characteristic of autophagic lysosomal reformation, and we confirmed the occurrence of autophagic lysosomal reformation after H2O2 treatment by demonstrating Rab7 dissociation from autolysosomes, recruitment of Phosphatidylinositol 4-phosphate (PI4P) and clathrin to the surface of autolysosomes, and the existence of tubular "pro-lysosome" structures extending from autolysosomes. Similar to starvation, H2O2 caused an initial deactivation and a subsequent reactivation for mTOR, and mTOR reactivation was essential for ALR. Our results provided a first non-starvation example of autophagic lysosomal reformation and provide evidence for its importance for some autophagic processes other than that of starvation.

  19. Critical role of bacterial isochorismatase in the autophagic process induced by Acinetobacter baumannii in mammalian cells

    PubMed Central

    Wang, Yang; Zhang, Kaiyu; Shi, Xiaochen; Wang, Chao; Wang, Feng; Fan, Junwen; Shen, Fengge; Xu, Jiancheng; Bao, Wanguo; Liu, Mingyuan; Yu, Lu

    2016-01-01

    A recent study reported that Acinetobacter baumannii could induce autophagy, but the recognition and clearance mechanism of intracytosolic A. baumannii in the autophagic process and the molecular mechanism of autophagy induced by the pathogen remains unknown. In this study, we first demonstrated that invading A. baumannii induced a complete, ubiquitin-mediated autophagic response that is dependent upon septins SEPT2 and SEPT9 in mammalian cells. We also demonstrated that autophagy induced by A. baumannii was Beclin-1 dependent via the AMPK/ERK/mammalian target of rapamycin pathway. Of interest, we found that the isochorismatase mutant strain had significantly decreased siderophore-mediated ferric iron acquisition ability and had a reduced the ability to induce autophagy. We verified that isochorismatase was required for the recognition of intracytosolic A. baumannii mediated by septin cages, ubiquitinated proteins, and ubiquitin-binding adaptor proteins p62 and NDP52 in autophagic response. We also confirmed that isochorismatase was required for the clearance of invading A. baumannii by autophagy in vitro and in the mouse model of infection. Together, these findings provide insight into the distinctive recognition and clearance of intracytosolic A. baumannii by autophagy in host cells, and that isochorismatase plays a critical role in the A. baumannii–induced autophagic process.—Wang, Y., Zhang, K., Shi, X., Wang, C., Wang, F., Fan, J., Shen, F., Xu, J., Bao, W., Liu, M., Yu, L. Critical role of bacterial isochorismatase in the autophagic process induced by Acinetobacter baumannii in mammalian cells. PMID:27432399

  20. Impaired autophagic flux is critically involved in drug-induced dopaminergic neuronal death.

    PubMed

    Lim, Junghyun; Lee, Yunsu; Jung, Shinae; Youdim, Moussa B H; Oh, Young J

    2014-01-01

    Autophagy is an evolutionarily conserved process that mediates the degradation of abnormal proteins and the removal of dysfunctional organelles. Recently, accumulating evidence has implicated the dysregulation of autophagy as underlying the pathophysiology of several neurodegenerative diseases. Using culture models of Parkinson's disease, we have investigated whether and how prototypic autophagic events occur upon exposure to N-methyl-4-phenylpyridinium, a dopaminergic neurotoxin, or nigericin, a K(+)/H(+) ionophore. From these independent studies, we have found that these drugs equally induce morphological and biochemical changes typical of autophagy, including accumulation of autophagic vacuoles, appearance of LC3-II forms, and alteration in the expression and distribution of p62. Further investigation has indicated that drug-induced autophagic phenomena are largely the consequences of an impaired autophagic flux. In these cell death paradigms, we have intriguingly found that Bak, a prototypic proapoptotic protein of the Bcl-2 family, exerts a protective role via reduction of the area occupied by swollen vacuoles and appearance of the LC3-II form, whereas silencing of Bak aggravates these phenomena. Further study has indicated that a protective role for Bak is primarily ascribed to its regulatory effect on the maintenance of autophagic flux and vacuole homeostasis. In this regard, a regulatory role for calcium has been proposed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. [Feverfew lactone induces autophagic death of hepatocellular carcinoma SMMC 7721 cells].

    PubMed

    Liu, Zhan-Pei; Li, Yan-Yan; Gao, Bo; Li, Jun; Gao, Jun-Ping; Lin, Ping

    2014-07-01

    To explore the effect and mechanism of feverfew lactone on inducing autophagic death of hepatocellular carcinoma. The proliferation of hepatocellular carcinoma SMMC 7721 cells treated with feverfew lactone was measured by MTT assay. The autophagy of SMMC 7721 induced with feverfew lactone was assessed by acridine orange staining, autophagic marker LC3 and p62 detecting and autophagic flows analyzing. In addition, a role of ROS in this process was stated by treatment with antioxidant agent N-acetyl-L-cysteine (NAC). The proliferation of SMMC 7721 cells were inhibited by feverfew lactone in a concentration dependence manner. The expression of LC3 and autophagic flows of SMMC 7721 cells were increased by feverfew lactone, while p62 was decreased, which implied that feverfew lactone could induce the autophagy of SMMC 7721 cells. Further more, the autophagy effect induced by feverfew lactone was declined obviously when treated with NAC suggested that ROS played an important role in this effect. Feverfew lactone induces autophagic death of SMMC 7721 cells by stimulating cells to produce ROS. The study will be helpful for the treatment of hepatocellular carcinoma and to provide theoretical basis for the clinical application of feverfew lactone.

  2. Increased autophagic response in a population of metastatic breast cancer cells.

    PubMed

    Li, Y I; Libby, Emily Falk; Lewis, Monica J; Liu, Jianzhong; Shacka, John J; Hurst, Douglas R

    2016-07-01

    Breast cancer cells are heterogeneous in their ability to invade and fully metastasize, and thus also in their capacity to survive the numerous stresses encountered throughout the multiple steps of the metastatic cascade. Considering the role of autophagy as a survival response to stress, the present study hypothesized that distinct populations of breast cancer cells may possess an altered autophagic capacity that influences their metastatic potential. It was observed that a metastatic breast cancer cell line, MDA-MB-231, that was sensitive to autophagic induction additionally possessed the ability to proliferate following nutrient deprivation. Furthermore, a selected subpopulation of these cells that survived multiple exposures to starvation conditions demonstrated a heightened response to autophagic induction compared to their parent cells. Although this subpopulation maintained a more grape-like pattern in three-dimensional culture compared to the extended spikes of the parent population, autophagic induction in this subpopulation elicited an invasive phenotype with extended spikes. Taken together, these results suggest that autophagic induction may contribute to the ability of distinct breast cancer cell populations to survive and invade.

  3. HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

    PubMed

    Aits, Sonja; Gustafsson, Lotta; Hallgren, Oskar; Brest, Patrick; Gustafsson, Mattias; Trulsson, Maria; Mossberg, Ann-Kristin; Simon, Hans-Uwe; Mograbi, Baharia; Svanborg, Catharina

    2009-03-01

    HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

  4. Comparison of the structure, function and autophagic maintenance of mitochondria in nigrostriatal and tuberoinfundibular dopamine neurons.

    PubMed

    Hawong, Hae-Young; Patterson, Joseph R; Winner, Brittany M; Goudreau, John L; Lookingland, Keith J

    2015-10-05

    A pathological hallmark of Parkinson׳s disease (PD) is progressive degeneration of nigrostriatal dopamine (NSDA) neurons, which underlies the motor symptoms of PD. While there is severe loss of midbrain NSDA neurons, tuberoinfundibular (TI) DA neurons in the mediobasal hypothalamus (MBH) remain intact. In the present study, confocal microscopic analysis revealed that mitochondrial content and numbers of mitophagosomes were lower in NSDA neuronal cell bodies in the substantia nigra pars compacta (SNpc) compared to TIDA neuronal cell bodies in the arcuate nucleus (ARC) of C57BL/6J male mice. Mitochondrial respiration, mass, membrane potential and morphology were determined using bioenergetic, flow cytometric and transmission electron microscopic analyses of synaptosomes isolated from discrete brain regions containing axon terminals of NSDA and TIDA neurons. Maximum and spare respiratory capacities, and mitochondrial mass were lower in synaptosomal mitochondria derived from the striatum (ST) as compared with the MBH, which correlated with lower numbers of mitochondria per synaptosome in these brain regions. In contrast, there was no regional difference in mitochondrial basal, maximum or spare respirations following inhibition of Complex I activity with rotenone. These results reveal that higher numbers of viable mitochondria are correlated with more extensive autophagic mitochondrial quality maintenance in TIDA neurons as compared with NSDA neurons.

  5. Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes.

    PubMed

    Piano, Ilaria; Novelli, Elena; Della Santina, Luca; Strettoi, Enrica; Cervetto, Luigi; Gargini, Claudia

    2016-01-01

    The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis.

  6. Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes

    PubMed Central

    Piano, Ilaria; Novelli, Elena; Della Santina, Luca; Strettoi, Enrica; Cervetto, Luigi; Gargini, Claudia

    2016-01-01

    The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis. PMID:26924963

  7. Autophagic myelin destruction by Schwann cells during Wallerian degeneration and segmental demyelination.

    PubMed

    Jang, So Young; Shin, Yoon Kyung; Park, So Young; Park, Joo Youn; Lee, Hye Jeong; Yoo, Young Hyun; Kim, Jong Kuk; Park, Hwan Tae

    2016-05-01

    As lysosomal hydrolysis has long been suggested to be responsible for myelin clearance after peripheral nerve injury, in this study, we investigated the possible role of autophagolysosome formation in myelin phagocytosis by Schwann cells and its final contribution to nerve regeneration. We found that the canonical formation of autophagolysosomes was induced in demyelinating Schwann cells after injury, and the inhibition of autophagy via Schwann cell-specific knockout of the atg7 gene or pharmacological intervention of lysosomal function caused a significant delay in myelin clearance. However, Schwann cell dedifferentiation, as demonstrated by extracellular signal-regulated kinase activation and c-Jun induction, and redifferentiation were not significantly affected, and thus the entire repair program progressed normally in atg7 knockout mice. Finally, autophagic Schwann cells were also found during segmental demyelination in a mouse model of inflammatory peripheral neuropathy. Together, our findings suggest that autophagy is the self-myelin destruction mechanism of Schwann cells, but mechanistically, it is a process distinct from Schwann cell plasticity for nerve repair. © 2015 Wiley Periodicals, Inc.

  8. Comparison of the structure, function and autophagic maintenance of mitochondria in nigrostriatal and tuberoinfundibular dopamine neurons

    PubMed Central

    Hawong, Hae-young; Patterson, Joseph R; Winner, Brittany M; Goudreau, John L; Lookingland, Keith J

    2015-01-01

    A pathological hallmark of Parkinson disease (PD) is progressive degeneration of nigrostriatal dopamine (NSDA) neurons, which underlies the motor symptoms of PD. While there is severe loss of midbrain NSDA neurons, tuberoinfundibular (TI) DA neurons in the mediobasal hypothalamus (MBH) remain intact. In the present study, confocal microscopic analysis revealed that mitochondrial content and numbers of mitophagosomes were lower in NSDA neuronal cell bodies in the substantia nigra pars compacta (SNpc) compared to TIDA neuronal cell bodies in the arcuate nucleus (ARC) of C57BL/6J male mice. Mitochondrial respiration, mass, membrane potential and morphology were determined using bioenergetic, flow cytometric and transmission electron microscopic analyses of synaptosomes isolated from discrete brain regions containing axon terminals of NSDA and TIDA neurons. Maximum and spare respiratory capacities, and mitochondrial mass were lower in synaptosomal mitochondria derived from the striatum (ST) as compared with the MBH, which correlated with lower numbers of mitochondria per synaptosome in these brain regions. In contrast, there was no regional difference in mitochondrial basal, maximum or spare respirations following inhibition of Complex I activity with rotenone. These results reveal that higher numbers of viable mitochondria are correlated with more extensive autophagic mitochondrial quality maintenance in TIDA neurons as compared with NSDA neurons. PMID:26141374

  9. TNFα Impairs Rhabdoviral Clearance by Inhibiting the Host Autophagic Antiviral Response

    PubMed Central

    Roca, Francisco J.; López-Muñoz, Azucena; Tyrkalska, Sylwia D.; Candel, Sergio; García-Moreno, Diana; Falco, Alberto; Meseguer, José

    2016-01-01

    TNFα is a pleiotropic pro-inflammatory cytokine with a key role in the activation of the immune system to fight viral infections. Despite its antiviral role, a few viruses might utilize the host produced TNFα to their benefit. Some recent reports have shown that anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor virus replication have not been identified. Here, a rhabdoviral infection model in zebrafish allowed us to identify the mechanism of action by which Tnfa has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa signals through its receptor Tnfr2 to enhance viral replication. Mechanistically, Tnfa does not affect viral adhesion and delivery from endosomes to the cytosol. In addition, the host interferon response was also unaffected by Tnfa levels. However, Tnfa blocks the host autophagic response, which is required for viral clearance. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish, and advances our understanding of the previously enigmatic deleterious role of TNFα in certain viral infections. PMID:27351838

  10. Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

    PubMed

    Jeong, Kwon; He, Baokun; Nohara, Kazunari; Park, Noheon; Shin, Youngmin; Kim, Seonghwa; Shimomura, Kazuhiro; Koike, Nobuya; Yoo, Seung-Hee; Chen, Zheng

    2015-07-31

    Circadian clocks orchestrate essential physiology in response to various cues, yet their mechanistic and functional plasticity remains unclear. Here, we investigated Clock(Δ19/+) heterozygous (Clk/+) mice, known to display lengthened periodicity and dampened amplitude, as a model of partially perturbed clocks. Interestingly, Clk/+ mice exhibited improved glycemic control and resistance to circadian period lengthening under high-fat diet (HFD). Furthermore, BMAL1 protein levels in Clk/+ mouse liver were upregulated compared with wild-type (WT) mice under HFD. Pharmacological and molecular studies showed that BMAL1 turnover entailed proteasomal and autophagic activities, and CLOCKΔ19 attenuated both processes. Consistent with an important role of BMAL1 in glycemic control, enhanced activation of insulin signaling was observed in Clk/+ mice relative to WT in HFD. Finally, transcriptome analysis revealed reprogramming of clock-controlled metabolic genes in Clk/+ mice. Our results demonstrate a novel role of autophagy in circadian regulation and reveal an unforeseen plasticity of circadian and metabolic networks.

  11. 46 CFR 58.01-35 - Main propulsion auxiliary machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Main propulsion auxiliary machinery. 58.01-35 Section 58... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-35 Main propulsion auxiliary machinery. Auxiliary machinery vital to the main propulsion system must be provided in duplicate unless the...

  12. 46 CFR 58.01-35 - Main propulsion auxiliary machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Main propulsion auxiliary machinery. 58.01-35 Section 58... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-35 Main propulsion auxiliary machinery. Auxiliary machinery vital to the main propulsion system must be provided in duplicate unless the...

  13. 46 CFR 58.01-35 - Main propulsion auxiliary machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Main propulsion auxiliary machinery. 58.01-35 Section 58... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-35 Main propulsion auxiliary machinery. Auxiliary machinery vital to the main propulsion system must be provided in duplicate unless the...

  14. 46 CFR 58.01-35 - Main propulsion auxiliary machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Main propulsion auxiliary machinery. 58.01-35 Section 58... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-35 Main propulsion auxiliary machinery. Auxiliary machinery vital to the main propulsion system must be provided in duplicate unless the...

  15. 46 CFR 58.01-35 - Main propulsion auxiliary machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Main propulsion auxiliary machinery. 58.01-35 Section 58... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-35 Main propulsion auxiliary machinery. Auxiliary machinery vital to the main propulsion system must be provided in duplicate unless the...

  16. 46 CFR 58.01-50 - Machinery space, noise.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Machinery space, noise. 58.01-50 Section 58.01-50... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-50 Machinery space, noise. (a) Each machinery space must be designed to minimize the exposure of personnel to noise in accordance with IMO A.468(XII...

  17. 46 CFR 42.15-35 - Machinery space openings.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Machinery space openings. 42.15-35 Section 42.15-35... BY SEA Conditions of Assignment of Freeboard § 42.15-35 Machinery space openings. (a) Machinery space..., funnel, or machinery space ventilators in an exposed position on the freeboard or superstructure deck...

  18. 46 CFR 174.195 - Bulkheads in machinery spaces.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Bulkheads in machinery spaces. 174.195 Section 174.195... in machinery spaces. (a) The bulkhead in each machinery space of each OSV must be watertight to the bulkhead deck. (b) Each penetration of, and each opening in, a bulkhead in a machinery space must— (1) Be...

  19. 46 CFR 45.149 - Machinery space openings.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Machinery space openings. 45.149 Section 45.149 Shipping... Assignment § 45.149 Machinery space openings. (a) Machinery space openings in position 1 or 2 must be framed... funnel or machinery space ventilator that must be kept open for the essential operations of the ship must...

  20. 46 CFR 45.149 - Machinery space openings.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Machinery space openings. 45.149 Section 45.149 Shipping... Assignment § 45.149 Machinery space openings. (a) Machinery space openings in position 1 or 2 must be framed... funnel or machinery space ventilator that must be kept open for the essential operations of the ship must...

  1. 46 CFR 45.149 - Machinery space openings.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Machinery space openings. 45.149 Section 45.149 Shipping... Assignment § 45.149 Machinery space openings. (a) Machinery space openings in position 1 or 2 must be framed... funnel or machinery space ventilator that must be kept open for the essential operations of the ship must...

  2. 46 CFR 174.195 - Bulkheads in machinery spaces.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Bulkheads in machinery spaces. 174.195 Section 174.195... in machinery spaces. (a) The bulkhead in each machinery space of each OSV must be watertight to the bulkhead deck. (b) Each penetration of, and each opening in, a bulkhead in a machinery space must— (1) Be...

  3. 46 CFR 42.15-35 - Machinery space openings.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Machinery space openings. 42.15-35 Section 42.15-35... BY SEA Conditions of Assignment of Freeboard § 42.15-35 Machinery space openings. (a) Machinery space..., funnel, or machinery space ventilators in an exposed position on the freeboard or superstructure deck...

  4. 46 CFR 42.15-35 - Machinery space openings.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Machinery space openings. 42.15-35 Section 42.15-35... BY SEA Conditions of Assignment of Freeboard § 42.15-35 Machinery space openings. (a) Machinery space..., funnel, or machinery space ventilators in an exposed position on the freeboard or superstructure deck...

  5. 46 CFR 174.195 - Bulkheads in machinery spaces.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Bulkheads in machinery spaces. 174.195 Section 174.195... in machinery spaces. (a) The bulkhead in each machinery space of each OSV must be watertight to the bulkhead deck. (b) Each penetration of, and each opening in, a bulkhead in a machinery space must— (1) Be...

  6. 46 CFR 58.01-50 - Machinery space, noise.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Machinery space, noise. 58.01-50 Section 58.01-50... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-50 Machinery space, noise. (a) Each machinery space must be designed to minimize the exposure of personnel to noise in accordance with IMO...

  7. 46 CFR 42.15-35 - Machinery space openings.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Machinery space openings. 42.15-35 Section 42.15-35... BY SEA Conditions of Assignment of Freeboard § 42.15-35 Machinery space openings. (a) Machinery space..., funnel, or machinery space ventilators in an exposed position on the freeboard or superstructure...

  8. 46 CFR 45.149 - Machinery space openings.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Machinery space openings. 45.149 Section 45.149 Shipping... Assignment § 45.149 Machinery space openings. (a) Machinery space openings in position 1 or 2 must be framed... funnel or machinery space ventilator that must be kept open for the essential operations of the ship...

  9. 46 CFR 42.15-35 - Machinery space openings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery space openings. 42.15-35 Section 42.15-35... BY SEA Conditions of Assignment of Freeboard § 42.15-35 Machinery space openings. (a) Machinery space..., funnel, or machinery space ventilators in an exposed position on the freeboard or superstructure...

  10. 46 CFR 45.149 - Machinery space openings.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery space openings. 45.149 Section 45.149 Shipping... Assignment § 45.149 Machinery space openings. (a) Machinery space openings in position 1 or 2 must be framed... funnel or machinery space ventilator that must be kept open for the essential operations of the ship...

  11. 46 CFR 174.195 - Bulkheads in machinery spaces.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Bulkheads in machinery spaces. 174.195 Section 174.195... in machinery spaces. (a) The bulkhead in each machinery space of each OSV must be watertight to the bulkhead deck. (b) Each penetration of, and each opening in, a bulkhead in a machinery space must— (1)...

  12. 46 CFR 174.195 - Bulkheads in machinery spaces.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Bulkheads in machinery spaces. 174.195 Section 174.195... in machinery spaces. (a) The bulkhead in each machinery space of each OSV must be watertight to the bulkhead deck. (b) Each penetration of, and each opening in, a bulkhead in a machinery space must— (1)...

  13. 46 CFR 58.01-50 - Machinery space, noise.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery space, noise. 58.01-50 Section 58.01-50... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-50 Machinery space, noise. (a) Each machinery space must be designed to minimize the exposure of personnel to noise in accordance with IMO...

  14. Study on musculoskeletal disorders in a machinery manufacturing plant.

    PubMed

    Xiao, Guo-Bing; Dempsey, Patrick G; Lei, Ling; Ma, Zao-Hua; Liang, You-Xin

    2004-04-01

    Musculoskeletal disorders and related risk factors in machinery manufacturing were investigated using interviews, postural analysis, and the revised National Institute for Occupational Safety & Health lifting equation. Sixty-nine workers involved in manual materials handling (Job A) and 51 machinery workers less involved with manual material-handling tasks (Job B) were studied. Low back pain (LBP) (at least one episode lasting for 24 hours or more in past 12 months) prevalence rates were 63.8% and 37.3% for Jobs A and B, respectively. Prevalence rates of LBP every day for a week or more attributed to lifting were 26.09% and 5.88% for Jobs A and B, respectively. Multiple regression analysis revealed that lifting repetitiveness and work age contributed to the occurrence of LBP. The "composite load" (object weight x activity repetitiveness) had a significant adverse effect on LBP.

  15. The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease.

    PubMed

    Nath, Samir R; Lieberman, Andrew P

    2017-01-01

    Polyglutamine disorders are chronic, progressive neurodegenerative diseases caused by expansion of a glutamine tract in widely expressed genes. Despite excellent models of disease, a well-documented clinical history and progression, and established genetic causes, there are no FDA approved, disease modifying treatments for these disorders. Downstream of the mutant protein, several divergent pathways of toxicity have been identified over the last several decades, supporting the idea that targeting only one of these pathways of toxicity is unlikely to robustly alleviate disease progression. As a result, a vast body of research has focused on eliminating the mutant protein to broadly prevent downstream toxicity, either by silencing mutant protein expression or leveraging the endogenous protein quality control machinery. In the latter approach, a focus has been placed on four critical components of mutant protein degradation that are active in the nucleus, a key site of toxicity: disaggregation, ubiquitination, deubiquitination, and proteasomal activity. These machineries have unique functional components, but work together as a cellular defense system that can be successfully leveraged to alleviate disease phenotypes in several models of polyglutamine toxicity. This review will highlight recent advances in understanding both the potential and role of these components of the protein quality control machinery in polyglutamine disease pathophysiology.

  16. The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease

    PubMed Central

    Nath, Samir R.; Lieberman, Andrew P.

    2017-01-01

    Polyglutamine disorders are chronic, progressive neurodegenerative diseases caused by expansion of a glutamine tract in widely expressed genes. Despite excellent models of disease, a well-documented clinical history and progression, and established genetic causes, there are no FDA approved, disease modifying treatments for these disorders. Downstream of the mutant protein, several divergent pathways of toxicity have been identified over the last several decades, supporting the idea that targeting only one of these pathways of toxicity is unlikely to robustly alleviate disease progression. As a result, a vast body of research has focused on eliminating the mutant protein to broadly prevent downstream toxicity, either by silencing mutant protein expression or leveraging the endogenous protein quality control machinery. In the latter approach, a focus has been placed on four critical components of mutant protein degradation that are active in the nucleus, a key site of toxicity: disaggregation, ubiquitination, deubiquitination, and proteasomal activity. These machineries have unique functional components, but work together as a cellular defense system that can be successfully leveraged to alleviate disease phenotypes in several models of polyglutamine toxicity. This review will highlight recent advances in understanding both the potential and role of these components of the protein quality control machinery in polyglutamine disease pathophysiology. PMID:28381987

  17. ADJUSTMENT, MAINTENANCE, AND REPAIR OF CROP HARVESTING MACHINERY. AGRICULTURAL MACHINERY--SERVICE OCCUPATIONS, MODULE NUMBER 11.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Center for Vocational and Technical Education.

    ONE OF A SERIES DESIGNED FOR HELPING TEACHERS PREPARE POSTSECONDARY-LEVEL STUDENTS FOR AGRICULTURAL MACHINERY SERVICE OCCUPATIONS AS PARTS MEN, MECHANICS, MECHANIC'S HELPERS, AND SERVICE SUPERVISORS, THIS GUIDE AIMS TO DEVELOP STUDENT COMPETENCY IN ADJUSTING, REPAIRING, AND MAINTAINING CROP HARVESTING MACHINERY. SUGGESTIONS FOR INTRODUCTION OF THE…

  18. AGRICULTURAL MACHINERY ASSEMBLY AND LUBRICATION. AGRICULTURAL MACHINERY--SERVICE OCCUPATIONS, MODULE NUMBER 7.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Center for Vocational and Technical Education.

    ONE OF A SERIES DESIGNED TO HELP TEACHERS PREPARE POSTSECONDARY STUDENTS FOR THE AGRICULTURAL MACHINERY SERVICE OCCUPATIONS AS PARTS MEN, MECHANICS, MECHANIC'S HELPERS, AND SERVICE SUPERVISORS, THIS GUIDE AIMS TO DEVELOP STUDENT UNDERSTANDING OF THE FUNCTIONS OF LUBRICANTS FOR AGRICULTURAL MACHINERY, SKILL IN THEIR SELECTION, AND UNDERSTANDING OF…

  19. 40 CFR Appendix A to Part 438 - Typical Products in Metal Products and Machinery Sectors

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Components Mining machinery & equipment, except oil field MOTOR VEHICLE Auto Exhaust System Repair Shops... Machinery Motors & Generators Oil Field Machinery & Equipment Packaging Machinery Paper Industries Machinery... Airports, Flying Fields, & Services BUS & TRUCK Bus Terminal & Service Facilities Courier Services, Except...

  20. 40 CFR Appendix A to Part 438 - Typical Products in Metal Products and Machinery Sectors

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Components Mining machinery & equipment, except oil field MOTOR VEHICLE Auto Exhaust System Repair Shops... Machinery Motors & Generators Oil Field Machinery & Equipment Packaging Machinery Paper Industries Machinery... Airports, Flying Fields, & Services BUS & TRUCK Bus Terminal & Service Facilities Courier Services, Except...

  1. 40 CFR Appendix A to Part 438 - Typical Products in Metal Products and Machinery Sectors

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Components Mining machinery & equipment, except oil field MOTOR VEHICLE Auto Exhaust System Repair Shops... Machinery Motors & Generators Oil Field Machinery & Equipment Packaging Machinery Paper Industries Machinery... Airports, Flying Fields, & Services BUS & TRUCK Bus Terminal & Service Facilities Courier Services, Except...

  2. 40 CFR Appendix A to Part 438 - Typical Products in Metal Products and Machinery Sectors

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Components Mining machinery & equipment, except oil field MOTOR VEHICLE Auto Exhaust System Repair Shops... Machinery Motors & Generators Oil Field Machinery & Equipment Packaging Machinery Paper Industries Machinery... Fields, & Services BUS & TRUCK Bus Terminal & Service Facilities Courier Services, Except by Air Freight...

  3. Rim15 and Sch9 kinases are involved in induction of autophagic degradation of ribosomes in budding yeast.

    PubMed

    Waliullah, Talukdar Muhammad; Yeasmin, Akter Mst; Kaneko, Atsuki; Koike, Naoki; Terasawa, Mashu; Totsuka, Takaya; Ushimaru, Takashi

    2017-02-01

    Autophagic degradation of ribosomes is promoted by nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1). Here we show that selective autophagic degradation of ribosomes (called ribophagy) after TORC1 inactivation requires the specific autophagy receptor Atg11. Rim15 protein kinase upregulated ribophagy, while it downregulated non-selective degradation of ribosomes.

  4. How to reduce the accumulation of autophagic vacuoles in NPC1-deficient neurons: a comparison of two pharmacological strategies.

    PubMed

    Meske, Volker; Priesnitz, Timm; Albert, Frank; Ohm, Thomas Georg

    2015-02-01

    A disturbed autophagic pathway leads to chronically increased levels of autophagic vacuoles in Niemann Pick Type-C 1 (NPC1) deficient neurons. Since these accumulations potentially contribute to neuronal cell death associated with the disease, we investigated two pharmacological strategies which potentially reduce the number of autophagic structures under following aspects: efficiency, sustainability and effect on neuronal cell viability. The strategies comprised (i) an interruption of the autophagic flux by the class III PI3K inhibitor 3-methyladenine (3-MA) and (ii) an acceleration of the autophagic execution by 2-hydroxypropyl-β-cyclodextrin (pCD). Our data show that the inhibition of autophagy with 3-MA only initially reduced the number of autophagic vacuoles in cultured neurons. Prolonged treatments with the PI3K-inhibitor reversed this lowering effect. The re-increase in the number of autophagic vacuoles was combined with a defect in the integrity of lysosomes which endangered further survival of cells. The treatment with pCD evoked a slow but sustained reduction of autophagic structures and had no negative effects on neuronal survival.

  5. The autophagic defect in Niemann-Pick disease type C neurons differs from somatic cells and reduces neuronal viability.

    PubMed

    Meske, Volker; Erz, Jennifer; Priesnitz, Timm; Ohm, Thomas-Georg

    2014-04-01

    Niemann-Pick disease type C (NPC) is a fatal, progressive neurovisceral disorder. Several studies report that the autophagic flux is disturbed in NPC1-deficient (NPC1-/-) cells. Since it has been suggested that the autophagic defect may contribute to the neurodegeneration, we used cell cultures of NPC1-deficient and NPC1-wildtype neurons to investigate whether the disturbance influences neuronal survival. We found a genotype-dependent difference in survival, when autophagy is induced during culturing. NPC1-deficient neurons are more sensitive to rapamycin treatment and starvation than wildtype neurons. The subsequent search for defects in regulatory components of the autophagic pathway and the autophagic flux brought up results which differ from previous reports on somatic cells in one essential aspect: we exclude that an enhanced formation of autophagosomes contributes to the imbalanced autophagic flux in NPC1 deficient neurons. We found that solely the clearance of autophagosomes is delayed in these cells, which leads to an accumulation of autophagic vacuoles within the lysosomal compartment. Lowering the abnormal lipid load of the acidic organelles with cyclodextrin is sufficient to correct the autophagic flux and prevents premature death of NPC1-/- neurons under autophagic stress. From our results, we conclude that a pharmacological intervention in the neuropathology of NPC-disease should focus on the restoration of the lysosomal degradation capacity of cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. The nascent polypeptide-associated complex is essential for autophagic flux

    PubMed Central

    Guo, Bin; Huang, Jie; Wu, Wenxian; Feng, Du; Wang, Xiaochen; Chen, Yingyu; Zhang, Hong

    2014-01-01

    The ribosome-associated nascent polypeptide-associated complex (NAC) is involved in multiple cotranslational processes, including protein transport into the ER and mitochondria, and also acts as a chaperone to assist protein folding. Here we demonstrated that NAC is also essential for autophagic degradation of a variety of protein aggregates in C. elegans. Loss of function of NAC impairs lysosome function, resulting in accumulation of autophagic substrates in enlarged autolysosomes. Knockdown of mammalian NAC also causes accumulation of nondegradative autolysosomes. Our study revealed that NAC plays an evolutionarily conserved role in the autophagy pathway and thus in maintaining protein homeostasis under physiological conditions. PMID:25126725

  7. Autophagic flux data in differentiated C2C12 myotubes following exposure to acetylcholine and caffeine.

    PubMed

    Bloemberg, Darin; Quadrilatero, Joe

    2016-06-01

    The C2C12 line of mouse myoblasts is a useful cell culture model in which to conduct in vitro analyses related to skeletal muscle. Here we present data regarding the autophagic response induced by two chemicals known to influence calcium release and contraction in skeletal muscles and C2C12 cells: acetylcholine and caffeine. More specifically, by concurrently administering acetylcholine or caffeine along with chloroquine to differentiated myotubes for various amounts of time and assessing the protein expression of LC3 and p62, we report data on the relative level of autophagic flux induced by these two calcium- and contraction-regulating chemicals.

  8. Fisetin induces autophagic cell death through suppression of mTOR signaling pathway in prostate cancer cells

    PubMed Central

    Suh, Yewseok; Afaq, Farrukh; Khan, Naghma; Johnson, Jeremy J.; Khusro, Fatima H.; Mukhtar, Hasan

    2010-01-01

    The mammalian target of rapamycin (mTOR) kinase is an important component of PTEN/PI3K/Akt signaling pathway, which is frequently deregulated in prostate cancer (CaP). Recent studies suggest that targeting PTEN/PI3K/Akt and mTOR signaling pathway could be an effective strategy for the treatment of hormone refractory CaP. Here, we show that the treatment of androgen-independent and PTEN-negative human CaP PC3 cells with fisetin, a dietary flavonoid, resulted in inhibition of mTOR kinase signaling pathway. Treatment of cells with fisetin inhibited mTOR activity and downregulated Raptor, Rictor, PRAS40 and GβL that resulted in loss of mTOR complexes (mTORC)1/2 formation. Fisetin also activated the mTOR repressor TSC2 through inhibition of Akt and activation of AMPK. Fisetin-mediated inhibition of mTOR resulted in hypophosphorylation of 4EBP1 and suppression of Cap-dependent translation. We also found that fisetin treatment leads to induction of autophagic-programmed cell death rather than cytoprotective autophagy as shown by small interfering RNA Beclin1-knockdown and autophagy inhibitor. Taken together, we provide evidence that fisetin functions as a dual inhibitor of mTORC1/2 signaling leading to inhibition of Cap-dependent translation and induction of autophagic cell death in PC3 cells. These results suggest that fisetin could be a useful chemotherapeutic agent in treatment of hormone refractory CaP. PMID:20530556

  9. Cancer gene therapy targeting cellular apoptosis machinery.

    PubMed

    Jia, Lin-Tao; Chen, Si-Yi; Yang, An-Gang

    2012-11-01

    The unraveling of cellular apoptosis machinery provides novel targets for cancer treatment, and gene therapy targeting this suicidal system has been corroborated to cause inflammation-free autonomous elimination of neoplastic cells. The apoptotic machinery can be targeted by introduction of a gene encoding an inducer, mediator or executioner of apoptotic cell death or by inhibition of anti-apoptotic gene expression. Strategies targeting cancer cells, which are achieved by selective gene delivery, specific gene expression or secretion of target proteins via genetic modification of autologous cells, dictate the outcome of apoptosis-based cancer gene therapy. Despite so far limited clinical success, gene therapy targeting the apoptotic machinery has great potential to benefit patients with threatening malignancies provided the availability of efficient and specific gene delivery and administration systems.

  10. The RNA polymerase I transcription machinery.

    PubMed

    Russell, Jackie; Zomerdijk, Joost C B M

    2006-01-01

    The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

  11. Mitochondrial Machineries for Protein Import and Assembly.

    PubMed

    Wiedemann, Nils; Pfanner, Nikolaus

    2017-03-15

    Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics. Expected final online publication date for the Annual Review of Biochemistry Volume 86 is June 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  12. Theranostic iridium(III) complexes as one- and two-photon phosphorescent trackers to monitor autophagic lysosomes.

    PubMed

    He, Liang; Tan, Cai-Ping; Ye, Rui-Rong; Zhao, Yi-Zhi; Liu, Ya-Hong; Zhao, Qiang; Ji, Liang-Nian; Mao, Zong-Wan

    2014-11-03

    During autophagy, the intracellular components are captured in autophagosomes and delivered to lysosomes for degradation and recycling. Changes in lysosomal trafficking and contents are key events in the regulation of autophagy, which has been implicated in many physiological and pathological processes. In this work, two iridium(III) complexes (LysoIr1 and LysoIr2) are developed as theranostic agents to monitor autophagic lysosomes. These complexes display lysosome-activated phosphorescence and can specifically label lysosomes with high photostability. Simultaneously, they can induce autophagy potently without initiating an apoptosis response. We demonstrate that LysoIr2 can effectively implement two functions, namely autophagy induction and lysosomal tracking, in the visualization of autophagosomal-lysosomal fusion. More importantly, they display strong two-photon excited fluorescence (TPEF), which is favorable for live cell imaging and in vivo applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Staurosporine-induced cell death in Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degeneration.

    PubMed

    Christensen, S T; Chemnitz, J; Straarup, E M; Kristiansen, K; Wheatley, D N; Rasmussen, L

    1998-01-01

    Staurosporine blocks signal transduction associated with cell survival, proliferation and chemosensory behaviour in the ciliated protozoan, Tetrahymena thermophila. Staurosporine inhibits cell proliferation and in vivo protein phosphorylation induced by phorbol ester. It also reduces the in vitro phosphorylation of the PKC-specific substrate, myelin basic protein fragment 4-14. Our results show that cell death in the presence of staurosporine is associated with morphological and ultrastructural changes similar to both apoptosis and autophagic degeneration, but these in turn can be postponed or prevented by 8-bromo-cyclic GMP, protoporphyrin IX, hemin or actinomycin D, although phorbol ester and insulin were ineffective. The results support the notion that staurosporine-induced cell death is an active process, associated with and/or requiring de novo RNA synthesis.

  14. Simulated ischemia/reperfusion-induced p65-Beclin 1-dependent autophagic cell death in human umbilical vein endothelial cells

    PubMed Central

    Zeng, Min; Wei, Xin; Wu, Zhiyong; Li, Wei; Zheng, Yin; Li, Bing; Meng, Xuqing; Fu, Xiuhong; Fei, Yi

    2016-01-01

    Myocardial ischemia/reperfusion (I/R) injury detrimentally alters the prognosis of patients undergoing revascularization after acute myocardial infarction. Our previous study demonstrated that NF-κB-induced autophagy plays a detrimental role in cardiac I/R injury using a rabbit myocardial I/R model. In this study, we sought to explore the specific mechanism of this autophagy-mediated cell damage in an in vitro simulated ischemia/reperfusion (sI/R) model using human umbilical vein endothelial cells. Our current study demonstrates that simulated I/R induces autophagy in a p65-Beclin 1-dependent manner, which can be suppressed with the inhibition of NF-κB. Furthermore, rapamycin which promotes autophagy, exacerbates sI/R-induced cell death. While 3-methyladenine rescues cell damage. Our data thus suggest that I/R promotes NF-κB p65 activity mediated Beclin 1-mediated autophagic flux, thereby exacerbating myocardial injury. PMID:27857190

  15. Viral strategies to subvert the mammalian translation machinery.

    PubMed

    Roberts, Lisa O; Jopling, Catherine L; Jackson, Richard J; Willis, Anne E

    2009-01-01

    Viruses do not carry their own protein biosynthesis machinery and the translation of viral proteins therefore requires that the virus usurps the machinery of the host cell. To allow optimal translation of viral proteins at the expense of cellular proteins, virus families have evolved a variety of methods to repress the host translation machinery, while allowing effective viral protein synthesis. Many viruses use noncanonical mechanisms that permit translation of their own RNAs under these conditions. Viruses have also developed mechanisms to evade host innate immune responses that would repress translation under conditions of viral infection, in particular PKR activation in response to double-stranded RNA (dsRNA). Importantly, the study of viral translation mechanisms has enormously enhanced our understanding of many aspects of the cellular protein biosynthesis pathway and its components. A number of unusual mechanisms of translation initiation that were first discovered in viruses have since been observed in cellular mRNAs, and it has become apparent that a diverse range of translation mechanisms operates in eukaryotes, allowing subtle regulation of this essential process.

  16. The machinery of mitochondrial inheritance and behavior.

    PubMed

    Yaffe, M P

    1999-03-05

    The distribution of mitochondria to daughter cells during cell division is an essential feature of cell proliferation. Until recently, it was commonly believed that inheritance of mitochondria and other organelles was a passive process, a consequence of their random diffusion throughout the cytoplasm. A growing recognition of the reticular morphology of mitochondria in many living cells, the association of mitochondria with the cytoskeleton, and the coordinated movements of mitochondria during cellular division and differentiation has illuminated the necessity for a cellular machinery that mediates mitochondrial behavior. Characterization of the underlying molecular components of this machinery is providing insight into mechanisms regulating mitochondrial morphology and distribution.

  17. Andrographolide alleviates imiquimod-induced psoriasis in mice via inducing autophagic proteolysis of MyD88.

    PubMed

    Shao, Fenli; Tan, Tao; Tan, Yang; Sun, Yang; Wu, Xingxin; Xu, Qiang

    2016-09-01

    Psoriasis is a chronic inflammatory skin disease with excessive activation of toll-like receptors (TLRs), which play important roles in developing psoriasis. Targeting TLR signaling remains a challenge for treating psoriasis. Here, we found that andrographolide (Andro), a small-molecule natural product, alleviated imiquimod- but not interleukin 23 (IL-23)-induced psoriasis in mice with reducing expressions of IL-23 and IL-1β in the skin. The improvement in imiquimod-induced psoriasis by Andro was not observed in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) knockout mice. Furthermore, Andro inhibited mRNA expressions of IL-23, IL-6 and IL-1β but not CD80 and CD86 in bone-marrow derived dendritic cells (BMDCs) treated with lipopolysaccharide (LPS) in a MAP1LC3B-dependent manner. In addition, Andro inhibited imiquimod-induced mRNA expressions of IL-23, IL-6, IL-1β, CD80 and CD86 in BMDCs from mice. Interestingly, Andro induced a degradation of myeloid differentiation factor 88 (MyD88) and blocked the recruitment of TNF receptor-associated factor 6 (TRAF6) to MyD88 upon LPS stimulation in BMDCs from mice. Blockade of autophagic proteolysis using NH4Cl or MAP1LC3B(-/-) BMDCs abolished the Andro-induced MyD88 degradation. In conclusion, Andro controls activation of MyD88-dependent cytokines and alleviates psoriasis in mice via inducing autophagic proteolysis of MyD88, which could be a novel strategy to treat psoriasis.

  18. Near-infrared photothermal therapy using EGFR-targeted gold nanoparticles increases autophagic cell death in breast cancer.

    PubMed

    Zhang, Meihua; Kim, Hoe Suk; Jin, Tiefeng; Moon, Woo Kyung

    2017-05-01

    Although triple negative breast cancer (TNBC) is a small percentage of all breast cancers, to date, TNBC is one of the most challenging types of breast cancer for basic and clinic research because TNBC patients display a high risk of relapse, shorter overall survival and limited therapeutic options after completion of conventional chemotherapy compared with patients with other breast cancer subtypes. The epidermal growth factor receptor (EGFR) is a promising target for TNBC treatment. Although near infrared-photothermal therapy (NIR-PTT) using anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs), has attracted considerable interest for non-invasive and targeted TNBC treatment through an activation of apoptotic pathway, it is unclear whether anti-EGFR-GNs-combined NIR-PTT modulates the induction of autophagy contributing to cell death. Therefore, we investigated the autophagic cell death in cultured TNBC cells and mouse xenograft tumors during anti-EGFR-GNs-combined NIR-PTT. We here found that the cytotoxicity induced by anti-EGFR-GNs-combined NIR-PTT was rescued by treatment with autophagy inhibitor, 3-methyladenine (3-MA). Anti-EGFR-GNs-combined NIR-PTT induced remarkable levels of autophagy activity as evidenced by a large number of autophagic vesicles and a significant increase in autophagy-specific proteins; microtubule-associated protein light chain 3 (LC3), p62, beclin-1, and autophagy-related gene5 (Atg5), accompanying the inhibition of AKT-mTOR signaling pathway responsible for inducing autophagy. Moreover, in mouse xenograft tumors, anti-EGFR-GNs-combined NIR-PTT also increased LC3 and beclin-1 levels. Our findings, for the first time, demonstrate that anti-EGFR-GNs-combined NIR-PTT remarkably induces autophagy leading to EGFR-targeted cancer cell death. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Paraquat, but Not Maneb, Induces Synucleinopathy and Tauopathy in Striata of Mice through Inhibition of Proteasomal and Autophagic Pathways

    PubMed Central

    Wills, Jonathan; Credle, Joel; Oaks, Adam W.; Duka, Valeriy; Lee, Jae-Hoon; Jones, Jessica; Sidhu, Anita

    2012-01-01

    SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau. PMID:22292029

  20. Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways.

    PubMed

    Wills, Jonathan; Credle, Joel; Oaks, Adam W; Duka, Valeriy; Lee, Jae-Hoon; Jones, Jessica; Sidhu, Anita

    2012-01-01

    SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.

  1. Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease

    PubMed Central

    Magalhaes, Joana; Gegg, Matthew E.; Migdalska-Richards, Anna; Doherty, Mary K.; Whitfield, Phillip D.; Schapira, Anthony H.V.

    2016-01-01

    Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD. PMID:27378698

  2. Formation and excretion of autophagic plastids (plastolysomes) in Brassica napus embryogenic microspores

    PubMed Central

    Parra-Vega, Verónica; Corral-Martínez, Patricia; Rivas-Sendra, Alba; Seguí-Simarro, Jose M.

    2015-01-01

    The change in developmental fate of microspores reprogrammed toward embryogenesis is a complex but fascinating experimental system where microspores undergo dramatic changes derived from the developmental switch. After 40 years of study of the ultrastructural changes undergone by the induced microspores, many questions are still open. In this work, we analyzed the architecture of DNA-containing organelles such as plastids and mitochondria in samples of B. napus isolated microspore cultures covering the different stages before, during, and after the developmental switch. Mitochondria presented a conventional oval or sausage-like morphology for all cell types studied, similar to that found in vivo in other cell types from vegetative parts. Similarly, plastids of microspores before induction and of non-induced cells showed conventional architectures. However, approximately 40% of the plastids of embryogenic microspores presented atypical features such as curved profiles, protrusions, and internal compartments filled with cytoplasm. Three-dimensional reconstructions confirmed that these plastids actually engulf cytoplasm regions, isolating them from the rest of the cell. Acid phosphatase activity was found in them, confirming the lytic activity of these organelles. In addition, digested plastid-like structures were found excreted to the apoplast. All these phenomena seemed transient, since microspore-derived embryos (MDEs) showed conventional plastids. Together, these results strongly suggested that under special circumstances, such as those of the androgenic switch, plastids of embryogenic microspores behave as autophagic plastids (plastolysomes), engulfing cytoplasm for digestion, and then are excreted out of the cytoplasm as part of a cleaning program necessary for microspores to become embryos. PMID:25745429

  3. Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease.

    PubMed

    Mankelow, Tosti J; Griffiths, Rebecca E; Trompeter, Sara; Flatt, Joanna F; Cogan, Nicola M; Massey, Edwin J; Anstee, David J

    2015-10-08

    During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 µm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia. © 2015 by The American Society of Hematology.

  4. Analysis of autophagic flux in response to sulforaphane in metastatic prostate cancer cells

    PubMed Central

    Watson, Gregory W; Wickramasekara, Samanthi; Fang, Yufeng; Palomera-Sanchez, Zoraya; Maier, Claudia S; Williams, David E; Dashwood, Roderick H; Perez, Viviana I; Ho, Emily

    2015-01-01

    Scope The phytochemical sulforaphane has been shown to decrease prostate cancer metastases in a genetic mouse model of prostate carcinogenesis, though the mechanism of action is not fully known. Sulforaphane has been reported to stimulate autophagy, and modulation of autophagy has been proposed to influence sulforaphane cytotoxicity; however, no conclusions about autophagy can be drawn without assessing autophagic flux, which has not been characterized in prostate cancer cells following sulforaphane treatment. Methods and Results We conducted an investigation to assess the impact of sulforaphane on autophagic flux in two metastatic prostate cancer cell lines at a concentration shown to decrease metastasis in vivo. Autophagic flux was assessed by multiple autophagy related proteins and substrates. We found that sulforaphane can stimulate autophagic flux and cell death only at high concentrations, above what has been observed in vivo. Conclusion These results suggest that sulforaphane does not directly stimulate autophagy or cell death in metastatic prostate cancer cells under physiologically relevant conditions, but instead supports the involvement of in vivo factors as important effectors of sulforaphane- mediated prostate cancer suppression. PMID:26108801

  5. IsaB inhibits autophagic flux to promote host transmission of methicillin-resistant Staphylococcus aureus

    PubMed Central

    Liu, Pei-Feng; Cheng, Jin-Shiung; Sy, Cheng-Len; Huang, Wei-Chun; Yang, Hsiu-Chen; Gallo, Richard L.; Huang, Chun-Ming; Shu, Chih-Wen

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major nosocomial pathogen that is widespread in both health care facilities and the community at large as a result of direct host-to-host transmission. Several virulence factors are associated with pathogen transmission to naive hosts. Immunodominant surface antigen B (IsaB) is a virulence factor that helps Staphylococcus aureus to evade the host defense system. However, the mechanism of IsaB on host transmissibility remains unclear. We found that IsaB expression was elevated in transmissible MRSA. Wild-type isaB strains inhibited autophagic flux to promote bacterial survival and elicit inflammation in THP-1 cells and mouse skin. MRSA isolates with increased IsaB expression showed decreased autophagic flux, and the MRSA isolate with the lowest IsaB expression showed increased autophagic flux. In addition, recombinant IsaB rescued the virulence of the isaB deletion strain and increased the Group A streptococcus (GAS) virulence in vivo. Together, these results reveal that IsaB diminishes autophagic flux, thereby allowing MRSA to evade host degradation. These findings suggest that IsaB is a suitable target for preventing or treating MRSA infection. PMID:26134948

  6. Granulovacuolar Degeneration Bodies of Alzheimer’s Disease Resemble Late-stage Autophagic Organelles

    PubMed Central

    Funk, Kristen E.; Mrak, Robert E.; Kuret, Jeff

    2010-01-01

    Aims Granulovacuolar degeneration involves the accumulation of large, double membrane-bound bodies within certain neurons during the course of Alzheimer’s disease and other adultonset dementias. Because of the two-layer membrane morphology, it has been proposed that the bodies are related to autophagic organelles. The aim of this study was to test this hypothesis, and determine the approximate stage at which the pathway stalled in Alzheimer’s disease. Methods Spatial colocalization of autophagic and endocytic markers with casein kinase 1 delta, a marker for GVD bodies, was evaluated in hippocampal sections prepared from postmortem Braak stage IV and V Alzheimer’s disease cases using double-label confocal fluorescence microscopy. Results GVD bodies colocalized weakly with early-stage autophagy markers LC3 and p62, but strongly with late-stage marker LAMP1 (lysosome-associated membrane protein 1), which decorated their surrounding membranes. GVD bodies also colocalized strongly with CHMP2B (charged multivesicular body protein 2B), which colocalized with the core granule, but less strongly with lysosomal marker cathepsin D. Conclusions The resultant immunohistochemical signature suggests that GVD bodies contain late-stage autophagic markers, and accumulate at the nexus of autophagic and endocytic pathways. . The data further suggest that failure to complete autolysosome formation may be an important correlate of GVD body accumulation. PMID:20946470

  7. N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca(2+) Mobilization.

    PubMed

    Law, Betty Y K; Mok, Simon W F; Chen, Juan; Michelangeli, Francesco; Jiang, Zhi-Hong; Han, Yu; Qu, Yuan Q; Qiu, Alena C L; Xu, Su-Wei; Xue, Wei-Wei; Yao, Xiao-Jun; Gao, Jia Y; Javed, Masood-Ul-Hassan; Coghi, Paolo; Liu, Liang; Wong, Vincent K W

    2017-01-01

    Resistance of cancer cells to chemotherapy remains a significant problem in oncology. Mechanisms regulating programmed cell death, including apoptosis, autophagy or necrosis, in the treatment of cancers have been extensively investigated over the last few decades. Autophagy is now emerging as an important pathway in regulating cell death or survival in cancer therapy. Recent studies demonstrated variety of natural small-molecules could induce autophagic cell death in apoptosis-resistant cancer cells, therefore, discovery of novel autophagic enhancers from natural products could be a promising strategy for treatment of chemotherapy-resistant cancer. By computational virtual docking analysis, biochemical assays, and advanced live-cell imaging techniques, we have identified N-desmethyldauricine (LP-4), isolated from rhizoma of Menispermum dauricum DC as a novel inducer of autophagy. LP-4 was shown to induce autophagy via the Ulk-1-PERK and Ca(2+)/Calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPK-mTOR signaling cascades, via mobilizing calcium release through inhibition of SERCA, and importantly, lead to autophagic cell death in a panel of cancer cells, apoptosis-defective and apoptosis-resistant cells. Taken together, this study provides detailed insights into the cytotoxic mechanism of a novel autophagic compound that targeting the apoptosis resistant cancer cells, and new implication on drug discovery from natural products for drug resistant cancer therapy.

  8. Natural small-molecule enhancers of autophagy induce autophagic cell death in apoptosis-defective cells.

    PubMed

    Law, Betty Yuen Kwan; Chan, Wai Kit; Xu, Su Wei; Wang, Jing Rong; Bai, Li Ping; Liu, Liang; Wong, Vincent Kam Wai

    2014-07-01

    Resistance of cancer cells to chemotherapy is a significant problem in oncology, and the development of sensitising agents or small-molecules with new mechanisms of action to kill these cells is needed. Autophagy is a cellular process responsible for the turnover of misfolded proteins or damaged organelles, and it also recycles nutrients to maintain energy levels for cell survival. In some apoptosis-resistant cancer cells, autophagy can also enhance the efficacy of anti-cancer drugs through autophagy-mediated mechanisms of cell death. Because the modulation of autophagic processes can be therapeutically useful to circumvent chemoresistance and enhance the effects of cancer treatment, the identification of novel autophagic enhancers for use in oncology is highly desirable. Many novel anti-cancer compounds have been isolated from natural products; therefore, we worked to discover natural, anti-cancer small-molecule enhancers of autophagy. Here, we have identified a group of natural alkaloid small-molecules that function as novel autophagic enhancers. These alkaloids, including liensinine, isoliensinine, dauricine and cepharanthine, stimulated AMPK-mTOR dependent induction of autophagy and autophagic cell death in a panel of apoptosis-resistant cells. Taken together, our work provides novel insights into the biological functions, mechanisms and potential therapeutic values of alkaloids for the induction of autophagy.

  9. 46 CFR 176.804 - Machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... watertight bulkhead pipe penetration valves; (h) Operational test of the means provided for pumping bilges... certification of a vessel, the owner or managing operator shall be prepared to conduct tests and have the vessel... the main propulsion machinery both ahead and astern; (b) Operational test and inspection of...

  10. 46 CFR 176.804 - Machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... watertight bulkhead pipe penetration valves; (h) Operational test of the means provided for pumping bilges... certification of a vessel, the owner or managing operator shall be prepared to conduct tests and have the vessel... the main propulsion machinery both ahead and astern; (b) Operational test and inspection of...

  11. 46 CFR 176.804 - Machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... watertight bulkhead pipe penetration valves; (h) Operational test of the means provided for pumping bilges... certification of a vessel, the owner or managing operator shall be prepared to conduct tests and have the vessel... the main propulsion machinery both ahead and astern; (b) Operational test and inspection of...

  12. AGRICULTURAL MACHINERY--POWER. TEACHERS COPY.

    ERIC Educational Resources Information Center

    HILL, DURWIN; VENABLE, BENNY MAC

    THE PURPOSE OF THIS DOCUMENT IS TO PROVIDE A STUDY GUIDE FOR STUDENTS PREPARING FOR AGRICULTURAL MACHINERY OCCUPATIONS IN A VOCATIONAL AGRICULTURE COOPERATIVE EDUCATION PROGRAM. THE MATERIAL WAS DESIGNED BY SUBJECT MATTER SPECIALISTS ON THE BASIS OF STATE ADVISORY COMMITTEE RECOMMENDATIONS, TRIED IN OPERATIONAL PROGRAMS, AND REFINED BY A TEACHER.…

  13. 46 CFR 176.804 - Machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... watertight bulkhead pipe penetration valves; (h) Operational test of the means provided for pumping bilges... certification of a vessel, the owner or managing operator shall be prepared to conduct tests and have the vessel... the main propulsion machinery both ahead and astern; (b) Operational test and inspection of...

  14. EARNINGS IN THE MACHINERY INDUSTRIES, MID-1966.

    ERIC Educational Resources Information Center

    BAUER, FREDERICK L.

    RESULTS OF A MID-1966 NATIONWIDE SURVEY BY THE BUREAU OF LABOR STATISTICS SHOWED THAT THE EARNINGS OF PRODUCTION AND RELATED NONELECTRICAL MACHINERY WORKERS IN 21 LARGE OCCUPATIONAL AREAS VARIED BY OCCUPATION, SIZE OF ESTABLISHMENT, AND COMMUNITY, INDUSTRY, LABOR-MANAGEMENT CONTRACT STATUS, AND LOCATION. THE AVERAGE HOURLY WAGE WAS $2.84. HIGHER…

  15. Heat shock factor 1 confers resistance to Hsp90 inhibitors through p62/SQSTM1 expression and promotion of autophagic flux.

    PubMed

    Samarasinghe, Buddhini; Wales, Christina T K; Taylor, Frederick R; Jacobs, Aaron T

    2014-02-01

    Heat shock protein 90 (Hsp90) has an important role in many cancers. Biochemical inhibitors of Hsp90 are in advanced clinical development for the treatment of solid and hematological malignancies. At the cellular level, their efficacy is diminished by the fact that Hsp90 inhibition causes activation of heat shock factor 1 (HSF1). We report a mechanism by which HSF1 activation diminishes the effect of Hsp90 inhibitors geldanamycin and 17-allylaminogeldanamycin (17-AAG, tanespimycin). Silencing HSF1 with siRNA or inhibiting HSF1 activity with KRIBB11 lowers the threshold for apoptosis in geldanamycin and 17-AAG-treated cancer cells. Autophagy also mitigates the actions of Hsp90 inhibitors. Blocking autophagy with 3-methyladenine (3-MA), bafilomycin A1, or beclin 1 siRNA also lower the threshold for apoptosis. Exploring a potential relationship between HSF1 and autophagy, we monitored autophagosome formation and autophagic flux in control and HSF1-silenced cells. Results show HSF1 is required for autophagy in Hsp90 inhibitor-treated cells. The reduced autophagy observed in HSF1-silenced cells correlates with enhanced cell death. To investigate how HSF1 promotes autophagy, we monitored the expression of genes involved in the autophagic cascade. These data show that sequestosome 1 (p62/SQSTM1), a protein involved in the delivery of autophagic substrates and nucleation of autophagosomes, is an HSF1-regulated gene. Gene silencing was used to evaluate the significance of p62/SQSTM1 in Hsp90 inhibitor resistance. Cells where p62/SQSTM1 was silenced showed a dramatic increase in sensitivity to Hsp90 inhibitors. Results highlight the importance of HSF1 and HSF1-dependent p62/SQSTM1 expression in resistance Hsp90 inhibitors, underscoring the potential of targeting HSF1 to improve the efficacy of Hsp90 inhibitors in cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. The eukaryotic gene transcription machinery.

    PubMed

    Kornberg, R D

    2001-08-01

    Seven purified proteins may be combined to reconstitute regulated, promoter-dependent RNA polymerase II transcription: five general transcription factors, Mediator, and RNA polymerase II. The entire system has been conserved across species from yeast to humans. The structure of RNA polymerase II, consisting of 10 polypeptides with a mass of about 500 kDa, has been determined at atomic resolution. On the basis of this structure, that of an actively transcribing RNA polymerase II complex has been determined as well.

  17. HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus.

    PubMed

    Page, Nicolas; Gros, Frédéric; Schall, Nicolas; Décossas, Marion; Bagnard, Dominique; Briand, Jean-Paul; Muller, Sylviane

    2011-05-01

    The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells. To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells. P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers. P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux. The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.

  18. HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus

    PubMed Central

    Page, Nicolas; Gros, Frédéric; Schall, Nicolas; Décossas, Marion; Bagnard, Dominique; Briand, Jean-Paul; Muller, Sylviane

    2011-01-01

    Background The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells. Objective To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells. Methods P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers. Results P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux. Conclusion The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice. PMID:21173017

  19. Cytosolic clearance of replication-deficient mutants reveals Francisella tularensis interactions with the autophagic pathway.

    PubMed

    Chong, Audrey; Wehrly, Tara D; Child, Robert; Hansen, Bryan; Hwang, Seungmin; Virgin, Herbert W; Celli, Jean

    2012-09-01

    Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival

  20. Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance

    PubMed Central

    Bingel, Corinna; Koeneke, Emily; Ridinger, Johannes; Bittmann, Annika; Sill, Martin; Peterziel, Heike; Wrobel, Jagoda K; Rettig, Inga; Milde, Till; Fernekorn, Uta; Weise, Frank; Schober, Andreas; Witt, Olaf; Oehme, Ina

    2017-01-01

    Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy. PMID:28837150

  1. Triggering autophagic cell death with a di-manganese(II) developmental therapeutic.

    PubMed

    Slator, Creina; Molphy, Zara; McKee, Vickie; Kellett, Andrew

    2017-02-04

    There is an unmet need for novel metal-based chemotherapeutics with alternative modes of action compared to clinical agents such as cisplatin and metallo-bleomycin. Recent attention in this field has focused on designing intracellular ROS-mediators as powerful cytotoxins of human cancers and identifying potentially unique toxic mechanisms underpinning their utility. Herein, we report the developmental di-manganese(II) therapeutic [Mn2(μ-oda)(phen)4(H2O)2][Mn2(μ-oda)(phen)4(oda)2]·4H2O (Mn-Oda) induces autophagy-promoted apoptosis in human ovarian cancer cells (SKOV3). The complex was initially identified to intercalate DNA by topoisomerase I unwinding and circular dichroism spectroscopy. Intracellular DNA damage, detected by γH2AX and the COMET assay, however, is not linked to direct Mn-Oda free radical generation, but is instead mediated through the promotion of intracellular reactive oxygen species (ROS) leading to autophagic vacuole formation and downstream nuclear degradation. To elucidate the cytotoxic profile of Mn-Oda, a wide range of biomarkers specific to apoptosis and autophagy including caspase release, mitochondrial membrane integrity, fluorogenic probe localisation, and cell cycle analysis were employed. Through these techniques, the activity of Mn-Oda was compared directly to i.) the pro-apoptotic clinical anticancer drug doxorubicin, ii.) the multimodal histone deacetylase inhibitor suberoyanilide hydroxamic acid, and iii.) the autophagy inducer rapamycin. In conjunction with ROS-specific trapping agents and established inhibitors of autophagy, we have identified autophagy-induction linked to mitochondrial superoxide production, with confocal image analysis of SKOV3 cells further supporting autophagosome formation.

  2. Autophagic clearance of mitochondria in the kidney copes with metabolic acidosis.

    PubMed

    Namba, Tomoko; Takabatake, Yoshitsugu; Kimura, Tomonori; Takahashi, Atsushi; Yamamoto, Takeshi; Matsuda, Jun; Kitamura, Harumi; Niimura, Fumio; Matsusaka, Taiji; Iwatani, Hirotsugu; Matsui, Isao; Kaimori, Junya; Kioka, Hidetaka; Isaka, Yoshitaka; Rakugi, Hiromi

    2014-10-01

    Metabolic acidosis, a common complication of CKD, causes mitochondrial stress by undefined mechanisms. Selective autophagy of impaired mitochondria, called mitophagy, contributes toward maintaining cellular homeostasis in various settings. We hypothesized that mitophagy is involved in proximal tubular cell adaptations to chronic metabolic acidosis. In transgenic mice expressing green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 (GFP-LC3), NH4Cl loading increased the number of GFP puncta exclusively in the proximal tubule. In vitro, culture in acidic medium produced similar results in proximal tubular cell lines stably expressing GFP-LC3 and facilitated the degradation of SQSTM1/p62 in wild-type cells, indicating enhanced autophagic flux. Upon acid loading, proximal tubule-specific autophagy-deficient (Atg5-deficient) mice displayed significantly reduced ammonium production and severe metabolic acidosis compared with wild-type mice. In vitro and in vivo, acid loading caused Atg5-deficient proximal tubular cells to exhibit reduced mitochondrial respiratory chain activity, reduced mitochondrial membrane potential, and fragmented morphology with marked swelling in mitochondria. GFP-LC3-tagged autophagosomes colocalized with ubiquitinated mitochondria in proximal tubular cells cultured in acidic medium, suggesting that metabolic acidosis induces mitophagy. Furthermore, restoration of Atg5-intact nuclei in Atg5-deficient proximal tubular cells increased mitochondrial membrane potential and ammoniagenesis. In conclusion, metabolic acidosis induces autophagy in proximal tubular cells, which is indispensable for maintaining proper mitochondrial functions including ammoniagenesis, and thus for adapted urinary acid excretion. Our results provide a rationale for the beneficial effect of alkali supplementation in CKD, a condition in which autophagy may be reduced, and suggest a new therapeutic option for acidosis by modulating autophagy. Copyright

  3. Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent

    PubMed Central

    Geng, Ying; Kohli, Latika; Klocke, Barbara J.; Roth, Kevin A.

    2010-01-01

    Glioblastoma (GBM) is a high-grade central nervous system malignancy and despite aggressive treatment strategies, GBM patients have a median survival time of just 1 year. Chloroquine (CQ), an antimalarial lysosomotropic agent, has been identified as a potential adjuvant in the treatment regimen of GBMs. However, the mechanism of CQ-induced tumor cell death is poorly defined. We and others have shown that CQ-mediated cell death may be p53-dependent and at least in part due to the intrinsic apoptotic death pathway. Here, we investigated the effects of CQ on 5 established human GBM lines, differing in their p53 gene status. CQ was found to induce a concentration-dependent death in each of these cell lines. Although CQ treatment increased caspase-3–like enzymatic activity in all 5 cell lines, a broad-spectrum caspase inhibitor did not significantly attenuate death. Moreover, CQ caused an accumulation of autophagic vacuoles in all cell lines and was found to affect the levels and subcellular distribution of cathepsin D, suggesting that altered lysosomal function may also play a role in CQ-induced cell death. Thus, CQ can induce p53-independent death in gliomas that do not require caspase-mediated apoptosis. To potentially identify more potent chemotherapeutics, various CQ derivatives and lysosomotropic compounds were tested on the GBM cells. Quinacrine and mefloquine were found to be more potent than CQ in killing GBM cells in vitro and given their superior blood–brain barrier penetration compared with CQ may prove more efficacious as chemotherapeutic agents for GBM patients. PMID:20406898

  4. A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects

    PubMed Central

    Schaffner, Adam; Fedick, Anastasia; Kaye, Lauren E.; Liao, Jun; Yachelevich, Naomi; Chu, Mary-Lynn; Boles, Richard G.; Moran, Ellen; Tokita, Mari; Gorman, Elizabeth; Zhang, Wei; Xia, Fan; Leduc, Magalie; Yang, Yaping; Eng, Christine; Wong, Lee-Jun; Schiffmann, Raphael; Diaz, George A.; Kornreich, Ruth; Thummel, Ryan; Wasserstein, Melissa; Yue, Zhenyu; Edelmann, Lisa

    2016-01-01

    Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway. PMID:27120463

  5. Autophagic Clearance of Mitochondria in the Kidney Copes with Metabolic Acidosis

    PubMed Central

    Namba, Tomoko; Takabatake, Yoshitsugu; Kimura, Tomonori; Takahashi, Atsushi; Yamamoto, Takeshi; Matsuda, Jun; Kitamura, Harumi; Niimura, Fumio; Matsusaka, Taiji; Iwatani, Hirotsugu; Matsui, Isao; Kaimori, Junya; Kioka, Hidetaka; Rakugi, Hiromi

    2014-01-01

    Metabolic acidosis, a common complication of CKD, causes mitochondrial stress by undefined mechanisms. Selective autophagy of impaired mitochondria, called mitophagy, contributes toward maintaining cellular homeostasis in various settings. We hypothesized that mitophagy is involved in proximal tubular cell adaptations to chronic metabolic acidosis. In transgenic mice expressing green fluorescent protein–tagged microtubule-associated protein 1 light chain 3 (GFP-LC3), NH4Cl loading increased the number of GFP puncta exclusively in the proximal tubule. In vitro, culture in acidic medium produced similar results in proximal tubular cell lines stably expressing GFP-LC3 and facilitated the degradation of SQSTM1/p62 in wild-type cells, indicating enhanced autophagic flux. Upon acid loading, proximal tubule–specific autophagy-deficient (Atg5-deficient) mice displayed significantly reduced ammonium production and severe metabolic acidosis compared with wild-type mice. In vitro and in vivo, acid loading caused Atg5-deficient proximal tubular cells to exhibit reduced mitochondrial respiratory chain activity, reduced mitochondrial membrane potential, and fragmented morphology with marked swelling in mitochondria. GFP-LC3–tagged autophagosomes colocalized with ubiquitinated mitochondria in proximal tubular cells cultured in acidic medium, suggesting that metabolic acidosis induces mitophagy. Furthermore, restoration of Atg5-intact nuclei in Atg5-deficient proximal tubular cells increased mitochondrial membrane potential and ammoniagenesis. In conclusion, metabolic acidosis induces autophagy in proximal tubular cells, which is indispensable for maintaining proper mitochondrial functions including ammoniagenesis, and thus for adapted urinary acid excretion. Our results provide a rationale for the beneficial effect of alkali supplementation in CKD, a condition in which autophagy may be reduced, and suggest a new therapeutic option for acidosis by modulating autophagy. PMID

  6. Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia

    PubMed Central

    Chen, Yu-Jen; Fang, Li-Wen; Su, Wen-Chi; Hsu, Wen-Yi; Yang, Kai-Chien; Huang, Huey-Lan

    2016-01-01

    Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her) superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL) cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA) partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of macrophagic differentiation in AML U937 cells by lapatinib. We also noted the synergistic effects of the use of lapatinib and cytotoxic drugs in U937 leukemia cells. These results indicate that lapatinib may have potential for development as a novel antileukemia agent. PMID:27499639

  7. A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects.

    PubMed

    Zhang, Jinglan; Lachance, Véronik; Schaffner, Adam; Li, Xianting; Fedick, Anastasia; Kaye, Lauren E; Liao, Jun; Rosenfeld, Jill; Yachelevich, Naomi; Chu, Mary-Lynn; Mitchell, Wendy G; Boles, Richard G; Moran, Ellen; Tokita, Mari; Gorman, Elizabeth; Bagley, Kaytee; Zhang, Wei; Xia, Fan; Leduc, Magalie; Yang, Yaping; Eng, Christine; Wong, Lee-Jun; Schiffmann, Raphael; Diaz, George A; Kornreich, Ruth; Thummel, Ryan; Wasserstein, Melissa; Yue, Zhenyu; Edelmann, Lisa

    2016-04-01

    Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.

  8. Disruption of the vacuolar-type H(+)-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes.

    PubMed

    Kissing, Sandra; Rudnik, Sönke; Damme, Markus; Lüllmann-Rauch, Renate; Ichihara, Atsuhiro; Kornak, Uwe; Eskelinen, Eeva-Liisa; Jabs, Sabrina; Heeren, Jörg; De Brabander, Jef K; Haas, Albert; Saftig, Paul

    2017-04-03

    The vacuolar-type H(+)-translocating ATPase (v-H(+)-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H(+)-ATPase and MTORC1, we destablilized v-H(+)-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H(+)-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H(+)-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H(+)-ATPase-mediated regulation of MTORC1.

  9. Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

    PubMed Central

    Jiang, H; Sun, J; Xu, Q; Liu, Y; Wei, J; Young, C Y F; Yuan, H; Lou, H

    2013-01-01

    We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome β5 and β1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF2α), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF2α and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as

  10. 46 CFR 58.01-40 - Machinery, angles of inclination.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-40 Machinery, angles of inclination. (a... angle of list up to and including 15°, and when the vessel is inclined under dynamic conditions (rolling...

  11. 46 CFR 58.01-40 - Machinery, angles of inclination.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-40 Machinery, angles of inclination. (a... angle of list up to and including 15°, and when the vessel is inclined under dynamic conditions (rolling...

  12. 54. West emergency brake in the south machinery room (interior ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    54. West emergency brake in the south machinery room (interior of both machinery rooms is identical). Facing west. - Henry Ford Bridge, Spanning Cerritos Channel, Los Angeles-Long Beach Harbor, Los Angeles, Los Angeles County, CA

  13. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... AND AUXILIARY MACHINERY AND RELATED SYSTEMS Refrigeration Machinery § 58.20-15 Installation of... refrigeration compressor spaces shall be effectively ventilated and drained and shall be separated from...

  14. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... AND AUXILIARY MACHINERY AND RELATED SYSTEMS Refrigeration Machinery § 58.20-15 Installation of... refrigeration compressor spaces shall be effectively ventilated and drained and shall be separated from...

  15. 52. Detail of electrical contacts in the south machinery room ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    52. Detail of electrical contacts in the south machinery room (interior of both machinery rooms is identical). Facing south. - Henry Ford Bridge, Spanning Cerritos Channel, Los Angeles-Long Beach Harbor, Los Angeles, Los Angeles County, CA

  16. 4. MACHINERY SHED AND STORAGE ROOM ADDITION, SOUTH AND WEST ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. MACHINERY SHED AND STORAGE ROOM ADDITION, SOUTH AND WEST WALL LOOKING NORTHEAST SEED STORAGE BUILDING (1963) BEHIND - Tucson Plant Material Center, Machinery Shed, 3241 North Romero Road, Tucson, Pima County, AZ

  17. 51. Electrical contacts and relays in the south machinery room ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    51. Electrical contacts and relays in the south machinery room (interior of both machinery rooms is identical). Facing south. - Henry Ford Bridge, Spanning Cerritos Channel, Los Angeles-Long Beach Harbor, Los Angeles, Los Angeles County, CA

  18. Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins

    PubMed Central

    Wu, An-Guo; Kam-Wai Wong, Vincent; Zeng, Wu; Liu, Liang; Yuen-Kwan Law, Betty

    2015-01-01

    With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae (RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-flight mass spectrometry {UHPLC-(Q)TOF-MS}. By exhibiting more potent autophagic effect in cells, the active fraction enhances the clearance of mutant huntingtin, and reduces protein level and aggregation of α-synuclein in a higher extent when compared with onjisaponin B. Here, we have reported for the first time the new application of cell-based CMC and UHPLC-(Q)TOF-MS analysis in identifying new autophagy inducers with neuroprotective effects from Chinese medicinal herb. This result has provided novel insights into the possible pharmacological actions of the active components present in the newly identified active fraction of RP, which may help to improve the efficacy of the traditional way of prescribing RP, and also provide new standard for the quality control of decoction of RP or its medicinal products in the future. PMID:26598009

  19. Autophagic kinases SmVPS34 and SmVPS15 are required for viability in the filamentous ascomycete Sordaria macrospora.

    PubMed

    Voigt, Oliver; Herzog, Britta; Jakobshagen, Antonia; Pöggeler, Stefanie

    2014-01-01

    Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae.

  20. 29 CFR 1910.214 - Cooperage machinery. [Reserved

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 5 2014-07-01 2014-07-01 false Cooperage machinery. 1910.214 Section 1910.214 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.214 Cooperage machinery....

  1. 46 CFR 58.01-25 - Means of stopping machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-25 Means of stopping machinery. Machinery driving forced-draft and induced-draft fans, fuel-oil transfer pumps, fuel-oil unit and service pumps, and... space concerned so that the fans or pumps may be stopped in case of fire in the compartment in which...

  2. 29 CFR 1915.165 - Ship's deck machinery.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Ship's deck machinery. 1915.165 Section 1915.165 Labor... (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Ship's Machinery and Piping Systems § 1915.165 Ship's deck machinery. (a) Before work is performed on the anchor windlass or any of...

  3. 29 CFR 1915.165 - Ship's deck machinery.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 7 2014-07-01 2014-07-01 false Ship's deck machinery. 1915.165 Section 1915.165 Labor... (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Ship's Machinery and Piping Systems § 1915.165 Ship's deck machinery. (a) Before work is performed on the anchor windlass or any of...

  4. 46 CFR 185.352 - Ventilation of gasoline machinery spaces.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Ventilation of gasoline machinery spaces. 185.352... (UNDER 100 GROSS TONS) OPERATIONS Miscellaneous Operating Requirements § 185.352 Ventilation of gasoline machinery spaces. The mechanical exhaust for the ventilation of a gasoline machinery space, required by...

  5. 46 CFR 185.352 - Ventilation of gasoline machinery spaces.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Ventilation of gasoline machinery spaces. 185.352... (UNDER 100 GROSS TONS) OPERATIONS Miscellaneous Operating Requirements § 185.352 Ventilation of gasoline machinery spaces. The mechanical exhaust for the ventilation of a gasoline machinery space, required...

  6. 46 CFR 185.352 - Ventilation of gasoline machinery spaces.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Ventilation of gasoline machinery spaces. 185.352... (UNDER 100 GROSS TONS) OPERATIONS Miscellaneous Operating Requirements § 185.352 Ventilation of gasoline machinery spaces. The mechanical exhaust for the ventilation of a gasoline machinery space, required...

  7. 46 CFR 97.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Accidents to machinery. 97.30-5 Section 97.30-5 Shipping... Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  8. 46 CFR 97.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Accidents to machinery. 97.30-5 Section 97.30-5 Shipping... Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  9. 46 CFR 196.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Accidents to machinery. 196.30-5 Section 196.30-5... Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  10. 46 CFR 196.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Accidents to machinery. 196.30-5 Section 196.30-5... Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  11. 46 CFR 196.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Accidents to machinery. 196.30-5 Section 196.30-5... Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  12. 46 CFR 97.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Accidents to machinery. 97.30-5 Section 97.30-5 Shipping... Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  13. 46 CFR 97.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Accidents to machinery. 97.30-5 Section 97.30-5 Shipping... Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  14. 46 CFR 196.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Accidents to machinery. 196.30-5 Section 196.30-5... Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  15. 46 CFR 196.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Accidents to machinery. 196.30-5 Section 196.30-5... Reports of Accidents, Repairs, and Unsafe Equipment § 196.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  16. 46 CFR 97.30-5 - Accidents to machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Accidents to machinery. 97.30-5 Section 97.30-5 Shipping... Reports of Accidents, Repairs, and Unsafe Equipment § 97.30-5 Accidents to machinery. (a) In the event of an accident to a boiler, unfired pressure vessel, or machinery tending to render the further use...

  17. 46 CFR 111.103-3 - Machinery space ventilation.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Machinery space ventilation. 111.103-3 Section 111.103-3...-GENERAL REQUIREMENTS Remote Stopping Systems § 111.103-3 Machinery space ventilation. (a) Each machinery space ventilation system must have two controls to stop the ventilation, one of which may be the supply...

  18. 46 CFR 171.095 - Machinery space bulkhead.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Machinery space bulkhead. 171.095 Section 171.095... PERTAINING TO VESSELS CARRYING PASSENGERS Additional Subdivision Requirements § 171.095 Machinery space... transverse watertight bulkheads to separate the machinery space from the remainder of the vessel. All...

  19. 46 CFR 171.095 - Machinery space bulkhead.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Machinery space bulkhead. 171.095 Section 171.095... PERTAINING TO VESSELS CARRYING PASSENGERS Additional Subdivision Requirements § 171.095 Machinery space... transverse watertight bulkheads to separate the machinery space from the remainder of the vessel. All...

  20. 33 CFR 157.39 - Machinery space bilges.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Machinery space bilges. 157.39... Vessel Operation § 157.39 Machinery space bilges. (a) A tank vessel may discharge an oily mixture from a machinery space bilge that is combined with an oil cargo residue if the vessel discharges in compliance with...

  1. 46 CFR 171.095 - Machinery space bulkhead.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Machinery space bulkhead. 171.095 Section 171.095... PERTAINING TO VESSELS CARRYING PASSENGERS Additional Subdivision Requirements § 171.095 Machinery space... transverse watertight bulkheads to separate the machinery space from the remainder of the vessel. All...

  2. 33 CFR 157.39 - Machinery space bilges.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Machinery space bilges. 157.39... Vessel Operation § 157.39 Machinery space bilges. (a) A tank vessel may discharge an oily mixture from a machinery space bilge that is combined with an oil cargo residue if the vessel discharges in compliance with...

  3. 46 CFR 111.103-3 - Machinery space ventilation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Machinery space ventilation. 111.103-3 Section 111.103-3...-GENERAL REQUIREMENTS Remote Stopping Systems § 111.103-3 Machinery space ventilation. (a) Each machinery space ventilation system must have two controls to stop the ventilation, one of which may be the supply...

  4. 33 CFR 157.39 - Machinery space bilges.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Machinery space bilges. 157.39... Vessel Operation § 157.39 Machinery space bilges. (a) A tank vessel may discharge an oily mixture from a machinery space bilge that is combined with an oil cargo residue if the vessel discharges in compliance with...

  5. 46 CFR 111.103-3 - Machinery space ventilation.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Machinery space ventilation. 111.103-3 Section 111.103-3...-GENERAL REQUIREMENTS Remote Stopping Systems § 111.103-3 Machinery space ventilation. (a) Each machinery space ventilation system must have two controls to stop the ventilation, one of which may be the supply...

  6. 46 CFR 185.352 - Ventilation of gasoline machinery spaces.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Ventilation of gasoline machinery spaces. 185.352... machinery spaces. The mechanical exhaust for the ventilation of a gasoline machinery space, required by... sufficient to insure at least one complete change of air in the space served. ...

  7. 29 CFR 1915.165 - Ship's deck machinery.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 7 2012-07-01 2012-07-01 false Ship's deck machinery. 1915.165 Section 1915.165 Labor... (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Ship's Machinery and Piping Systems § 1915.165 Ship's deck machinery. (a) Before work is performed on the anchor windlass or any of...

  8. 29 CFR 1915.165 - Ship's deck machinery.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 7 2013-07-01 2013-07-01 false Ship's deck machinery. 1915.165 Section 1915.165 Labor... (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Ship's Machinery and Piping Systems § 1915.165 Ship's deck machinery. (a) Before work is performed on the anchor windlass or any of...

  9. 30 CFR 57.14205 - Machinery, equipment, and tools.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery, equipment, and tools. 57.14205... and Equipment Safety Practices and Operational Procedures § 57.14205 Machinery, equipment, and tools. Machinery, equipment, and tools shall not be used beyond the design capacity intended by the...

  10. 30 CFR 56.14205 - Machinery, equipment, and tools.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Machinery, equipment, and tools. 56.14205... Equipment Safety Practices and Operational Procedures § 56.14205 Machinery, equipment, and tools. Machinery, equipment, and tools shall not be used beyond the design capacity intended by the manufacturer where...

  11. 30 CFR 56.14205 - Machinery, equipment, and tools.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Machinery, equipment, and tools. 56.14205... Equipment Safety Practices and Operational Procedures § 56.14205 Machinery, equipment, and tools. Machinery, equipment, and tools shall not be used beyond the design capacity intended by the manufacturer where...

  12. 30 CFR 57.14205 - Machinery, equipment, and tools.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Machinery, equipment, and tools. 57.14205... and Equipment Safety Practices and Operational Procedures § 57.14205 Machinery, equipment, and tools. Machinery, equipment, and tools shall not be used beyond the design capacity intended by the...

  13. 46 CFR 109.205 - Inspection of boilers and machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Inspection of boilers and machinery. 109.205 Section 109... OPERATIONS Tests, Drills, and Inspections § 109.205 Inspection of boilers and machinery. The chief engineer or engineer in charge, before he assumes charge of the boilers and machinery of a unit shall inspect...

  14. 46 CFR 109.205 - Inspection of boilers and machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Inspection of boilers and machinery. 109.205 Section 109... OPERATIONS Tests, Drills, and Inspections § 109.205 Inspection of boilers and machinery. The chief engineer or engineer in charge, before he assumes charge of the boilers and machinery of a unit shall inspect...

  15. 46 CFR 109.205 - Inspection of boilers and machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Inspection of boilers and machinery. 109.205 Section 109... OPERATIONS Tests, Drills, and Inspections § 109.205 Inspection of boilers and machinery. The chief engineer or engineer in charge, before he assumes charge of the boilers and machinery of a unit shall inspect...

  16. 46 CFR 109.205 - Inspection of boilers and machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Inspection of boilers and machinery. 109.205 Section 109... OPERATIONS Tests, Drills, and Inspections § 109.205 Inspection of boilers and machinery. The chief engineer or engineer in charge, before he assumes charge of the boilers and machinery of a unit shall inspect...

  17. 29 CFR 1915.165 - Ship's deck machinery.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 7 2011-07-01 2011-07-01 false Ship's deck machinery. 1915.165 Section 1915.165 Labor... (CONTINUED) OCCUPATIONAL SAFETY AND HEALTH STANDARDS FOR SHIPYARD EMPLOYMENT Ship's Machinery and Piping Systems § 1915.165 Ship's deck machinery. (a) Before work is performed on the anchor windlass or any...

  18. 46 CFR 252.33 - Hull and machinery insurance.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Hull and machinery insurance. 252.33 Section 252.33... Subsidy Rates § 252.33 Hull and machinery insurance. (a) Subsidy items. The fair and reasonable net premium costs (including stamp taxes) of hull and machinery, increased value, excess general average...

  19. 46 CFR 282.23 - Hull and machinery insurance.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Hull and machinery insurance. 282.23 Section 282.23... COMMERCE OF THE UNITED STATES Calculation of Subsidy Rates § 282.23 Hull and machinery insurance. (a) Subsidy items. The fair and reasonable net premium costs (including stamp taxes) of hull and machinery...

  20. 29 CFR 1910.214 - Cooperage machinery. [Reserved

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 5 2013-07-01 2013-07-01 false Cooperage machinery. 1910.214 Section 1910.214 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.214 Cooperage machinery....

  1. 33 CFR 157.39 - Machinery space bilges.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 2 2011-07-01 2011-07-01 false Machinery space bilges. 157.39... Vessel Operation § 157.39 Machinery space bilges. (a) A tank vessel may discharge an oily mixture from a machinery space bilge that is combined with an oil cargo residue if the vessel discharges in compliance...

  2. 46 CFR 111.103-3 - Machinery space ventilation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Machinery space ventilation. 111.103-3 Section 111.103-3...-GENERAL REQUIREMENTS Remote Stopping Systems § 111.103-3 Machinery space ventilation. (a) Each machinery space ventilation system must have two controls to stop the ventilation, one of which may be the...

  3. 46 CFR 171.095 - Machinery space bulkhead.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Machinery space bulkhead. 171.095 Section 171.095... PERTAINING TO VESSELS CARRYING PASSENGERS Additional Subdivision Requirements § 171.095 Machinery space... transverse watertight bulkheads to separate the machinery space from the remainder of the vessel....

  4. 46 CFR 111.103-3 - Machinery space ventilation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Machinery space ventilation. 111.103-3 Section 111.103-3...-GENERAL REQUIREMENTS Remote Stopping Systems § 111.103-3 Machinery space ventilation. (a) Each machinery space ventilation system must have two controls to stop the ventilation, one of which may be the...

  5. 33 CFR 157.39 - Machinery space bilges.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Machinery space bilges. 157.39... Vessel Operation § 157.39 Machinery space bilges. (a) A tank vessel may discharge an oily mixture from a machinery space bilge that is combined with an oil cargo residue if the vessel discharges in compliance...

  6. 46 CFR 171.095 - Machinery space bulkhead.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Machinery space bulkhead. 171.095 Section 171.095... PERTAINING TO VESSELS CARRYING PASSENGERS Additional Subdivision Requirements § 171.095 Machinery space... transverse watertight bulkheads to separate the machinery space from the remainder of the vessel....

  7. 46 CFR 185.352 - Ventilation of gasoline machinery spaces.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Ventilation of gasoline machinery spaces. 185.352... machinery spaces. The mechanical exhaust for the ventilation of a gasoline machinery space, required by... sufficient to insure at least one complete change of air in the space served....

  8. 29 CFR 1910.215 - Abrasive wheel machinery.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 5 2012-07-01 2012-07-01 false Abrasive wheel machinery. 1910.215 Section 1910.215 Labor... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.215 Abrasive wheel machinery. (a) General requirements—(1) Machine guarding. Abrasive wheels shall be used only on machines provided...

  9. 29 CFR 1910.215 - Abrasive wheel machinery.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 5 2014-07-01 2014-07-01 false Abrasive wheel machinery. 1910.215 Section 1910.215 Labor... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.215 Abrasive wheel machinery. (a) General requirements—(1) Machine guarding. Abrasive wheels shall be used only on machines provided...

  10. 29 CFR 1910.215 - Abrasive wheel machinery.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 5 2013-07-01 2013-07-01 false Abrasive wheel machinery. 1910.215 Section 1910.215 Labor... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.215 Abrasive wheel machinery. (a) General requirements—(1) Machine guarding. Abrasive wheels shall be used only on machines provided...

  11. 29 CFR 1910.215 - Abrasive wheel machinery.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 5 2011-07-01 2011-07-01 false Abrasive wheel machinery. 1910.215 Section 1910.215 Labor... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.215 Abrasive wheel machinery. (a) General requirements—(1) Machine guarding. Abrasive wheels shall be used only on machines provided...

  12. 29 CFR 1910.215 - Abrasive wheel machinery.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 5 2010-07-01 2010-07-01 false Abrasive wheel machinery. 1910.215 Section 1910.215 Labor... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.215 Abrasive wheel machinery. (a) General requirements—(1) Machine guarding. Abrasive wheels shall be used only on machines provided...

  13. 46 CFR 58.01-20 - Machinery guards.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Machinery guards. 58.01-20 Section 58.01-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-20 Machinery guards. Gears, couplings, flywheels and...

  14. 46 CFR 58.01-20 - Machinery guards.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Machinery guards. 58.01-20 Section 58.01-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-20 Machinery guards. Gears, couplings, flywheels and...

  15. 46 CFR 58.01-20 - Machinery guards.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Machinery guards. 58.01-20 Section 58.01-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-20 Machinery guards. Gears, couplings, flywheels and...

  16. 46 CFR 58.01-20 - Machinery guards.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Machinery guards. 58.01-20 Section 58.01-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-20 Machinery guards. Gears, couplings, flywheels and...

  17. 46 CFR 58.01-20 - Machinery guards.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Machinery guards. 58.01-20 Section 58.01-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS General Requirements § 58.01-20 Machinery guards. Gears, couplings, flywheels and...

  18. 29 CFR 1910.214 - Cooperage machinery. [Reserved

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 5 2011-07-01 2011-07-01 false Cooperage machinery. 1910.214 Section 1910.214 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.214 Cooperage machinery....

  19. Opportunities for on-line machinery sensing

    SciTech Connect

    Murray, S.F.; Ling, F.F.

    1996-11-01

    While most engineers were concerned with progress in the development of robots and robotic devices, there has been a revolution going on in sensor technology. Micro-sensor devices, so small that they could be encapsulated easily, and so light they could resist high g forces and impacts, are being developed for a variety of applications especially aerospace, automotive and consumer durables. In the field of tribology, the ability of sensors to gather and process information promises real advances in monitoring and sensing potential problems with machinery and with tools. Yet, there are many critical applications where no failure detection devices are being used or even considered. There is a need for more training and better dissemination of know-how for the various types of sensors. This paper is a review of the recent literature on in-situ micro-sensors that might have relevance to machinery condition sensing.

  20. Thermal spray manual for machinery components

    SciTech Connect

    Travis, R.; Ginther, C.; Herbstritt, M.; Herbstritt, J.

    1995-12-31

    The Thermal Spray Manual For Machinery Components is a National Shipbuilding Research (SP-7) Project. This Manual is being developed by Puget Sound Naval Shipyard with the help of other government thermal spray facilities and SP-7 panel members. The purpose of the manual is to provide marine repair facilities with a ``how to do`` document that will be ``user friendly`` and known to be technically sound through production experience. The manual`s intent is to give marine repair facilities the ability to maximize the thermal spray process as a repair method for machinery components and to give these facilities guidelines on how to become qualified to receive certification that they meet the requirements of Military Standard 1687A.

  1. Laser-optic instruments improve machinery alignment

    SciTech Connect

    Bloch, H.P.

    1987-10-12

    Laser-optic alignment systems are fast becoming cost-effective devices that improve the accuracy and speed of machinery shaft alignment. Because of the difficulty, if not impossibility, of aligning operating machinery, cold alignment specifications must be determined to compensate for thermal growth so that the shaft alignment remains within tolerances when the machine reaches normal operating temperature. Some methods for accomplishing this are reviewed here. Three years' field experience with laser-optic alignment systems shows that many of these limitations can be eliminated, resulting in a more accurate alignment in less time. Some actual field alignments are given as examples of the improvement achieved by the use of laser equipment, and a procedure is given that shows how the laser-optic system may be used to determine running alignment changes caused by thermal growth.

  2. Machinery vibration: Origins, impressions and cures

    SciTech Connect

    Haq, I. )

    1995-01-01

    The current trend toward high performance (speed, power, flow, etc.) and low eight are causing new machinery dynamics problems. Vibration diagnostics engineering of rotor-bearing-casing systems must consider both internal and external influences to effectively predict and diagnose these problems. It is assumed that machinery vibration data are free from ambiguity, error, conform to a standard and clearly identify the physical cause(s) responsible for vibration. Rotor vibration due to internal forces are described: unbalance force characteristics; response characteristics; and rules of rotor fundamental response. Rotor vibration due to external forces include: rotating aerodynamic stall; oil whirl; oil whip; structural resonance; vane/blade passing vibration; misalignment; rotor rubbing; gear mesh vibrations; and shaft crack. These are also discussed.

  3. GPCR signalling to the translation machinery.

    PubMed

    Musnier, Astrid; Blanchot, Benoît; Reiter, Eric; Crépieux, Pascale

    2010-05-01

    G protein-coupled receptors (GPCRs) are involved in most physiological processes, many of them being engaged in fully differentiated cells. These receptors couple to transducers of their own, primarily G proteins and beta-arrestins, which launch intracellular signalling cascades. Some of these signalling events regulate the translational machinery to fine-tune general cell metabolism or to alter protein expression pattern. Though extensively documented for tyrosine kinase receptors, translational regulation by GPCRs is still poorly appreciated. The objective of this review paper is to address the following questions: i) is there a "GPCR signature" impacting on the translational machinery, and ultimately on the type of mRNA translated? ii) are the regulatory networks involved similar as those utilized by tyrosine kinase receptors? In particular, we will discuss the specific features of translational control mediated by GPCRs and highlight the intrinsic properties of GPCRs these mechanisms could rely on.

  4. Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

    2014-12-01

    Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung

  5. Materials Research for Superconducting Machinery-IV

    DTIC Science & Technology

    1975-09-01

    LABORATORIES Preparation of a Handbook on Mechanical , Thermal, Electrical, and Magnetic properties of Materials for Superconducting Machinery. Eldridge, E. A...Properties of Structural Materials Program Area Mechanical Properties 1. Fracture and Fitigue a. Materials Group Second Year Program (FY 75...crack growth rate tests from 4-300 K on structural alloys, and the effects of stress level and frequency. Mechanical , magnetic, electrical loss

  6. The exportomer: the peroxisomal receptor export machinery.

    PubMed

    Platta, Harald W; Hagen, Stefanie; Erdmann, Ralf

    2013-04-01

    Peroxisomes constitute a dynamic compartment of almost all eukaryotic cells. Depending on environmental changes and cellular demands peroxisomes can acquire diverse metabolic roles. The compartmentalization of peroxisomal matrix enzymes is a prerequisite to carry out their physiologic function. The matrix proteins are synthesized on free ribosomes in the cytosol and are ferried to the peroxisomal membrane by specific soluble receptors. Subsequent to cargo release into the peroxisomal matrix, the receptors are exported back to the cytosol to facilitate further rounds of matrix protein import. This dislocation step is accomplished by a remarkable machinery, which comprises enzymes required for the ubiquitination as well as the ATP-dependent extraction of the receptor from the membrane. Interestingly, receptor ubiquitination and dislocation are the only known energy-dependent steps in the peroxisomal matrix protein import process. The current view is that the export machinery of the receptors might function as molecular motor not only in the dislocation of the receptors but also in the import step of peroxisomal matrix protein by coupling ATP-dependent removal of the peroxisomal import receptor with cargo translocation into the organelle. In this review we will focus on the architecture and function of the peroxisomal receptor export machinery, the peroxisomal exportomer.

  7. Machinery Vibration Monitoring Program at the Savannah River Site

    SciTech Connect

    Potvin, M.M.

    1990-01-01

    The Reactor Maintenance's Machinery Vibration Monitoring Program (MVMP) plays an essential role in ensuring the safe operation of the three Production Reactors at the Westinghouse Savannah River Company (WRSC) Savannah River Site (SRS). This program has increased machinery availability and reduced maintenance cost by the early detection and determination of machinery problems. This paper presents the Reactor Maintenance's Machinery Vibration Monitoring Program, which has been documented based on Electric Power Research Institute's (EPRI) NP-5311, Utility Machinery Monitoring Guide, and some examples of the successes that it has enjoyed.

  8. Low Back Pain in Port Machinery Operators

    NASA Astrophysics Data System (ADS)

    BOVENZI, M.; PINTO, I.; STACCHINI, N.

    2002-05-01

    The occurrence of several types of low back pain (LBP) was investigated by a standardized questionnaire in a group of 219 port machinery operators exposed to whole-body vibration (WBV) and postural load and in a control group of 85 maintenance workers employed at the same transport company. The group of port machinery operators included 85 straddle carrier drivers, 88 fork-lift truck drivers, and 46 crane operators. The vector sum of the frequency-weighted r.m.s. acceleration of vibration measured on the seatpan of port vehicles and machines averaged 0·90 m/s2 for fork-lift trucks, 0·48 m/s2 for straddle carriers, 0·53 m/s2 for mobile cranes, and 0·22 m/s2 for overhead cranes. The 12-month prevalence of low back symptoms (LBP, sciatic pain, treated LBP, sick leave due to LBP) was significantly greater in the fork-lift truck drivers than in the controls and the other two groups of port machinery operators. After adjusting for potential confounders, the prevalence of low back symptoms was found to increase with the increase of WBV exposure expressed as duration of exposure (driving years), equivalent vibration magnitude (m/s2), or cumulative vibration exposure (yr m2/s4). An excess risk for lumbar disc herniation was observed in the port machinery operators with prolonged driving experience. In both the controls and the port machinery operators, low back complaints were strongly associated with perceived postural load assessed in terms of frequency and/or duration of awkward postures at work. Multivariate analysis showed that vibration exposure and postural load were independent predictors of LBP. Even though the cross-sectional design of the present study does not permit firm conclusions on the relationship between WBV exposure and low back disorders, the findings of this investigation provide additional epidemiological evidence that seated WBV exposure combined with non-neutral trunk postures, as while driving, is associated with an increased risk of long

  9. Turnover of Lipidated LC3 and Autophagic Cargoes in Mammalian Cells.

    PubMed

    Rodríguez-Arribas, M; Yakhine-Diop, S M S; González-Polo, R A; Niso-Santano, M; Fuentes, J M

    2017-01-01

    Macroautophagy (usually referred to as autophagy) is the most important degradation system in mammalian cells. It is responsible for the elimination of protein aggregates, organelles, and other cellular content. During autophagy, these materials (i.e., cargo) must be engulfed by a double-membrane structure called an autophagosome, which delivers the cargo to the lysosome to complete its degradation. Autophagy is a very dynamic pathway called autophagic flux. The process involves all the steps that are implicated in cargo degradation from autophagosome formation. There are several techniques to monitor autophagic flux. Among them, the method most used experimentally to assess autophagy is the detection of LC3 protein processing and p62 degradation by Western blotting. In this chapter, we provide a detailed and straightforward protocol for this purpose in cultured mammalian cells, including a brief set of notes concerning problems associated with the Western-blotting detection of LC3 and p62.

  10. Could plant lectins become promising anti-tumour drugs for causing autophagic cell death?

    PubMed

    Liu, Z; Luo, Y; Zhou, T-T; Zhang, W-Z

    2013-10-01

    Plant lectins, a group of highly diverse carbohydrate-binding proteins of non-immune origin, are ubiquitously distributed through a variety of plant species, and have recently drawn rising attention due to their remarkable ability to kill tumour cells using mechanisms implicated in autophagy. In this review, we provide a brief outline of structures of some representative plant lectins such as concanavalin A, Polygonatum cyrtonema lectin and mistletoe lectins. These can target autophagy by modulating BNIP-3, ROS-p38-p53, Ras-Raf and PI3KCI-Akt pathways, as well as Beclin-1, in many types of cancer cells. In addition, we further discuss how plant lectins are able to kill cancer cells by modulating autophagic death, for therapeutic purposes. Together, these findings provide a comprehensive perspective concerning plant lectins as promising new anti-tumour drugs, with respect to autophagic cell death in future cancer therapeutics.

  11. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease

    PubMed Central

    Filimonenko, Maria; Stuffers, Susanne; Raiborg, Camilla; Yamamoto, Ai; Malerød, Lene; Fisher, Elizabeth M.C.; Isaacs, Adrian; Brech, Andreas; Stenmark, Harald; Simonsen, Anne

    2007-01-01

    The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations. PMID:17984323

  12. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease.

    PubMed

    Filimonenko, Maria; Stuffers, Susanne; Raiborg, Camilla; Yamamoto, Ai; Malerød, Lene; Fisher, Elizabeth M C; Isaacs, Adrian; Brech, Andreas; Stenmark, Harald; Simonsen, Anne

    2007-11-05

    The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.

  13. Induction of Cell Death by Betulinic Acid through Induction of Apoptosis and Inhibition of Autophagic Flux in Microglia BV-2 Cells.

    PubMed

    Seo, Jeongbin; Jung, Juneyoung; Jang, Dae Sik; Kim, Joungmok; Kim, Jeong Hee

    2017-03-10

    Betulinic acid (BA), a natural pentacyclic triterpene found in many medicinal plants is known to have various biological activity including tumor suppression and anti-inflammatory effects. In this study, the cell-death induction effect of BA was investigated in BV-2 microglia cells. BA was cytotoxic to BV-2 cells with IC₅₀ of approximately 2.0 μM. Treatment of BA resulted in a dosedependent chromosomal DNA degradation, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that BA-treated BV-2 cells showed hypodiploid DNA content. BA treatment triggered apoptosis by decreasing Bcl-2 levels, activation of capase-3 protease and cleavage of PARP. In addition, BA treatment induced the accumulation of p62 and the increase in conversion of LC3-I to LC3-II, which are important autophagic flux monitoring markers. The increase in LC3-II indicates that BA treatment induced autophagosome formation, however, accumulation of p62 represents that the downstream autophagy pathway is blocked. It is demonstrated that BA induced cell death of BV-2 cells by inducing apoptosis and inhibiting autophagic flux. These data may provide important new information towards understanding the mechanisms by which BA induce cell death in microglia BV-2 cells.

  14. cAMP and EPAC Are Key Players in the Regulation of the Signal Transduction Pathway Involved in the α-Hemolysin Autophagic Response

    PubMed Central

    Mestre, María Belén; Colombo, María Isabel

    2012-01-01

    Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla) is the S. aureus–secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus–containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell. PMID:22654658

  15. Nucleolus-like compartmentalization of the transcription machinery in fast-growing bacterial cells.

    PubMed

    Jin, Ding Jun; Mata Martin, Carmen; Sun, Zhe; Cagliero, Cedric; Zhou, Yan Ning

    2017-02-01

    We have learned a great deal about RNA polymerase (RNA Pol), transcription factors, and the transcriptional regulation mechanisms in prokaryotes for specific genes, operons, or transcriptomes. However, we have only begun to understand how the transcription machinery is three-dimensionally (3D) organized into bacterial chromosome territories to orchestrate the transcription process and to maintain harmony with the replication machinery in the cell. Much progress has been made recently in our understanding of the spatial organization of the transcription machinery in fast-growing Escherichia coli cells using state-of-the-art superresolution imaging techniques. Co-imaging of RNA polymerase (RNA Pol) with DNA and transcription elongation factors involved in ribosomal RNA (rRNA) synthesis, and ribosome biogenesis has revealed similarities between bacteria and eukaryotes in the spatial organization of the transcription machinery for growth genes, most of which are rRNA genes. Evidence supports the notion that RNA Pol molecules are concentrated, forming foci at the clustering of rRNA operons resembling the eukaryotic nucleolus. RNA Pol foci are proposed to be active transcription factories for both rRNA genes expression and ribosome biogenesis to support maximal growth in optimal growing conditions. Thus, in fast-growing bacterial cells, RNA Pol foci mimic eukaryotic Pol I activity, and transcription factories resemble nucleolus-like compartmentation. In addition, the transcription and replication machineries are mostly segregated in space to avoid the conflict between the two major cellular functions in fast-growing cells.

  16. Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

    PubMed

    Cordani, Marco; Oppici, Elisa; Dando, Ilaria; Butturini, Elena; Dalla Pozza, Elisa; Nadal-Serrano, Mercedes; Oliver, Jordi; Roca, Pilar; Mariotto, Sofia; Cellini, Barbara; Blandino, Giovanni; Palmieri, Marta; Di Agostino, Silvia; Donadelli, Massimo

    2016-08-01

    Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.

  17. Looking at the metabolic consequences of the colchicine-based in vivo autophagic flux assay

    PubMed Central

    Seiliez, Iban; Belghit, Ikram; Gao, Yujie; Skiba-Cassy, Sandrine; Dias, Karine; Cluzeaud, Marianne; Rémond, Didier; Hafnaoui, Nordine; Salin, Bénédicte; Camougrand, Nadine; Panserat, Stéphane

    2016-01-01

    ABSTRACT Monitoring autophagic flux in vivo or in organs remains limited and the ideal methods relative to the techniques possible with cell culture may not exist. Recently, a few papers have demonstrated the feasibility of measuring autophagic flux in vivo by intraperitoneal (IP) injection of pharmacological agents (chloroquine, leupeptin, vinblastine, and colchicine). However, the metabolic consequences of the administration of these drugs remain largely unknown. Here, we report that 0.8 mg/kg/day IP colchicine increased LC3-II protein levels in the liver of fasted trout, supporting the usefulness of this drug for studying autophagic flux in vivo in our model organism. This effect was accompanied by a decrease of plasma glucose concentration associated with a fall in the mRNA levels of gluconeogenesis-related genes. Concurrently, triglycerides and lipid droplets content in the liver increased. In contrast, transcript levels of β-oxidation-related gene Cpt1a dropped significantly. Together, these results match with the reported role of autophagy in the regulation of glucose homeostasis and intracellular lipid stores, and highlight the importance of considering these effects when using colchicine as an in vivo “autophagometer.” PMID:26902586

  18. Use of pHlurorin-mKate2-human LC3 to Monitor Autophagic Responses.

    PubMed

    Tanida, I; Ueno, T; Uchiyama, Y

    2017-01-01

    In this chapter, we introduce the usage of pHluorin-mKate2-human LC3 for monitoring autophagy. Using EGFP and RFP, tandem fluorescent protein-tagged LC3 has been generated for monitoring autophagic structures. A critical point for this purpose is the sensitivity of the green fluorescent protein to acidic pH. A super-ecliptic pHluorin is most sensitive to acidic pH among EGFP, mWasabi, and pHluorin, indicating pHluorin is most suitable for monitoring autophagic structures. During autophagy, green-positive and red-positive fluorescent puncta of pHluorin-mKate2-human LC3 indicate signals of preautophagosomes and autophagosomes. After fusion of autophagosomes with lysosomes to form autolysosomes, green fluorescence of this intraautophagosomal protein is abolished according to acidification of autolysosomes. Therefore, these green-negative and red-positive fluorescent puncta reflect autolysosomes, in which intraluminal proteins are finally degraded by lysosomal proteases. To monitor autophagic flux, the accumulation of its green-negative and red-positive fluorescent puncta is monitored by inhibiting major lysosomal proteases, cathepsins. In addition, a mutant pHluorin-mKate2-human LC3△G is also introduced as a negative control probe.

  19. Memantine Induces NMDAR1-Mediated Autophagic Cell Death in Malignant Glioma Cells

    PubMed Central

    Yoon, Wan-Soo; Yeom, Mi-Young; Kang, Eun-Sun; Chung, Yong-An; Chung, Dong-Sup; Jeun, Sin-Soo

    2017-01-01

    Objective Autophagy is one of the key responses of cells to programmed cell death. Memantine, an approved anti-dementia drug, has an antiproliferative effect on cancer cells but the mechanism is poorly understood. The aim of the present study was to test the possibility of induction of autophagic cell death by memantine in glioma cell lines. Methods Glioma cell lines (T-98 G and U-251 MG) were used for this study. Results The antiproliferative effect of memantine was shown on T-98 G cells, which expressed N-methyl-D-aspartate 1 receptor (NMDAR1). Memantine increased the autophagic-related proteins as the conversion ratio of light chain protein 3-II (LC3-II)-/LC3-I and the expression of beclin-1. Memantine also increased formation of autophagic vacuoles observed under a transmission electron microscope. Transfection of small interfering RNA (siRNA) to knock down NMDAR1 in the glioma cells induced resistance to memantine and decreased the LC3-II/LC3-I ratio in T-98 G cells. Conclusion Our study demonstrates that in glioma cells, memantine inhibits proliferation and induces autophagy mediated by NMDAR1. PMID:28264232

  20. Interaction of Chlamydia trachomatis serovar L2 with the host autophagic pathway.

    PubMed

    Al-Younes, Hesham M; Brinkmann, Volker; Meyer, Thomas F

    2004-08-01

    Chlamydiae are obligate intracellular pathogens that replicate within a membrane-bound compartment (the inclusion) and are associated with important human diseases, such as trachoma, pneumonia, and atherosclerosis. We have examined the interaction of the host autophagic pathway with Chlamydia trachomatis serovar L2 by using the specific autophagosomal stain monodansylcadaverine, antibodies to autophagosome-associated markers, and traditionally used autophagic inhibitors, particularly 3-methyladenine and amino acids. Chlamydial inclusions did not sequester monodansylcadaverine, suggesting absence of fusion with autophagosomes. Interestingly, exposure of cultures infected for 19 h to 3-methyladenine or single amino acids until the end of infection (44 h) caused various degrees of abnormalities in the inclusion maturation and in the progeny infectivity. Incubation of host cells with chemicals throughout the entire period of infection modulated the growth of Chlamydia even more dramatically. Remarkably, autophagosomal markers MAP-LC3 and calreticulin were redistributed to the inclusion of Chlamydia, a process that appears to be sensitive to 3-methyladenine and some amino acids. The present data indicate the lack of autophagosomal fusion with the inclusion because it was devoid of monodansylcadaverine and no distinct rim of autophagosomal protein-specific staining around the inclusion could be observed. However, high sensitivity of Chlamydia to conditions that could inhibit host autophagic pathway and the close association of MAP-LC3 and calreticulin with the inclusion membrane still suggest a potential role of host autophagy in the pathogenesis of Chlamydia.