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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. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

    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.

  11. 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

  12. 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

  13. Staphylococcal lipoteichoic acid promotes osteogenic differentiation of mouse mesenchymal stem cells by increasing autophagic activity.

    PubMed

    Liu, Xin; Wang, Yuan; Cao, Zhen; Dou, Ce; Bai, Yun; Liu, Chuan; Dong, Shiwu; Fei, Jun

    2017-02-16

    This study sought to explore the effect of staphylococcal lipoteichoic acid (LTA) on autophagy in mouse mesenchymal stem cells (MSCs), and then influence osteogenesis through the change of autophagy. C3H10T1/2 cells were induced by osteogenic medium with the treatment of LTA at different concentrations (1, 5, 10 μg/mL); 3-methyladenine (3-MA) were used as the autophagy inhibitor, and rapamycin (rapamycin, Rap) were used to activate autophagy; the effects on osteogenesis were detected by alkaline phosphatase staining, alizarin red staining, real-time quantitative PCR, and western blotting; autophagic activity was investigated by the expression of LC3-Ⅱand p62 proteins. Compared with control group, the expression of osteogenesis markers was significantly up-regulated with the LTA treatment on the mRNA and protein level; the positive rate of alkaline phosphatase was enhanced in the LTA groups; and the formation of calcium nodules was increased simultaneously. The expression of LC3-Ⅱ protein was increased in LTA groups, while the expression of p62 protein was decreased. Inhibition of autophagy significantly reduced the effect of LTA on osteogenesis of MSCs; the promotion of LTA on osteogenic differentiation was further enhanced when adding rapamycin to activate autophagic activity. It provides new insight of prevention and treatment for bone infection.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  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. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  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. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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…

  1. 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…

  2. [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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. SWCNTs induced autophagic cell death in human bronchial epithelial cells.

    PubMed

    Park, Eun-Jung; Zahari, Nur Elida M; Lee, Eun-Woo; Song, Jaewhan; Lee, Jae-Hyeok; Cho, Myung-Haing; Kim, Jae-Ho

    2014-04-01

    Carbon nanotubes are being actively introduced in electronics, computer science, aerospace, and other industries. Thus, the urgent need for toxicological studies on CNTs is mounting. In this study, we investigated the alterations in cellular response with morphological changes induced by single-walled carbon nanotubes (SWCNTs) in BEAS-2B cells, a human bronchial epithelial cell line. At 24h after exposure, SWCNTs rapidly decreased ATP production and cell viability as well a slight increase in the number of cells in the subG1 and G1 phases. In addition, SWCNTs increased the expression of superoxide dismutase (SOD)-1, but not SOD-2, and the number of cells generating ROS. The concentration of Cu and Zn ions also increased in a dose-dependent manner in cells exposed to SWCNTs. SWCNTs significantly enhanced the release of nitric oxide, interleukin (IL)-6, and IL-8 and up-regulated the expression of chemokine- and cytokine-related genes. Furthermore, the levels of autophagy-related genes, especially the DRAM1 gene, and the autophagosome formation-related proteins, were clearly up-regulated together with an increase of autophagosome-like vacuoles. Based on these results, we suggest that SWCNTs induce autophagic cell death through mitochondrial dysfunction and cytosolic damage in human bronchial epithelial cells.

  13. 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.

  14. 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.

  15. 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.

  16. [Autophagic processes in plants: mechanisms, regulation and function].

    PubMed

    Guiboileau, Anne; Masclaux-Daubresse, Céline

    2012-06-01

    Large numbers of publications investigating the molecular details, the regulation and the physiological roles of autophagic processes have appeared over the last few years, dealing with animals, plants and unicellular eukaryotic organisms. This strong interest is caused by the fact that autophagic processes are ubiquitous in eukaryotic organisms. They are involved in the adaptation of organisms to their environment and to stressful conditions, thereby contributing to cell and organism survival and longevity. This review article aims to describe the discovery of autophagy, the molecular details of this complex process, its regulation, and its specific functions in plants.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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

  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. 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

    2017-03-05

    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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... specific location of fires that are not in machinery spaces need not be indicated. (d) Fire pumps. (1) The ECC must include control of the main machinery space fire pumps. (2) Remote control of a required...

  12. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... specific location of fires that are not in machinery spaces need not be indicated. (d) Fire pumps. (1) The ECC must include control of the main machinery space fire pumps. (2) Remote control of a required...

  13. 46 CFR 62.50-20 - Additional requirements for minimally attended machinery plants.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... specific location of fires that are not in machinery spaces need not be indicated. (d) Fire pumps. (1) The ECC must include control of the main machinery space fire pumps. (2) Remote control of a required...

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

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... specific location of fires that are not in machinery spaces need not be indicated. (d) Fire pumps. (1) The ECC must include control of the main machinery space fire pumps. (2) Remote control of a required...

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

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... maintenance, a roving watch, or similar activities in the machinery spaces but not at the ECC, both alarms and... specific location of fires that are not in machinery spaces need not be indicated. (d) Fire pumps. (1) The ECC must include control of the main machinery space fire pumps. (2) Remote control of a required...

  16. 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.

  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. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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).

  7. 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.

  8. 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.

  9. 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.

  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. 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

  12. 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.

  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. 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.

  15. 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)

  16. 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…

  17. 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.

  18. 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.

  19. 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

  20. 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

  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.

  2. 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....

  3. 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....

  4. 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....

  5. 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....

  6. 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...

  7. 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...

  8. 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....

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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).

  15. 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

  16. 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

  17. 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.

  18. 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.

  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. 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.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) Machinery compartments containing equipment for ammonia shall be fitted with a sprinkler system providing an... AND AUXILIARY MACHINERY AND RELATED SYSTEMS Refrigeration Machinery § 58.20-15 Installation...

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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)

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  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)...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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)...

  4. 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)...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. 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)...

  18. 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)...

  19. 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...

  20. 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.

  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 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

  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. 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

  6. 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.

  7. 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

  8. 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.

  9. 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…

  10. 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…

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

    PubMed

    Chung, Kyung Min; Park, Hyunhee; Jung, Seonghee; Ha, Shinwon; Yoo, Seung-Jun; Woo, Hanwoong; Lee, Hyang Ju; Kim, Seong Who; Kim, Eun-Kyoung; Moon, Cheil; Yu, Seong-Woon

    2015-10-01

    Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca(2+) concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca(2+) levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death.

  1. 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.

  2. 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…

  3. 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.…

  4. 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...

  5. 46 CFR 176.804 - Machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Skeletal muscle myotubes of the severely obese exhibit altered ubiquitin-proteasome and autophagic/lysosomal proteolytic flux

    PubMed Central

    Bollinger, Lance M.; Powell, Jonathan J. S.; Houmard, Joseph A.; Witczak, Carol A.; Brault, Jeffrey J.

    2015-01-01

    Objective Whole-body protein metabolism is dysregulated with obesity. Our goal was to determine if activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity. Methods We utilized primary Human Skeletal Muscle cell (HSkM) cultures since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean (BMI ≤ 26.0 kg/m2) and 8 severely obese (BMI ≥ 39.0) women were examined basally and when stimulated to atrophy (serum and amino acid starvation). Results HSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin-proteasome system diverged according to obesity status, with a decrease in the lean and an increase in HSkM from obese subjects. HSkMC from the obese also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from lean and obese individuals under basal and starved conditions. Conclusions Our data indicate that muscle cells of the lean and severely obese have innate differences in management of protein degradation, which may explain their metabolic differences. PMID:26010327

  15. Herpes Simplex Virus and Interferon Signaling Induce Novel Autophagic Clusters in Sensory Neurons

    PubMed Central

    Katzenell, Sarah

    2016-01-01

    ABSTRACT Herpes simplex virus 1 (HSV-1) establishes lifelong infection in the neurons of trigeminal ganglia (TG), cycling between productive infection and latency. Neuronal antiviral responses are driven by type I interferon (IFN) and are crucial to controlling HSV-1 virulence. Autophagy also plays a role in this neuronal antiviral response, but the mechanism remains obscure. In this study, HSV-1 infection of murine TG neurons triggered unusual clusters of autophagosomes, predominantly in neurons lacking detectable HSV-1 antigen. Treatment of neurons with IFN-β induced a similar response, and cluster formation by infection or IFN treatment was dependent upon an intact IFN-signaling pathway. The autophagic clusters were decorated with both ISG15, an essential effecter of the antiviral response, and p62, a selective autophagy receptor. The autophagic clusters were not induced by rapamycin or starvation, consistent with a process of selective autophagy. While clusters were triggered by other neurotropic herpesviruses, infection with unrelated viruses failed to induce this response. Following ocular infection in vivo, clusters formed exclusively in the infected ophthalmic branch of the TG. Taken together, our results show that infection with HSV and antiviral signaling in TG neurons produce an unorthodox autophagic response. This autophagic clustering is associated with antiviral signaling, the presence of viral genome, and the absence of HSV protein expression and may therefore represent an important neuronal response to HSV infection and the establishment of latency. IMPORTANCE Herpes simplex virus type 1 (HSV-1) is a ubiquitous virus and a significant cause of morbidity and some mortality. It is the causative agent of benign cold sores, but it can also cause blindness and life-threatening encephalitis. The success of HSV-1 is largely due to its ability to establish lifelong latent infections in neurons and to occasionally reactivate. The exact mechanisms by which

  16. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 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...

  17. 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

  18. 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

  19. 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

  20. Autophagic induction of amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase 1 G93A mutant in NSC34 cells.

    PubMed

    Wei, Yanming

    2014-01-01

    Previous studies have confirmed that the beclin 1 complex plays a key role in the initial stage of autophagy and deregulated autophagy might involve in amyotrophic lateral sclerosis. However, the mechanism underlying altered autophagy associated with the beclin 1 complex remains unclear. In this study, we transfected the Cu/Zn superoxide dismutase 1 G93A mutant protein into the motor neuron-like cell line NSC34 cultured in vitro. Western blotting and co-immunoprecipitation showed that the Cu/Zn superoxide dismutase 1 G93A mutant enhanced the turnover of autophagic marker microtubule-associated protein light chain 3II (LC3II) and stimulated the conversion of EGFP-LC3I to EGFP-LC3II, but had little influence on the binding capacity of the autophagy modulators ATG14L, rubicon, UVRAG, and hVps34 to beclin 1 during autophagosome formation. These results suggest that the amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase 1 G93A mutant can upregulate autophagic activity in NSC34 cells, but that this does not markedly affect beclin 1 complex components.

  1. Assessing Basal and Acute Autophagic Responses in the Adult Drosophila Nervous System: The Impact of Gender, Genetics and Diet on Endogenous Pathway Profiles

    PubMed Central

    Molina, Brandon; Mauntz, Ruth E.; Gonzalez, Arysa; Barekat, Ayeh; El-Mecharrafie, Nadja; Garza, Shannon; Gurney, Michael A.; Achal, Madhulika; Linton, Phyllis-Jean; Harris, Greg L.; Finley, Kim D.

    2016-01-01

    The autophagy pathway is critical for the long-term homeostasis of cells and adult organisms and is often activated during periods of stress. Reduced pathway efficacy plays a central role in several progressive neurological disorders that are associated with the accumulation of cytotoxic peptides and protein aggregates. Previous studies have shown that genetic and transgenic alterations to the autophagy pathway impacts longevity and neural aggregate profiles of adult Drosophila. In this study, we have identified methods to measure the acute in vivo induction of the autophagy pathway in the adult fly CNS. Our findings indicate that the genotype, age, and gender of adult flies can influence pathway responses. Further, we demonstrate that middle-aged male flies exposed to intermittent fasting (IF) had improved neuronal autophagic profiles. IF-treated flies also had lower neural aggregate profiles, maintained more youthful behaviors and longer lifespans, when compared to ad libitum controls. In summary, we present methodology to detect dynamic in vivo changes that occur to the autophagic profiles in the adult Drosophila CNS and that a novel IF-treatment protocol improves pathway response in the aging nervous system. PMID:27711219

  2. Retinoic Acid Induced-Autophagic Flux Inhibits ER-Stress Dependent Apoptosis and Prevents Disruption of Blood-Spinal Cord Barrier after Spinal Cord Injury

    PubMed Central

    Zhou, Yulong; Zhang, Hongyu; Zheng, Binbin; Ye, Libing; Zhu, Sipin; Johnson, Noah R; Wang, Zhouguang; Wei, Xiaojie; Chen, Daqing; Cao, Guodong; Fu, Xiaobing; Li, Xiaokun; Xu, Hua-Zi; Xiao, Jian

    2016-01-01

    Spinal cord injury (SCI) induces the disruption of the blood-spinal cord barrier (BSCB) which leads to infiltration of blood cells, an inflammatory response, and neuronal cell death, resulting spinal cord secondary damage. Retinoic acid (RA) has a neuroprotective effect in both ischemic brain injury and SCI, however the relationship between BSCB disruption and RA in SCI is still unclear. In this study, we demonstrated that autophagy and ER stress are involved in the protective effect of RA on the BSCB. RA attenuated BSCB permeability and decreased the loss of tight junction (TJ) molecules such as P120, β-catenin, Occludin and Claudin5 after injury in vivo as well as in Brain Microvascular Endothelial Cells (BMECs). Moreover, RA administration improved functional recovery in the rat model of SCI. RA inhibited the expression of CHOP and caspase-12 by induction of autophagic flux. However, RA had no significant effect on protein expression of GRP78 and PDI. Furthermore, combining RA with the autophagy inhibitor chloroquine (CQ) partially abolished its protective effect on the BSCB via exacerbated ER stress and subsequent loss of tight junctions. Taken together, the neuroprotective role of RA in recovery from SCI is related to prevention of of BSCB disruption via the activation of autophagic flux and the inhibition of ER stress-induced cell apoptosis. These findings lay the groundwork for future translational studies of RA for CNS diseases, especially those related to BSCB disruption. PMID:26722220

  3. Bioinert Anodic Alumina Nanotubes for Targeting of Endoplasmic Reticulum Stress and Autophagic Signaling: A Combinatorial Nanotube-Based Drug Delivery System for Enhancing Cancer Therapy.

    PubMed

    Wang, Ye; Kaur, Gagandeep; Chen, Yuting; Santos, Abel; Losic, Dusan; Evdokiou, Andreas

    2015-12-16

    Although nanoparticle-based targeted delivery systems have gained promising achievements for cancer therapy, the development of sophisticated strategies with effective combinatorial therapies remains an enduring challenge. Herein, we report the fabrication of a novel nanomaterial, so-called anodic alumina nanotubes (AANTs) for proof-of-concept cancer therapy by targeting cell signaling networks. This strategy is to target autophagic and endoplasmic reticulum (ER) stress signaling by using thapsigargin (TG)-loaded AANTs cotreated with an autophagy inhibitor 3-methyladenine (3-MA). We first show that AANTs are nontoxic and can activate autophagy in different cell types including human fibroblast cells (HFF), human monocyte cells (THP-1), and human breast cancer cells (MDA-MB 231-TXSA). Treatment with 3-MA at a nontoxic dose reduced the level of autophagy induced by AANTs, and consequently sensitized breast cancer cells to AANTs-induced cellular stresses. To target autophagic and ER stress signaling networking, breast cancer cells were treated with 3-MA together with AANTs loaded with the prototype ER stress inducer TG. We demonstrated that 3-MA enhanced the cancer cell killing effect of AANTs loaded with TG. This effect was associated with enhanced ER stress signaling due to the combination effect of TG and 3-MA. These findings not only demonstrate the excellent biocompatibility of AANTs as novel biomaterials but also provide new opportunities for developing ER- and autophagy-targeted delivery systems for future clinical cancer therapy.

  4. 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....

  5. 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....

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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....

  12. 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...

  13. 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....

  14. 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....

  15. 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...

  16. 46 CFR 58.01-25 - Means of stopping machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 46 CFR 109.205 - Inspection of boilers and machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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....

  5. 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....

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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....

  14. 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...

  15. 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...

  16. 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....

  17. 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....

  18. Betanin-Enriched Red Beetroot (Beta vulgaris L.) Extract Induces Apoptosis and Autophagic Cell Death in MCF-7 Cells.

    PubMed

    Nowacki, Laëtitia; Vigneron, Pascale; Rotellini, Laura; Cazzola, Hélène; Merlier, Franck; Prost, Elise; Ralanairina, Robert; Gadonna, Jean-Pierre; Rossi, Claire; Vayssade, Muriel

    2015-12-01

    Recent studies have pointed out the preventive role of beetroot extracts against cancers and their cytotoxic activity on cancer cells. Among many different natural compounds, these extracts contained betanin and its stereoisomer isobetanin, which belongs to the betalain group of highly bioavailable antioxidants. However, a precise identification of the molecules responsible for this tumor-inhibitory effect was still required. We isolated a betanin/isobetanin concentrate from fresh beetroots, corresponding to the highest purified betanin extract used for studying anticancer activities of these molecules. The cytotoxicity of this betanin-enriched extract was then characterized on cancer and normal cells and we highlighted the death signalling pathways involved. Betanin/isobetanin concentrate significantly decreased cancer cell proliferation and viability. Particularly in MCF-7-treated cells, the expressions of apoptosis-related proteins (Bad, TRAILR4, FAS, p53) were strongly increased and the mitochondrial membrane potential was altered, demonstrating the involvement of both intrinsic and extrinsic apoptotic pathways. Autophagosome vesicles in MCF-7-treated cells were observed, also suggesting autophagic cell death upon betanin/isobetanin treatment. Importantly, the betanin-enriched extract had no obvious effect towards normal cell lines. Our data bring new insight to consider the betanin/isobetanin mix as therapeutic anticancer compound, alone or in combination with classical chemotherapeutic drugs, especially in functional p53 tumors.

  19. Dual attenuation of proteasomal and autophagic BMAL1 degradation in ClockΔ19/+ mice contributes to improved glucose homeostasis

    PubMed Central

    Jeong, Kwon; He, Baokun; Nohara, Kazunari; Park, Noheon; Shin, Youngmin; Kim, Seonghwa; Shimomura, Kazuhiro; Koike, Nobuya; Yoo, Seung-Hee; Chen, Zheng

    2015-01-01

    Circadian clocks orchestrate essential physiology in response to various cues, yet their mechanistic and functional plasticity remains unclear. Here, we investigated ClockΔ19/+ 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. PMID:26228022

  20. Molecular Basis of Autophagic Cell Death in Prostate Cancer

    DTIC Science & Technology

    2009-03-01

    Hsiung JW, Huang SA, Chien S. The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle ...lipid-binding proteinwith high affinity for phosphatidic acid (PA) and cardiolipin (CL). Previously, it has been shown that PA directly interacted...lyceride), PI (phosphatidylinositol), DAG (diacylglycerol), PI4P (PtdIns(4)P), PA ( phosphatidic acid ), PI4,5P2 (PtdIns(4,5)P2), PS (phosphatidylserine

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  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. Glucagon-like Peptide-1 Protects Pancreatic Beta-cells from Death by Increasing Autophagic Flux and Restoring Lysosomal Function.

    PubMed

    Zummo, Francesco P; Cullen, Kirsty S; Honkanen-Scott, Minna; Shaw, James Am; Lovat, Penny E; Arden, Catherine

    2017-02-23

    Studies in animal models of type 2 diabetes have shown that glucagon-like peptide-1 (GLP-1) receptor agonists prevent β-cell loss. Whether GLP-1 mediates β-cell survival via the key lysosomal-mediated process of autophagy is unknown.Here we report that treatment of INS-1E β-cells and primary islets with glucolipotoxicity (0.5mmol/l palmitate, 25mmol/l glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilisation (LMP) and release of Cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Co-treatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. siRNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4.Collectively, our data highlights lysosomal dysfunction as a critical mediator of β-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy / lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signalling pathway as a potential focus.

  10. Icariside II-induced mitochondrion and lysosome mediated apoptosis is counterbalanced by an autophagic salvage response in hepatoblastoma.

    PubMed

    Geng, Ya-di; Zhang, Chao; Shi, Ya-Min; Xia, Yuan-Zheng; Guo, Chao; Yang, Lei; Kong, Ling-Yi

    2015-09-28

    In this study, the anti-cancer effect of Icariside II (IS), a natural plant flavonoid, against hepatoblastoma cells and the underlying mechanisms were investigated. The in vitro and in vivo studies show that IS decreased the viability of human hepatoblastoma HepG2 cells in a concentration- and time-dependent manner and inhibited tumor growth in mice transplanted with H22 liver carcinomas. IS impaired mitochondria and lysosomes as evidenced by signs of induced mitochondrial and lysosomal membrane permeabilization, resulting in caspase activation and apoptosis. SQSTM1 up-regulation and autophagic flux measurements demonstrated that IS exposure also impaired autophagosome degradation which resulted in autophagosome accumulation, which plays a pro-survival role as the genetic knockdown of LC3B further sensitized the IS-treated cells. Electron microscopy images showed that autophagosome engulfs IS-impaired mitochondria and lysosomes, thus blocking cytotoxicity induced by further leakage of the hydrolases from lysosomes and pro-apoptosis members from mitochondria. In conclusion, these data suggest that IS plays multiple roles as a promising chemotherapeutic agent that induces cell apoptosis involving both mitochondrial and lysosomal damage.

  11. Huaier Extract Induces Autophagic Cell Death by Inhibiting the mTOR/S6K Pathway in Breast Cancer Cells

    PubMed Central

    Li, Yaming; Zhang, Ning; Dong, Lun; Sun, Mingjuan; Cun, Jinjing; Zhang, Yan; Lv, Shangge; Yang, Qifeng

    2015-01-01

    Huaier extract is attracting increased attention due to its biological activities, including antitumor, anti-parasite and immunomodulatory effects. Here, we investigated the role of autophagy in Huaier-induced cytotoxicity in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cells. Huaier treatment inhibited cell viability in all three cell lines and induced various large membranous vacuoles in the cytoplasm. In addition, electron microscopy, MDC staining, accumulated expression of autophagy markers and flow cytometry revealed that Huaier extract triggered autophagy. Inhibition of autophagy attenuated Huaier-induced cell death. Furthermore, Huaier extract inhibited the mammalian target of the rapamycin (mTOR)/S6K pathway in breast cancer cells. After implanting MDA-MB-231 cells subcutaneously into the right flank of BALB/c nu/nu mice, Huaier extract induced autophagy and effectively inhibited xenograft tumor growth. This study is the first to show that Huaier-induced cytotoxicity is partially mediated through autophagic cell death in breast cancer cells through suppression of the mTOR/S6K pathway. PMID:26134510

  12. Interactions between autophagic and endo-lysosomal markers in endothelial cells.

    PubMed

    Oeste, Clara L; Seco, Esther; Patton, Wayne F; Boya, Patricia; Pérez-Sala, Dolores

    2013-05-01

    Autophagic and endo-lysosomal degradative pathways are essential for cell homeostasis. Availability of reliable tools to interrogate these pathways is critical to unveil their involvement in physiology and pathophysiology. Although several probes have been recently developed to monitor autophagic or lysosomal compartments, their specificity has not been validated through co-localization studies with well-known markers. Here, we evaluate the selectivity and interactions between one lysosomal (Lyso-ID) and one autophagosomal (Cyto-ID) probe under conditions modulating autophagy and/or endo-lysosomal function in live cells. The probe for acidic compartments Lyso-ID was fully localized inside vesicles positive for markers of late endosome-lysosomes, including Lamp1-GFP and GFP-CINCCKVL. Induction of autophagy by amino acid deprivation in bovine aortic endothelial cells caused an early and potent increase in the fluorescence of the proposed autophagy dye Cyto-ID. Cyto-ID-positive compartments extensively co-localized with the autophagosomal fluorescent reporter RFP-LC3, although the time and/or threshold for organelle detection was different for each probe. Interestingly, use of Cyto-ID in combination with Lysotracker Red or Lyso-ID allowed the observation of structures labeled with either one or both probes, the extent of co-localization increasing upon treatment with protease inhibitors. Inhibition of the endo-lysosomal pathway with chloroquine or U18666A resulted in the formation of large Cyto-ID and Lyso-ID-positive compartments. These results constitute the first assessment of the selectivity of Cyto-ID and Lyso-ID as probes for the autophagic and lysosomal pathways, respectively. Our observations show that these probes can be used in combination with protein-based markers for monitoring the interactions of both pathways in live cells.

  13. Infection with Usutu Virus Induces an Autophagic Response in Mammalian Cells

    PubMed Central

    Blázquez, Ana-Belén; Escribano-Romero, Estela; Merino-Ramos, Teresa; Saiz, Juan-Carlos; Martín-Acebes, Miguel A.

    2013-01-01

    Usutu virus (USUV) is an African mosquito-borne flavivirus closely related to West Nile virus and Japanese encephalitis virus, which host range includes mainly mosquitoes and birds, although infections in humans have been also documented, thus warning about USUV as a potential health threat. Circulation of USUV in Africa was documented more than 50 years ago, but it was not until the last decade that it emerged in Europe causing episodes of avian mortality and some human severe cases. Since autophagy is a cellular pathway that can play important roles on different aspects of viral infections and pathogenesis, the possible implication of this pathway in USUV infection has been examined using Vero cells and two viral strains of different origin. USUV infection induced the unfolded protein response, revealed by the splicing of Xbp-1 mRNA. Infection with USUV also stimulated the autophagic process, which was demonstrated by an increase in the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), a marker of autophagosome formation. In addition to this, an increase in the lipidated form of LC3, that is associated with autophagosome formation, was noticed following infection. Pharmacological modulation of the autophagic pathway with the inductor of autophagy rapamycin resulted in an increase in virus yield. On the other hand, treatment with 3-methyladenine or wortmannin, two distinct inhibitors of phosphatidylinositol 3-kinases involved in autophagy, resulted in a decrease in virus yield. These results indicate that USUV virus infection upregulates the cellular autophagic pathway and that drugs that target this pathway can modulate the infection of this virus, thus identifying a potential druggable pathway in USUV-infection. PMID:24205422

  14. Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

    PubMed Central

    Baskaran, Sulochanadevi; Carlson, Lars-Anders; Stjepanovic, Goran; Young, Lindsey N; Kim, Do Jin; Grob, Patricia; Stanley, Robin E; Nogales, Eva; Hurley, James H

    2014-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001 PMID:25490155

  15. Pictet–Spengler reaction-based biosynthetic machinery in fungi

    PubMed Central

    Yan, Wei; Ge, Hui Ming; Wang, Gang; Jiang, Nan; Mei, Ya Ning; Jiang, Rong; Li, Sui Jun; Chen, Chao Jun; Jiao, Rui Hua; Xu, Qiang; Ng, Seik Weng; Tan, Ren Xiang

    2014-01-01

    The Pictet–Spengler (PS) reaction constructs plant alkaloids such as morphine and camptothecin, but it has not yet been noticed in the fungal kingdom. Here, a silent fungal Pictet–Spenglerase (FPS) gene of Chaetomium globosum 1C51 residing in Epinephelus drummondhayi guts is described and ascertained to be activable by 1-methyl-l-tryptophan (1-MT). The activated FPS expression enables the PS reaction between 1-MT and flavipin (fungal aldehyde) to form “unnatural” natural products with unprecedented skeletons, of which chaetoglines B and F are potently antibacterial with the latter inhibiting acetylcholinesterase. A gene-implied enzyme inhibition (GIEI) strategy has been introduced to address the key steps for PS product diversifications. In aggregation, the work designs and validates an innovative approach that can activate the PS reaction-based fungal biosynthetic machinery to produce unpredictable compounds of unusual and novel structure valuable for new biology and biomedicine. PMID:25425666

  16. Ursodeoxycholic acid effectively kills drug-resistant gastric cancer cells through induction of autophagic death.

    PubMed

    Lim, Sung-Chul; Han, Song Iy

    2015-09-01

    Carcinoma cells that have acquired drug resistance often exhibit cross-resistance to various other cytotoxic stimuli. Here, we investigated the effects of ursodeoxycholic acid (UDCA), a gastrointestinal tumor-suppressor, on a cisplatin‑resistant SNU601 gastric cancer subline (SNU601/R). While other anticancer drugs, including L-OHP, etoposide, and death ligand TRAIL, had minimal effects on the viability of these resistant cells, they were sensitive to UDCA. The UDCA‑induced reduction in the viability of the SNU601/R cells was accomplished through autophagy while the primary means of cell death in the parental SNU601 cells (SNU601/WT) was apoptosis. Previously, we demonstrated that the UDCA-triggered apoptosis of gastric cancer cells was regulated by a cell surface death receptor, TRAIL-R2/DR5, which was upregulated and re-distributed on lipid rafts. The UDCA stimulation of TRAIL-R2/DR5 also occurred in the SNU601/R cells despite the lack of apoptosis. In the present study, we found that CD95/Fas, another cell surface death receptor, was also translocated into lipid rafts in response to UDCA although it was not involved in the decrease in cell viability. Specifically, raft relocalization of CD95/Fas was triggered by UDCA in the SNU601/WT cells in which apoptosis occurred, but not in the SNU601/R cells where autophagic death occurred. Notably, UDCA reduced ATG5 levels, an essential component of autophagy, in the SNU601/WT, but not in the SNU601/R cell line. Moreover, in CD95/Fas-silenced SNU601/WT cells, UDCA did not decrease ATG5 levels and induced autophagic cell death rather than apoptosis. These results imply that raft‑distributed CD95/Fas may support UDCA-induced apoptosis via downregulation of ATG5 levels, preventing the autophagic pathway. Taken together, these results suggest that UDCA induces both apoptotic and autophagic cell death depending on the intracellular signaling environment, thereby conferring the advantage to overcome drug resistance

  17. 46 CFR 30.10-42 - Machinery space-TB/ALL.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Machinery space-TB/ALL. 30.10-42 Section 30.10-42...-42 Machinery space—TB/ALL. The term machinery space means any space that contains machinery and related equipment including Category A machinery spaces, propelling machinery, boilers, oil fuel...

  18. 46 CFR 30.10-42 - Machinery space-TB/ALL.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 1 2012-10-01 2012-10-01 false Machinery space-TB/ALL. 30.10-42 Section 30.10-42...-42 Machinery space—TB/ALL. The term machinery space means any space that contains machinery and related equipment including Category A machinery spaces, propelling machinery, boilers, oil fuel...

  19. 46 CFR 30.10-42 - Machinery space-TB/ALL.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Machinery space-TB/ALL. 30.10-42 Section 30.10-42...-42 Machinery space—TB/ALL. The term machinery space means any space that contains machinery and related equipment including Category A machinery spaces, propelling machinery, boilers, oil fuel...

  20. 46 CFR 30.10-42 - Machinery space-TB/ALL.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Machinery space-TB/ALL. 30.10-42 Section 30.10-42...-42 Machinery space—TB/ALL. The term machinery space means any space that contains machinery and related equipment including Category A machinery spaces, propelling machinery, boilers, oil fuel...

  1. 46 CFR 30.10-42 - Machinery space-TB/ALL.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Machinery space-TB/ALL. 30.10-42 Section 30.10-42...-42 Machinery space—TB/ALL. The term machinery space means any space that contains machinery and related equipment including Category A machinery spaces, propelling machinery, boilers, oil fuel...

  2. Failure Accommodation Tested in Magnetic Suspension Systems for Rotating Machinery

    NASA Technical Reports Server (NTRS)

    Provenza, Andy J.

    2000-01-01

    The NASA Glenn Research Center at Lewis Field and Texas A&M University are developing techniques for accommodating certain types of failures in magnetic suspension systems used in rotating machinery. In recent years, magnetic bearings have become a viable alternative to rolling element bearings for many applications. For example, industrial machinery such as machine tool spindles and turbomolecular pumps can today be bought off the shelf with magnetically supported rotating components. Nova Gas Transmission Ltd. has large gas compressors in Canada that have been running flawlessly for years on magnetic bearings. To help mature this technology and quiet concerns over the reliability of magnetic bearings, NASA researchers have been investigating ways of making the bearing system tolerant to faults. Since the potential benefits from an oil-free, actively controlled bearing system are so attractive, research that is focused on assuring system reliability and safety is justifiable. With support from the Fast Quiet Engine program, Glenn's Structural Mechanics and Dynamics Branch is working to demonstrate fault-tolerant magnetic suspension systems targeted for aerospace engine applications. The Flywheel Energy Storage Program is also helping to fund this research.

  3. Adaptive feature extraction using sparse coding for machinery fault diagnosis

    NASA Astrophysics Data System (ADS)

    Liu, Haining; Liu, Chengliang; Huang, Yixiang

    2011-02-01

    In the signal processing domain, there has been growing interest in sparse coding with a learned dictionary instead of a predefined one, which is advocated as an effective mathematical description for the underlying principle of mammalian sensory systems in processing information. In this paper, sparse coding is introduced as a feature extraction technique for machinery fault diagnosis and an adaptive feature extraction scheme is proposed based on it. The two core problems of sparse coding, i.e., dictionary learning and coefficients solving, are discussed in detail. A natural extension of sparse coding, shift-invariant sparse coding, is also introduced. Then, the vibration signals of rolling element bearings are taken as the target signals to verify the proposed scheme, and shift-invariant sparse coding is used for vibration analysis. With the purpose of diagnosing the different fault conditions of bearings, features are extracted following the proposed scheme: basis functions are separately learned from each class of vibration signals trying to capture the defective impulses; a redundant dictionary is built by merging all the learned basis functions; based on the redundant dictionary, the diagnostic information is made explicit in the solved sparse representations of vibration signals; sparse features are formulated in terms of activations of atoms. The multiclass linear discriminant analysis (LDA) classifier is used to test the discriminability of the extracted sparse features and the adaptability of the learned atoms. The experiments show that sparse coding is an effective feature extraction technique for machinery fault diagnosis.

  4. Towards a comprehensive understanding of brain machinery by correlative microscopy

    NASA Astrophysics Data System (ADS)

    Allegra Mascaro, Anna Letizia; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco S.

    2015-06-01

    Unraveling the complexity of brain structure and function is the biggest challenge of contemporary science. Due to their flexibility, optical techniques are the key to exploring this intricate network. However, a single imaging technique can reveal only a small part of this machinery due to its inherent multilevel organization. To obtain a more comprehensive view of brain functionality, complementary approaches have been combined. For instance, brain activity was monitored simultaneously on different spatiotemporal scales with functional magnetic resonance imaging and calcium imaging. On the other hand, dynamic information on the structural plasticity of neuronal networks has been contextualized in a wider framework combining two-photon and light-sheet microscopy. Finally, synaptic features have been revealed on previously in vivo imaged samples by correlative light-electron microscopy. Although these approaches have revealed important features of brain machinery, they provided small bridges between specific spatiotemporal scales, lacking an omni-comprehensive view. In this perspective, we briefly review the state of the art of correlative techniques and propose a wider methodological framework fusing multiple levels of brain investigation.

  5. Structural insights into the bacterial carbon-phosphorus lyase machinery

    PubMed Central

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J.; Passmore, Lori A.; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E.

    2015-01-01

    Summary Phosphorous is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use organic phosphonate compounds, which require specialised enzymatic machinery for breaking the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolises phosphonate remain unknown. Here we determine the crystal structure of the 240 kDa Escherichia coli C-P lyase core complex (PhnGHIJ) and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that likely couple organic phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy and show that it binds to PhnJ via a conserved insertion domain. Our results provide a structural basis for understanding microbial phosphonate breakdown. PMID:26280334

  6. Structural insights into the bacterial carbon-phosphorus lyase machinery.

    PubMed

    Seweryn, Paulina; Van, Lan Bich; Kjeldgaard, Morten; Russo, Christopher J; Passmore, Lori A; Hove-Jensen, Bjarne; Jochimsen, Bjarne; Brodersen, Ditlev E

    2015-09-03

    Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.

  7. End users "Feedback" to improve ergonomic design of machinery.

    PubMed

    Strambi, F; Bartalini, M; Boy, S; Gauthy, R; Landozzi, R; Novelli, D; Stanzani, C

    2012-01-01

    This paper describes the Feedback method designed to collect the contribution of users for the reconstruction and comprehension of the actual work and real activity for the improvement of the technical standards, design, manufacturing and use of machinery. The Feedback method has since now been applied successfully - in collaboration with public authorities, market surveillance bodies, social partners organization and technical institutes - to five different types of machines: woodworking machinery, forklift trucks, angle grinder and combine harvester. After ten years of experimentation in seven European countries Feedback has proved to be trans-nationally comparable and has attracted the interest of as much as 250 expert users - mostly workers, but also employers and technicians - who have shared their knowledge and experience by taking part in almost 30 working groups. The information collected with the Feedback method can be used by: -CEN and ISO standardization committees and working groups to become aware of the problems relating to the real use of specific machines in different work contexts, and thus to be able to draw up new or to revise existing standards accordingly; - Designers and manufacturers to produce better, more comfortable and safer machines and to provide precise instructions for use; - Employers, users and workers for training purposes and for defining appropriate work procedures; - Inspection bodies to enhance their knowledge and improve the efficiency of their interventions and advice.

  8. Characterization of HJ-PI01 as a novel Pim-2 inhibitor that induces apoptosis and autophagic cell death in triple-negative human breast cancer

    PubMed Central

    Zhao, Yu-qian; Yin, Yi-qiong; Liu, Jie; Wang, Gui-hua; Huang, Jian; Zhu, Ling-juan; Wang, Jin-hui

    2016-01-01

    Aim: Pim-2 is a short-lived serine/threonine kinase, which plays a key role in metastasis of breast cancer through persistent activation of STAT3. Although the crystal structure of Pim-2 has been reported, but thus far no specific Pim-2-targeted compounds have been reported. In this study, we identified a novel Pim-2 inhibitor, HJ-PI01, by in silico analysis and experimental validation. Methods: The protein-protein interaction (PPI) network, chemical synthesis, molecular docking, and molecular dynamics (MD) simulations were used to design and discover the new Pim-2 inhibitor HJ-PI01. The anti-tumor effects of HJ-PI01 were evaluated in human breast MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7 cells in vitro and in MDA-MB-231 xenograft mice, which were treated with HJ-PI01 (40 mg·kg−1·d−1, ig) with or without lienal polypeptide (50 mg·kg−1·d−1, ip) for 10 d. The apoptosis/autophage-inducing mechanisms of HJ-PI01 were elucidated using Western blots, immunoblots, flow cytometry, transmission electron microscopy and fluorescence microscopy. Results: Based on the PrePPI network, the potential partners interacting with Pim-2 in regulating apoptosis (160 protein pairs) and autophagy (47 protein pairs) were identified. Based on the structural characteristics of Pim-2, a total of 15 compounds (HJ-PI01 to HJ-P015) were synthesized, which showed moderate or remarkable anti-proliferative potency in the human breast cancer cell lines tested. The most effective compound HJ-PI01 exerted a robust inhibition on MDA-MB-231 cells compared with chlorpromazine and the pan-Pim inhibitor PI003. Molecular dynamics (MD) simulation revealed that HJ-PI01 had a good binding score with Pim-2. Moreover, HJ-PI01 (300 nmol/L) induced death receptor-dependent and mitochondrial apoptosis as well as autophagic death in MDA-MB-231 cells. In MDA-MB-231 xenograft mice, administration of HJ-PI01 remarkably inhibited the tumor growth and induced tumor cell apoptosis in vivo. Co-administration of

  9. Local Geometrical Machinery for Complexity and Control

    NASA Astrophysics Data System (ADS)

    Ivancevic, Vladimir G.; Reid, Darryn J.

    2015-11-01

    In this Chapter, we present local geometrical machinery for studying complexity and control, consisting of dynamics on Kähler manifolds, which combine three geometrical structures-Riemannian, symplectic and complex (Hermitian)-in a mutually compatible way. In other words, every Kähler manifold is simultaneously Riemannian, symplectic and complex (Hermitian). It is well known that Riemannian manifolds represent the stage on which Lagrangian dynamics is set, symplectic manifolds represent the stage for Hamiltonian dynamics, and complex (Hermitian) varieties comprise the stage for quantum dynamics. Therefore, Kähler manifolds represent the richest dynamical stage available where Lagrangian, Hamiltonian, and quantum dynamics all dance together.

  10. Apigenin induces autophagic cell death in human papillary thyroid carcinoma BCPAP cells.

    PubMed

    Zhang, Li; Cheng, Xian; Gao, Yanyan; Zheng, Jie; Xu, Qiang; Sun, Yang; Guan, Haixia; Yu, Huixin; Sun, Zhen

    2015-11-01

    Apigenin, abundantly present in fruits and vegetables, is recognized as a flavonoid with anti-inflammatory, antioxidant and anticancer properties. In this study, we first investigated the anti-neoplastic effects of apigenin on papillary thyroid carcinoma (PTC) cell line BCPAP cells. Our results show that apigenin inhibited the viability of BCPAP cells in a dose-dependent manner. A large body of evidence demonstrates that autophagy contributes to cell death in certain contexts. In the present study, autophagy was induced by apigenin treatment in BCPAP cells, as evidenced by Beclin-1 accumulation, conversion of LC3 protein, p62 degradation as well as the significantly increased formation of acidic vesicular organelles (AVOs) compared to the control group. 3-MA, an autophagy inhibitor, rescued the cells from apigenin-induced cell death. Notably, apigenin enhanced production of reactive oxygen species (ROS), and subsequent induction of significant DNA damage as monitored by the TUNEL assay. In addition, apigenin treatment caused a significant accumulation of cells in the G2/M phase via down-regulation of Cdc25C expression. Our findings reveal that apigenin inhibits papillary thyroid cancer cell viability by the stimulation of reactive oxygen species (ROS) production, induction of DNA damage, leading to G2/M cell cycle arrest followed by autophagic cell death. Thus, our results provide new insights into the molecular mechanisms underlying apigenin-mediated autophagic cell death and suggest apigenin as a potential chemotherapeutic agent which is able to fight against papillary thyroid cancer.

  11. Features of autophagic cell death in Plasmodium liver-stage parasites.

    PubMed

    Eickel, Nina; Kaiser, Gesine; Prado, Monica; Burda, Paul-Christian; Roelli, Matthias; Stanway, Rebecca R; Heussler, Volker T

    2013-04-01

    Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.

  12. Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

    SciTech Connect

    Johnson-Lyles, Denise N.; Peifley, Kimberly; Lockett, Stephen; Neun, Barry W.; Hansen, Matthew; Clogston, Jeffrey; Stern, Stephan T.; McNeil, Scott E.

    2010-11-01

    Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C{sub 60}OH{sub x}), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

  13. Intermittent fasting is neuroprotective in focal cerebral ischemia by minimizing autophagic flux disturbance and inhibiting apoptosis.

    PubMed

    Jeong, Ji Heun; Yu, Kwang Sik; Bak, Dong Ho; Lee, Je Hun; Lee, Nam Seob; Jeong, Young Gil; Kim, Dong Kwan; Kim, Jwa-Jin; Han, Seung-Yun

    2016-11-01

    Previous studies have demonstrated that autophagy induced by caloric restriction (CR) is neuroprotective against cerebral ischemia. However, it has not been determined whether intermittent fasting (IF), a variation of CR, can exert autophagy-related neuroprotection against cerebral ischemia. Therefore, the neuroprotective effect of IF was evaluated over the course of two weeks in a rat model of focal cerebral ischemia, which was induced by middle cerebral artery occlusion and reperfusion (MCAO/R). Specifically, the role of autophagy modulation as a potential underlying mechanism for this phenomenon was investigated. It was demonstrated that IF reduced infarct volume and brain edema, improved neurobehavioral deficits, and rescued neuronal loss after MCAO/R. Furthermore, neuronal apoptosis was decreased by IF in the rat cortex. An increase in the number of autophagosomes (APs) was demonstrated in the cortices of IF-treated rats, using immunofluorescence staining and transmission electron microscopy. Using immunoblots, an IF-induced increase was detected in microtubule-associated protein 1 light chain 3 (LC3)-II, Rab7, and cathepsin D protein levels, which corroborated previous morphological studies. Notably, IF reduced the accumulation of APs and p62, demonstrating that IF attenuated the MCAO/R-induced disturbance of autophagic flux in neurons. The findings of the present study suggest that IF-induced neuroprotection in focal cerebral ischemia is due, at least in part, to the minimization of autophagic flux disturbance and inhibition of apoptosis.

  14. Autophagic flux, a possible mechanism for delayed gentamicin-induced ototoxicity

    PubMed Central

    Kim, Yeon Ju; Tian, Chunjie; Kim, Jangho; Shin, Beomyong; Choo, Oak-Sung; Kim, You-Sun; Choung, Yun-Hoon

    2017-01-01

    Aminoglycoside antibiotics including gentamicin (GM) induce delayed ototoxic effects such as hearing loss after long-term use, unlike the early-onset ototoxicity caused by cisplatin. The purpose of the study was to identify the mechanism of the delayed GM-induced ototoxicity by exploring the role of autophagy in vitro and in vivo. Treating HEI-OC1 auditory cells with GM led to a time-dependent increase of the autophagosome marker LC3-II, which was accompanied by cell death. In contrast, cisplatin and penicillin caused a rapid increase and had no effect on LC3-II levels, respectively. LC3-II-expressing autophagosomes co-localized with the labeled GM. GM-treated autophagosomes expressed reduced levels of Rab7, which is necessary for the fusion of autophagosomes with lysosomes. When the autophagic flux enhancer rapamycin was applied to GM-treated cells, Rab7 and the lysosomal enzyme cathepsin D were upregulated, and increased cell survival was observed. In animal studies, the intraperitoneal injection of GM worsened hearing thresholds and induced the accumulation of LC3 in the organ of Corti. This hearing impairment was attenuated by rapamycin. These findings suggest that the delayed onset-ototoxicity of GM may be closely related to the accumulation of autophagosomes via impaired autophagy. This GM-induced auditory cell death could be inhibited by enhancing autophagic flux. PMID:28145495

  15. Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants

    PubMed Central

    Hebbar, Sarita; Sahoo, Ishtapran; Matysik, Artur; Argudo Garcia, Irene; Osborne, Kathleen Amy; Papan, Cyrus; Torta, Federico; Narayanaswamy, Pradeep; Fun, Xiu Hui; Wenk, Markus R; Shevchenko, Andrej; Schwudke, Dominik; Kraut, Rachel

    2015-01-01

    Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death. PMID:26639035

  16. Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates

    PubMed Central

    Korac, Jelena; Schaeffer, Veronique; Kovacevic, Igor; Clement, Albrecht M.; Jungblut, Benno; Behl, Christian; Terzic, Janos; Dikic, Ivan

    2013-01-01

    SUMMARY Aggregation of misfolded proteins and the associated loss of neurons are considered as a hallmark of numerous neurodegenerative diseases. Optineurin is present in protein inclusions observed in various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, Creutzfeld-Jacob disease and Pick’s disease. Optineurin deletion mutations have also been described in ALS patients. However, the role of optineurin in mechanisms of protein aggregation remains unclear. In this report, we demonstrate that optineurin recognized various protein aggregates via its C-terminal coiled-coil domain in a ubiquitin-independent manner. We also show that optineurin depletion significantly increase protein aggregation in HeLa cells and morpholino-silencing of the optineurin ortholog in zebrafish causes the motor axonopathy phenotype similar to a zebrafish model of ALS. A more severe phenotype is observed when optineurin is depleted in zebrafish carrying ALS mutations. Furthermore, TANK1 binding kinase 1 (TBK1) is co-localized with optineurin on protein aggregates and is important in clearance of protein aggregates through the autophagy-lysosome pathway. TBK1 phosphorylates optineurin at position Ser-177 and regulates its ability to interact with autophagy modifiers. This study provides evidence for a ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates as well as additional relevance for TBK1 as an upstream regulator of the autophagic pathway. PMID:23178947

  17. Precise temporal regulation of roughest is required for correct salivary gland autophagic cell death in Drosophila.

    PubMed

    Simon, Claudio R; Moda, Livia M R; Octacilio-Silva, Shirlei; Anhezini, Lucas; Machado-Gitai, Luciana C H; Ramos, Ricardo Guelerman P

    2009-07-01

    The Drosophila roughest (rst) locus encodes an immunoglobulin superfamily transmembrane glycoprotein implicated in a variety of embryonic and postembryonic developmental processes. Here we demonstrate a previously unnoticed role for this gene in the autophagic elimination of larval salivary glands during early pupal stages by showing that overexpression of the Rst protein ectodomain in early pupa leads to persistence of salivary glands up to at least 12 hours after head eversion, although with variable penetrance. The same phenotype is observed in individuals carrying the dominant regulatory allele rst(D), but not in loss of function alleles. Analysis of persistent glands at the ultrastructural level showed that programmed cell death starts at the right time but is arrested at an early stage of the process. Finally we describe the expression pattern and intracellular distribution of Rst in wild type and rst(D) mutants, showing that its downregulation in salivary glands at the beginning of pupal stage is an important factor in the correct implementation of the autophagic program of this tissue in space and time.

  18. The ATG12-Conjugating Enzyme ATG10 Is Essential for Autophagic Vesicle Formation in Arabidopsis thaliana

    PubMed Central

    Phillips, Allison R.; Suttangkakul, Anongpat; Vierstra, Richard D.

    2008-01-01

    Autophagy is an important intracellular recycling system in eukaryotes that utilizes small vesicles to traffic cytosolic proteins and organelles to the vacuole for breakdown. Vesicle formation requires the conjugation of the two ubiquitin-fold polypeptides ATG8 and ATG12 to phosphatidylethanolamine and the ATG5 protein, respectively. Using Arabidopsis thaliana mutants affecting the ATG5 target or the ATG7 E1 required to initiate ligation of both ATG8 and ATG12, we previously showed that the ATG8/12 conjugation pathways together are important when plants encounter nutrient stress and during senescence. To characterize the ATG12 conjugation pathway specifically, we characterized a null mutant eliminating the E2-conjugating enzyme ATG10 that, similar to plants missing ATG5 or ATG7, cannot form the ATG12-ATG5 conjugate. atg10-1 plants are hypersensitive to nitrogen and carbon starvation and initiate senescence and programmed cell death (PCD) more quickly than wild type, as indicated by elevated levels of senescence- and PCD-related mRNAs and proteins during carbon starvation. As detected with a GFP-ATG8a reporter, atg10-1 and atg5-1 mutant plants fail to accumulate autophagic bodies inside the vacuole. These results indicate that ATG10 is essential for ATG12 conjugation and that the ATG12-ATG5 conjugate is necessary to form autophagic vesicles and for the timely progression of senescence and PCD in plants. PMID:18245858

  19. Hydrogen peroxide impairs autophagic flux in a cell model of nonalcoholic fatty liver disease

    SciTech Connect

    Jiang, Pengtao; Huang, Zhen; Zhao, Hong; Wei, Taotao

    2013-04-19

    Highlights: •Free fatty acids exposure induces elevated autophagy. •H{sub 2}O{sub 2} inhibits autophagic flux through impairing the fusion between autophagosomes and lysosomes. •Inhibition of autophagy potentiates H{sub 2}O{sub 2}-induced cell death. -- Abstract: Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, but the pathogenesis of NAFLD is not fully clear. The aim of this study was to determine whether autophagy plays a role in the pathogenesis of NAFLD. We found that the levels of autophagy were elevated in hepatoma cells upon exposure to free fatty acids, as confirmed by the increase in the number of autophagosomes. However, exposure of hepatoma cells to H{sub 2}O{sub 2} and TNF-α, two typical “second hit” factors, increased the initiation of autophagy but inhibited the autophagic flux. The inhibition of autophagy sensitized cells to pro-apoptotic stimuli. Taken together, our results suggest that autophagy acts as a protective mechanism in the pathogenesis of NAFLD and that impairment of autophagy might induce more severe lesions of the liver. These findings will be a benefit to the understanding of the pathogenesis of NAFLD and might suggest a strategy for the prevention and cure of NAFLD.

  20. Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor-Mediated Signaling in Neuroblastoma.

    PubMed

    Soori, Mehrnoosh; Lu, Guizhen; Mason, Robert W

    2016-02-01

    Inhibition of the major lysosomal proteases, cathepsins B, D, and L, impairs growth of several cell types but leads to apoptosis in neuroblastoma. The goal of this study was to examine the mechanisms by which enzyme inhibition could cause cell death. Cathepsin inhibition caused cellular accumulation of fragments of the insulin growth factor 1 (IGF-1) receptor. The fragments were located in dense organelles that were characterized as autophagosomes. This novel discovery provides the first clear link between lysosomal function, autophagy, and IGF-1- mediated cell proliferation. A more in-depth analysis of the IGF1 signaling pathway revealed that the mitogen-activated protein kinase (MAPK) cell-proliferation pathway was impaired in inhibitor treated cells, whereas the Akt cell survival pathway remained functional. Shc, an adapter protein that transmits IGF-1 signaling through the MAPK pathway, was sequestered in autophagosomes; whereas IRS-2, an adapter protein that transmits IGF-1 signaling through the Akt pathway, was unaffected by cathepsin inhibition. Furthermore, Shc was sequestered in autophagosomes as its active form, indicating that autophagy is a key mechanism for downregulating IGF-1-induced cell proliferation. Cathepsin inhibition had a greater effect on autophagic sequestration of the neuronal specific adapter protein, Shc-C, than ubiquitously expressed Shc-A, providing mechanistic support for the enhanced sensitivity of neuronally derived tumor cells. We also observed impaired activation of MAPK by epidermal growth factor treatment in inhibitor-treated cells. The Shc adapter proteins are central to transducing proliferation signaling by a range of receptor tyrosine kinases; consequently, cathepsin inhibition may become an important therapeutic approach for treating neuroblastoma and other tumors of neuronal origin.

  1. Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor–Mediated Signaling in Neuroblastoma

    PubMed Central

    Soori, Mehrnoosh; Lu, Guizhen

    2016-01-01

    Inhibition of the major lysosomal proteases, cathepsins B, D, and L, impairs growth of several cell types but leads to apoptosis in neuroblastoma. The goal of this study was to examine the mechanisms by which enzyme inhibition could cause cell death. Cathepsin inhibition caused cellular accumulation of fragments of the insulin growth factor 1 (IGF-1) receptor. The fragments were located in dense organelles that were characterized as autophagosomes. This novel discovery provides the first clear link between lysosomal function, autophagy, and IGF-1– mediated cell proliferation. A more in-depth analysis of the IGF1 signaling pathway revealed that the mitogen-activated protein kinase (MAPK) cell-proliferation pathway was impaired in inhibitor treated cells, whereas the Akt cell survival pathway remained functional. Shc, an adapter protein that transmits IGF-1 signaling through the MAPK pathway, was sequestered in autophagosomes; whereas IRS-2, an adapter protein that transmits IGF-1 signaling through the Akt pathway, was unaffected by cathepsin inhibition. Furthermore, Shc was sequestered in autophagosomes as its active form, indicating that autophagy is a key mechanism for downregulating IGF-1-induced cell proliferation. Cathepsin inhibition had a greater effect on autophagic sequestration of the neuronal specific adapter protein, Shc-C, than ubiquitously expressed Shc-A, providing mechanistic support for the enhanced sensitivity of neuronally derived tumor cells. We also observed impaired activation of MAPK by epidermal growth factor treatment in inhibitor-treated cells. The Shc adapter proteins are central to transducing proliferation signaling by a range of receptor tyrosine kinases; consequently, cathepsin inhibition may become an important therapeutic approach for treating neuroblastoma and other tumors of neuronal origin. PMID:26660229

  2. Calcitonin gene-related peptide inhibits autophagic-lysosomal proteolysis through cAMP/PKA signaling in rat skeletal muscles.

    PubMed

    Machado, Juliano; Manfredi, Leandro H; Silveira, Wilian A; Gonçalves, Dawit A P; Lustrino, Danilo; Zanon, Neusa M; Kettelhut, Isis C; Navegantes, Luiz C

    2016-03-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 μg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation.

  3. The Mendelian disorders of the epigenetic machinery

    PubMed Central

    Bjornsson, Hans Tomas

    2015-01-01

    The Mendelian disorders of the epigenetic machinery are genetic disorders that involve disruption of the various components of the epigenetic machinery (writers, erasers, readers, and remodelers) and are thus expected to have widespread downstream epigenetic consequences. Studying this group may offer a unique opportunity to learn about the role of epigenetics in health and disease. Among these patients, neurological dysfunction and, in particular, intellectual disability appears to be a common phenotype; however, this is often seen in association with other more specific features in respective disorders. The specificity of some of the clinical features raises the question whether specific cell types are particularly sensitive to the loss of these factors. Most of these disorders demonstrate dosage sensitivity as loss of a single allele appears to be sufficient to cause the observed phenotypes. Although the pathogenic sequence is unknown for most of these disorders, there are several examples where disrupted expression of downstream target genes accounts for a substantial portion of the phenotype; hence, it may be useful to systematically map such disease-relevant target genes. Finally, two of these disorders (Rubinstein-Taybi and Kabuki syndromes) have shown post-natal rescue of markers of the neurological dysfunction with drugs that lead to histone deacetylase inhibition, indicating that some of these disorders may be treatable causes of intellectual disability. PMID:26430157

  4. Hormonal control of the metabolic machinery of hepatocellular carcinoma.

    PubMed

    Wong, Carmen Chak-Lui; Wong, Chun-Ming; Ng, Irene Oi-Lin

    2016-06-01

    Hepatocellular carcinoma (HCC) is one of the most fatal malignancies worldwide. It is an aggressive cancer with low cure rate, frequent metastasis, and highly resistant to conventional chemotherapies. Better knowledge regarding the molecular and metabolic alterations in HCC will be instrumental to the development of novel therapeutic interventions against HCC. In the August 2015 issue of Hepatology, Nie et al. reports an important molecular pathway that contributes to the Warburg Effect in HCC. They have beautifully demonstrated that the loss of a component of a hormonal system, the mineralocorticoid receptor (MR), reprogrammed the metabolic machinery of HCC cells to aerobic glycolysis through the miR-338-3p-PKL/R axis. The implication could be that in addition to drugs that directly target the metabolic enzymes in cancer cells, more translational efforts could be focused on the development of drugs that involve the activation of the MR-aldosterone system or other hormonal systems to target the Warburg effect.

  5. Synergism between upregulation of Rab7 and inhibition of autophagic degradation caused by mycoplasma facilitates intracellular mycoplasma infection.

    PubMed

    Hu, Xiaopeng; Yu, Jie; Zhou, Xiang; Li, Zhaoming; Xia, Yun; Luo, Zhiyong; Wu, Yaqun

    2014-03-01

    Following fusion of a mycoplasma with a host cell membrane, the inserted components of mycoplasma may then be transported through the endocytic pathway. However, the effects of mycoplasmas on the host cell endomembrane system are largely unknown. In this study, mycoplasma‑induced changes in the dynamics of endocytic and autophagic systems were investigated. Endocytosis and autophagy are two major processes involved in the survival of intracellular prokaryotic pathogens. It was found that, immediately following infection, mycoplasmas induce endocytosis in the host cell; however, in the long term the mycoplasmas suppress turnover of the components of the endocytic pathway. Immunofluorescence microscopy revealed that Rab7 and LC3‑II are recruited to the intracellular mycoplasma‑containing compartments. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) showed that mycoplasmas increase expression of Rab7 by upregulating transcription, but increase levels of LC3‑II and p62 by post‑translational regulation. Furthermore, it was demonstrated that mycoplasma infection causes inhibition of autophagic degradation of LC3‑II and p62. In addition, it was found that upregulation of Rab7 and inhibition of autophagic degradation synergistically contributes to intracellular mycoplasma accumulation. In conclusion, these findings suggest that mycoplasmas may manipulate host cell endosomal and autophagic systems in order to facilitate intracellular infection.

  6. Cholinergic Machinery as Relevant Target in Acute Lymphoblastic T Leukemia.

    PubMed

    Dobrovinskaya, Oxana; Valencia-Cruz, Georgina; Castro-Sánchez, Luis; Bonales-Alatorre, Edgar O; Liñan-Rico, Liliana; Pottosin, Igor

    2016-01-01

    Various types of non-neuronal cells, including tumors, are able to produce acetylcholine (ACh), which acts as an autocrine/paracrine growth factor. T lymphocytes represent a key component of the non-neuronal cholinergic system. T cells-derived ACh is involved in a stimulation of their activation and proliferation, and acts as a regulator of immune response. The aim of the present work was to summarize the data about components of cholinergic machinery in T lymphocytes, with an emphasis on the comparison of healthy and leukemic T cells. Cell lines derived from acute lymphoblastic leukemias of T lineage (T-ALL) were found to produce a considerably higher amount of ACh than healthy T lymphocytes. Additionally, ACh produced by T-ALL is not efficiently hydrolyzed, because acetylcholinesterase (AChE) activity is drastically decreased in these cells. Up-regulation of muscarinic ACh receptors was also demonstrated at expression and functional level, whereas nicotinic ACh receptors seem to play a less important role and not form functional channels in cells derived from T-ALL. We hypothesized that ACh over-produced in T-ALL may act as an autocrine growth factor and play an important role in leukemic clonal expansion through shaping of intracellular Ca(2+) signals. We suggest that cholinergic machinery may be attractive targets for new drugs against T-ALL. Specifically, testing of high affinity antagonists of muscarinic ACh receptors as well as antagomiRs, which interfere with miRNAs involved in the suppression of AChE expression, may be the first choice options.

  7. Cholinergic Machinery as Relevant Target in Acute Lymphoblastic T Leukemia

    PubMed Central

    Dobrovinskaya, Oxana; Valencia-Cruz, Georgina; Castro-Sánchez, Luis; Bonales-Alatorre, Edgar O.; Liñan-Rico, Liliana; Pottosin, Igor

    2016-01-01

    Various types of non-neuronal cells, including tumors, are able to produce acetylcholine (ACh), which acts as an autocrine/paracrine growth factor. T lymphocytes represent a key component of the non-neuronal cholinergic system. T cells-derived ACh is involved in a stimulation of their activation and proliferation, and acts as a regulator of immune response. The aim of the present work was to summarize the data about components of cholinergic machinery in T lymphocytes, with an emphasis on the comparison of healthy and leukemic T cells. Cell lines derived from acute lymphoblastic leukemias of T lineage (T-ALL) were found to produce a considerably higher amount of ACh than healthy T lymphocytes. Additionally, ACh produced by T-ALL is not efficiently hydrolyzed, because acetylcholinesterase (AChE) activity is drastically decreased in these cells. Up-regulation of muscarinic ACh receptors was also demonstrated at expression and functional level, whereas nicotinic ACh receptors seem to play a less important role and not form functional channels in cells derived from T-ALL. We hypothesized that ACh over-produced in T-ALL may act as an autocrine growth factor and play an important role in leukemic clonal expansion through shaping of intracellular Ca2+ signals. We suggest that cholinergic machinery may be attractive targets for new drugs against T-ALL. Specifically, testing of high affinity antagonists of muscarinic ACh receptors as well as antagomiRs, which interfere with miRNAs involved in the suppression of AChE expression, may be the first choice options. PMID:27630569

  8. Delayed apoptosis allows islet β-cells to implement an autophagic mechanism to promote cell survival

    PubMed Central

    Hayes, Heather L.; Peterson, Brett S.; Haldeman, Jonathan M.; Newgard, Christopher B.; Hohmeier, Hans E.

    2017-01-01

    Increased β-cell death coupled with the inability to replicate existing β-cells drives the decline in β-cell mass observed in the progression of both major forms of diabetes. Understanding endogenous mechanisms of islet cell survival could have considerable value for the development of novel strategies to limit β-cell loss and thereby promote β-cell recovery. Insulinoma cells have provided useful insight into β-cell death pathways but observations made in cell lines sometimes fail to translate to primary islets. Here, we report dramatic differences in the temporal regulation and engagement of the apoptotic program in primary rodent islets relative to the INS-1 derived 832/13 cell line. As expected, 832/13 cells rapidly induced cell stress markers in response to ER stress or DNA damage and were fully committed to apoptosis, resulting in >80% cell death within 24 h. In contrast, primary rat islets were largely refractory to cell death in response to ER stress and DNA damage, despite rapid induction of stress markers, such as XBP-1(s), CHOP, and PUMA. Gene expression profiling revealed a general suppression of pro-apoptotic machinery, such as Apaf-1 and caspase 3, and sustained levels of pro-survival factors, such as cIAP-1, cIAP-2, and XIAP, in rat islets. Furthermore, we observed sustained induction of autophagy following chronic ER stress and found that inhibition of autophagy rendered islet β-cells highly vulnerable to ER stress-induced cell death. We propose that islet β-cells dampen the apoptotic response to delay the onset of cell death, providing a temporal window in which autophagy can be activated to limit cellular damage and promote survival. PMID:28212395

  9. Autophagy: molecular machinery for self-eating

    PubMed Central

    Yorimitsu, T; Klionsky, DJ

    2006-01-01

    Autophagy is a highly conserved process in eukaryotes in which the cytoplasm, including excess or aberrant organelles, is sequestered into double-membrane vesicles and delivered to the degradative organelle, the lysosome/vacuole, for breakdown and eventual recycling of the resulting macromolecules. This process has an important role in various biological events such as adaptation to changing environmental conditions, cellular remodeling during development and differentiation, and determination of lifespan. Auto-phagy is also involved in preventing certain types of disease, although it may contribute to some pathologies. Recent studies have identified many components that are required to drive this complicated cellular process. Autophagy-related genes were first identified in yeast, but homologs are found in all eukaryotes. Analyses in a range of model systems have provided huge advances toward understanding the molecular basis of autophagy. Here we review our current knowledge on the machinery and molecular mechanism of autophagy. PMID:16247502

  10. Impairment of Atg5-dependent autophagic flux promotes paraquat- and MPP⁺-induced apoptosis but not rotenone or 6-hydroxydopamine toxicity.

    PubMed

    Garcia-Garcia, Aracely; Anandhan, Annandurai; Burns, Michaela; Chen, Han; Zhou, You; Franco, Rodrigo

    2013-11-01

    Controversial reports on the role of autophagy as a survival or cell death mechanism in dopaminergic cell death induced by parkinsonian toxins exist. We investigated the alterations in autophagic flux and the role of autophagy protein 5 (Atg5)-dependent autophagy in dopaminergic cell death induced by parkinsonian toxins. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP⁺) and rotenone, the pesticide paraquat, and the dopamine analog 6-hydroxydopamine (6-OHDA) was paralleled by increased autophagosome accumulation. However, when compared with basal autophagy levels using chloroquine, autophagosome accumulation was a result of impaired autophagic flux. Only 6-OHDA induced an increase in autophagosome formation. Overexpression of a dominant negative form of Atg5 increased paraquat- and MPP⁺-induced cell death. Stimulation of mammalian target of rapamycin (mTOR)-dependent signaling protected against cell death induced by paraquat, whereas MPP⁺-induced toxicity was enhanced by wortmannin, a phosphoinositide 3-kinase class III inhibitor, rapamycin, and trehalose, an mTOR-independent autophagy activator. Modulation of autophagy by either pharmacological or genetic approaches had no effect on rotenone or 6-OHDA toxicity. Cell death induced by parkinsonian neurotoxins was inhibited by the pan caspase inhibitor (Z-VAD), but only caspase-3 inhibition was able to decrease MPP⁺-induced cell death. Finally, inhibition of the lysosomal hydrolases, cathepsins, increased the toxicity by paraquat and MPP⁺, supporting a protective role of Atg5-dependent autophagy and lysosomes degradation pathways on dopaminegic cell death. These results demonstrate that in dopaminergic cells, Atg5-dependent autophagy acts as a protective mechanism during apoptotic cell death induced by paraquat and MPP⁺ but not during rotenone or 6-OHDA toxicity.

  11. A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

    PubMed Central

    Young, Christopher NJ; Sinadinos, Anthony; Lefebvre, Alexis; Chan, Philippe; Arkle, Stephen; Vaudry, David; Gorecki, Dariusz C

    2015-01-01

    P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmdmdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca2+ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy. PMID:25700737

  12. A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90.

    PubMed

    Young, Christopher N J; Sinadinos, Anthony; Lefebvre, Alexis; Chan, Philippe; Arkle, Stephen; Vaudry, David; Gorecki, Dariusz C

    2015-01-01

    P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd(mdx) mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca(2+) influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome--autophagy--and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.

  13. Regulation of Host Translational Machinery by African Swine Fever Virus

    PubMed Central

    Castelló, Alfredo; Quintas, Ana; Sánchez, Elena G.; Sabina, Prado; Nogal, Marisa; Carrasco, Luis; Revilla, Yolanda

    2009-01-01

    African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host's defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2α, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV

  14. Regulation of host translational machinery by African swine fever virus.

    PubMed

    Castelló, Alfredo; Quintas, Ana; Sánchez, Elena G; Sabina, Prado; Nogal, Marisa; Carrasco, Luis; Revilla, Yolanda

    2009-08-01

    African swine fever virus (ASFV), like other complex DNA viruses, deploys a variety of strategies to evade the host's defence systems, such as inflammatory and immune responses and cell death. Here, we analyse the modifications in the translational machinery induced by ASFV. During ASFV infection, eIF4G and eIF4E are phosphorylated (Ser1108 and Ser209, respectively), whereas 4E-BP1 is hyperphosphorylated at early times post infection and hypophosphorylated after 18 h. Indeed, a potent increase in eIF4F assembly is observed in ASFV-infected cells, which is prevented by rapamycin treatment. Phosphorylation of eIF4E, eIF4GI and 4E-BP1 is important to enhance viral protein production, but is not essential for ASFV infection as observed in rapamycin- or CGP57380-treated cells. Nevertheless, eIF4F components are indispensable for ASFV protein synthesis and virus spread, since eIF4E or eIF4G depletion in COS-7 or Vero cells strongly prevents accumulation of viral proteins and decreases virus titre. In addition, eIF4F is not only activated but also redistributed within the viral factories at early times of infection, while eIF4G and eIF4E are surrounding these areas at late times. In fact, other components of translational machinery such as eIF2alpha, eIF3b, eIF4E, eEF2 and ribosomal P protein are enriched in areas surrounding ASFV factories. Notably, the mitochondrial network is polarized in ASFV-infected cells co-localizing with ribosomes. Thus, translation and ATP synthesis seem to be coupled and compartmentalized at the periphery of viral factories. At later times after ASFV infection, polyadenylated mRNAs disappear from the cytoplasm of Vero cells, except within the viral factories. The distribution of these pools of mRNAs is similar to the localization of viral late mRNAs. Therefore, degradation of cellular polyadenylated mRNAs and recruitment of the translation machinery to viral factories may contribute to the inhibition of host protein synthesis, facilitating ASFV

  15. The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells.

    PubMed

    Jeon, Seung-Hyun; Kim, Se Hyun; Kim, Yeni; Kim, Yong Sik; Lim, Yoongho; Lee, Young Han; Shin, Soon Young

    2011-09-23

    In this study, we investigated the antitumor effects of the tricyclic antidepressant 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (imipramine) on glioma cells. We found that exposure of U-87MG cells to imipramine resulted in the inhibition of PI3K/Akt/mTOR signaling, reduction of clonogenicity, and induction of cell death. Imipramine stimulated the formation of acidic vesicular organelles, the conversion of LC3-I to LC3-II, and the redistribution of LC3 to autophagosomes, suggesting that it stimulates the progression of autophagy. It did not, however, induce apoptosis. We further showed that knockdown of Beclin-1 using siRNA abrogated imipramine-induced cell death. These results suggest that imipramine exerts antitumor effects on PTEN-null U-87MG human glioma cells by inhibiting PI3K/Akt/mTOR signaling and by inducing autophagic cell death.

  16. The autophagic tumor stroma model of cancer or “battery-operated tumor growth”

    PubMed Central

    Martinez-Outschoorn, Ubaldo E; Whitaker-Menezes, Diana; Pavlides, Stephanos; Chiavarina, Barbara; Bonuccelli, Gloria; Trimmer, Casey; Tsirigos, Aristotelis; Migneco, Gemma; Witkiewicz, Agnieszka K; Balliet, Renee; Mercier, Isabelle; Wang, Chengwang; Flomenberg, Neal; Howell, Anthony; Lin, Zhao; Caro, Jaime; Pestell, Richard G

    2010-01-01

    The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the “Autophagy Paradox.” We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Cell Metabolism” or “Battery-Operated Tumor Growth.” In this sense, autophagy in the tumor stroma serves as a “battery” to fuel tumor growth, progression and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients—both effectively “starving” cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy by the upregulation of natural, endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor

  17. Caspase-independent autophagic cytotoxicity in etoposide-treated CaSki cervical carcinoma cells.

    PubMed

    Lee, Seung-Baek; Tong, Seo-Yun; Kim, Jung-Jin; Um, Soo-Jong; Park, Jong-Sup

    2007-10-01

    We studied the in vitro mechanism of etoposide-induced cell death in cervical cancer cells. Etoposide is cytotoxic to these cells, causing cell death by both apoptosis and autophagy, which has recently been described as a possible mechanism for nonapoptotic cell death. Electron microscopy revealed that autophagosomes/autolysosomes exhibited an autophagic appearance in the presence of etoposide. When autophagy was blocked by inhibitors of autophagy, including 3-methyladenine, both the expression of beclin 1 protein and the antitumor effect of etoposide were suppressed. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, reduced etoposide-induced cytotoxicity in CaSki cells. Hence, autophagy and apoptosis likely occur concurrently in etoposide-treated cervical cancer cells.

  18. Interactions with LC3 and polyubiquitin chains link nbr1 to autophagic protein turnover.

    PubMed

    Waters, Sarah; Marchbank, Katie; Solomon, Ellen; Whitehouse, Caroline; Gautel, Mathias

    2009-06-18

    Nbr1, a ubiquitous kinase scaffold protein, contains a PB1, and a ubiquitin-associated (UBA) domain. We show here that the nbr1 UBA domain binds to lysine-48 and -63 linked polyubiquitin-B chains. Nbr1 also binds to the autophagic effector protein LC3-A via a novel binding site. Ubiquitin-binding, but not PB1-mediated p62/SQSTM1 interaction, is required to target nbr1 to LC3 and polyubiquitin-positive bodies. Nbr1 binds additionally to proteins implicated in ubiquitin-mediated protein turnover and vesicle trafficking: ubiquitin-specific peptidases USP8, and the endosomal transport regulator p14/Robld3. Nbr1 thus contributes to specific steps in protein turnover regulation disrupted in several hereditary human diseases.

  19. Methods to Study the BECN1 Interactome in the Course of Autophagic Responses.

    PubMed

    Antonioli, M; Ciccosanti, F; Dengjel, J; Fimia, G M

    2017-01-01

    Autophagy is an extremely dynamic process that mediates the rapid degradation of intracellular components in response to different stress conditions. The autophagic response is executed by specific protein complexes, whose function is regulated by posttranslational modifications and interactions with positive and negative regulators. A comprehensive analysis of how autophagy complexes are temporally modified upon stress stimuli is therefore particularly relevant to understand how this pathway is regulated. Here, we describe a method to define the protein-protein interaction network of a central complex involved in autophagy induction, the Beclin 1 complex. This method is based on the quantitative comparison of protein complexes immunopurified at different time points using a stable isotope labeling by amino acids in cell culture approach. Understanding how the Beclin 1 complex dynamically changes in response to different stress stimuli may provide useful insights to disclose novel molecular mechanisms responsible for the dysregulation of autophagy in pathological conditions, such as cancer, neurodegeneration, and infections.

  20. Resveratrol Enhances Autophagic Flux and Promotes Ox-LDL Degradation in HUVECs via Upregulation of SIRT1

    PubMed Central

    Zhang, Yanlin; Cao, Xueqin; Zhu, Wawa; Liu, Zhihua; Liu, Huihui; Zhou, Yande; Cao, Yongjun; Liu, Chunfeng; Xie, Ying

    2016-01-01

    Oxidized low-density lipoprotein- (Ox-LDL-) induced autophagy dysfunction in human vascular endothelial cells contributes to the development of atherosclerosis (AS). Resveratrol (RSV) protects against Ox-LDL-induced endothelium injury. The objective of this study was to determine the mechanisms underlying Ox-LDL-induced autophagy dysfunction and RSV-mediated protection in human umbilical vein endothelial cells (HUVECs). The results showed that Ox-LDL suppressed the expression of sirtuin 1 (SIRT1) and increased LC3-II and sequestosome 1 (p62) protein levels without altering p62 mRNA levels in HUVECs. Pretreatment with bafilomycin A1 (BafA1) to inhibit lysosomal degradation abrogated the Ox-LDL-induced increase in LC3-II protein level. Ox-LDL increased colocalization of GFP and RFP puncta in mRFP-GFP-tandem fluorescent LC3- (tf-LC3-) transfected cells. Moreover, Ox-LDL decreased the expression of mature cathepsin D and attenuated cathepsin D activity. Pretreatment with RSV increased the expression of SIRT1 and LC3-II and increased p62 protein degradation. RSV induced RFP-LC3 aggregation more than GFP-LC3 aggregation. RSV restored lysosomal function and promoted Ox-LDL degradation in HUVECs. All the protective effects of RSV were blocked after SIRT1 was knocked down. These findings demonstrated that RSV upregulated the expression of SIRT1, restored lysosomal function, enhanced Ox-LDL-induced impaired autophagic flux, and promoted Ox-LDL degradation through the autophagy-lysosome degradation pathway in HUVECs. PMID:27069532

  1. Varicella-Zoster Virus Infectious Cycle: ER Stress, Autophagic Flux, and Amphisome-Mediated Trafficking

    PubMed Central

    Grose, Charles; Buckingham, Erin M.; Carpenter, John E.; Kunkel, Jeremy P.

    2016-01-01

    Varicella-zoster virus (VZV) induces abundant autophagy. Of the nine human herpesviruses, the VZV genome is the smallest (~124 kbp), lacking any known inhibitors of autophagy, such as the herpes simplex virus ICP34.5 neurovirulence gene. Therefore, this review assesses the evidence for VZV-induced cellular stress, endoplasmic-reticulum-associated degradation (ERAD), and autophagic flux during the VZV infectious cycle. Even though VZV is difficult to propagate in cell culture, the biosynthesis of the both N- and O-linked viral glycoproteins was found to be abundant. In turn, this biosynthesis provided evidence of endoplasmic reticulum (ER) stress, including a greatly enlarged ER and a greatly diminished production of cellular glycoproteins. Other signs of ER stress following VZV infection included detection of the alternatively spliced higher-molecular-weight form of XBP1 as well as CHOP. VZV infection in cultured cells leads to abundant autophagosome production, as was visualized by the detection of the microtubule-associated protein 1 light chain 3-II (LC3-II). The degree of autophagy induced by VZV infection is comparable to that induced in uninfected cells by serum starvation. The inhibition of autophagic flux by chemicals such as 3-methyladenine or ATG5 siRNA, followed by diminished virus spread and titers, has been observed. Since the latter observation pointed to the virus assembly/trafficking compartments, we purified VZ virions by ultracentrifugation and examined the virion fraction for components of the autophagy pathway. We detected LC3-II protein (an autophagy marker) as well as Rab11 protein, a component of the endosomal pathway. We also observed that the virion-containing vesicles were single-walled; thus, they are not autophagosomes. These results suggested that some VZ virions after secondary envelopment were transported to the outer cell membrane in a vesicle derived from both the autophagy and endosomal pathways, such as an amphisome. Thus, these

  2. Hypercalcemia induces targeted autophagic degradation of aquaporin-2 at the onset of nephrogenic diabetes insipidus.

    PubMed

    Khositseth, Sookkasem; Charngkaew, Komgrid; Boonkrai, Chatikorn; Somparn, Poorichaya; Uawithya, Panapat; Chomanee, Nusara; Payne, D Michael; Fenton, Robert A; Pisitkun, Trairak

    2017-01-27

    Hypercalcemia can cause renal dysfunction such as nephrogenic diabetes insipidus (NDI), but the mechanisms underlying hypercalcemia-induced NDI are not well understood. To elucidate the early molecular changes responsible for this disorder, we employed mass spectrometry-based proteomic analysis of inner medullary collecting ducts (IMCD) isolated from parathyroid hormone-treated rats at onset of hypercalcemia-induced NDI. Forty-one proteins, including the water channel aquaporin-2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the downregulated proteins were associated with cytoskeletal protein binding, regulation of actin filament polymerization, and cell-cell junctions. Targeted LC-MS/MS and immunoblot studies confirmed the downregulation of 16 proteins identified in the initial proteomic analysis and in additional experiments using a vitamin D treatment model of hypercalcemia-induced NDI. Evaluation of transcript levels and estimated half-life of the downregulated proteins suggested enhanced protein degradation as the possible regulatory mechanism. Electron microscopy showed defective intercellular junctions and autophagy in the IMCD cells from both vitamin D- and parathyroid hormone-treated rats. A significant increase in the number of autophagosomes was confirmed by immunofluorescence labeling of LC3. Colocalization of LC3 and Lamp1 with aquaporin-2 and other downregulated proteins was found in both models. Immunogold electron microscopy revealed aquaporin-2 in autophagosomes in IMCD cells from both hypercalcemia models. Finally, parathyroid hormone withdrawal reversed the NDI phenotype, accompanied by termination of aquaporin-2 autophagic degradation and normalization of both nonphoshorylated and S256-phosphorylated aquaporin-2 levels. Thus, enhanced autophagic degradation of proteins plays an important role in the initial mechanism of hypercalcemic-induced NDI.

  3. Disruption in the autophagic process underlies the sensory neuropathy in dystonia musculorum mice.

    PubMed

    Ferrier, Andrew; De Repentigny, Yves; Lynch-Godrei, Anisha; Gibeault, Sabrina; Eid, Walaa; Kuo, Daniel; Zha, Xiaohui; Kothary, Rashmi

    2015-01-01

    A homozygous mutation in the DST (dystonin) gene causes a newly identified lethal form of hereditary sensory and autonomic neuropathy in humans (HSAN-VI). DST loss of function similarly leads to sensory neuron degeneration and severe ataxia in dystonia musculorum (Dst(dt)) mice. DST is involved in maintaining cytoskeletal integrity and intracellular transport. As autophagy is highly reliant upon stable microtubules and motor proteins, we assessed the influence of DST loss of function on autophagy using the Dst(dt-Tg4) mouse model. Electron microscopy (EM) revealed an accumulation of autophagosomes in sensory neurons from these mice. Furthermore, we demonstrated that the autophagic flux was impaired. Levels of LC3-II, a marker of autophagosomes, were elevated. Consequently, Dst(dt-Tg4) sensory neurons displayed impaired protein turnover of autophagosome substrate SQTSM1/p62 and of polyubiquitinated proteins. Interestingly, in a previously described Dst(dt-Tg4) mouse model that is partially rescued by neuronal specific expression of the DST-A2 isoform, autophagosomes, autolysosomes, and damaged organelles were reduced when compared to Dst(dt-Tg4) mutant mice. LC3-II, SQTSM1, polyubiquitinated proteins and autophagic flux were also restored to wild-type levels in the rescued mice. Finally, a significant decrease in DNAIC1 (dynein, axonemal, intermediate chain 1; the mouse ortholog of human DNAI1), a member of the DMC (dynein/dynactin motor complex), was noted in Dst(dt-Tg4) dorsal root ganglia and sensory neurons. Thus, DST-A2 loss of function perturbs late stages of autophagy, and dysfunctional autophagy at least partially underlies Dst(dt) pathogenesis. We therefore conclude that the DST-A2 isoform normally facilitates autophagy within sensory neurons to maintain cellular homeostasis.

  4. 29 CFR 1910.215 - Abrasive wheel machinery.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 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 with... omitted; and (ii) The spindle end, nut, and outer flange may be exposed on machines designed as...

  5. 29 CFR 1910.213 - Woodworking machinery requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ..., DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.213 Woodworking machinery requirements. (a) Machine construction general. (1) Each machine shall be so constructed... with the saw. (9) All belts, pulleys, gears, shafts, and moving parts shall be guarded in...

  6. 29 CFR 1910.213 - Woodworking machinery requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.213 Woodworking machinery requirements. (a) Machine construction general. (1) Each machine shall be so constructed... with the saw. (9) All belts, pulleys, gears, shafts, and moving parts shall be guarded in...

  7. 29 CFR 1910.213 - Woodworking machinery requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.213 Woodworking machinery requirements. (a) Machine construction general. (1) Each machine shall be so constructed... with the saw. (9) All belts, pulleys, gears, shafts, and moving parts shall be guarded in...

  8. 29 CFR 1910.213 - Woodworking machinery requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.213 Woodworking machinery requirements. (a) Machine construction general. (1) Each machine shall be so constructed... with the saw. (9) All belts, pulleys, gears, shafts, and moving parts shall be guarded in...

  9. 29 CFR 1910.213 - Woodworking machinery requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ..., DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Machinery and Machine Guarding § 1910.213 Woodworking machinery requirements. (a) Machine construction general. (1) Each machine shall be so constructed... with the saw. (9) All belts, pulleys, gears, shafts, and moving parts shall be guarded in...

  10. 46 CFR 169.315 - Ventilation (other than machinery spaces).

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Ventilation (other than machinery spaces). 169.315... SCHOOL VESSELS Construction and Arrangement Hull Structure § 169.315 Ventilation (other than machinery... provide adequate ventilation in all ordinary weather conditions. Provided that paragraph (a) of...

  11. 46 CFR 169.315 - Ventilation (other than machinery spaces).

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Ventilation (other than machinery spaces). 169.315... SCHOOL VESSELS Construction and Arrangement Hull Structure § 169.315 Ventilation (other than machinery... provide adequate ventilation in all ordinary weather conditions. Provided that paragraph (a) of...

  12. 46 CFR 169.315 - Ventilation (other than machinery spaces).

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Ventilation (other than machinery spaces). 169.315... SCHOOL VESSELS Construction and Arrangement Hull Structure § 169.315 Ventilation (other than machinery... provide adequate ventilation in all ordinary weather conditions. Provided that paragraph (a) of...

  13. Oblique view of southeast machinery house, SF 109, with NE ...

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

    Oblique view of southeast machinery house, SF 109, with NE machinery house in background, at downstream end of lock, view towards north northwest - St. Lucie Canal, St. Lucie Lock No. 1, St. Lucie, Cross State Canal, Okeechobee Intracoastal Waterway, Stuart, Martin County, FL

  14. 46 CFR 185.208 - 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. 185.208 Section 185.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) OPERATIONS Marine Casualties and Voyage Records § 185.208 Accidents to machinery. The owner,...

  15. 46 CFR 185.208 - 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. 185.208 Section 185.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) OPERATIONS Marine Casualties and Voyage Records § 185.208 Accidents to machinery. The owner,...

  16. 46 CFR 122.208 - 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. 122.208 Section 122.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS CARRYING MORE THAN 150... Voyage Records § 122.208 Accidents to machinery. The owner, managing operator, or master shall...

  17. 46 CFR 78.33-5 - Accidents to machinery.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 3 2010-10-01 2010-10-01 false Accidents to machinery. 78.33-5 Section 78.33-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 78.33-5 Accidents to machinery. (a) In the event of an...

  18. 46 CFR 78.33-5 - Accidents to machinery.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 3 2014-10-01 2014-10-01 false Accidents to machinery. 78.33-5 Section 78.33-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 78.33-5 Accidents to machinery. (a) In the event of an...

  19. 46 CFR 185.208 - 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. 185.208 Section 185.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) OPERATIONS Marine Casualties and Voyage Records § 185.208 Accidents to machinery. The owner,...

  20. 46 CFR 78.33-5 - Accidents to machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 3 2013-10-01 2013-10-01 false Accidents to machinery. 78.33-5 Section 78.33-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 78.33-5 Accidents to machinery. (a) In the event of an...

  1. 46 CFR 122.208 - 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. 122.208 Section 122.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS CARRYING MORE THAN 150... Voyage Records § 122.208 Accidents to machinery. The owner, managing operator, or master shall...

  2. 46 CFR 185.208 - 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. 185.208 Section 185.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) OPERATIONS Marine Casualties and Voyage Records § 185.208 Accidents to machinery. The owner,...

  3. 46 CFR 78.33-5 - Accidents to machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 3 2011-10-01 2011-10-01 false Accidents to machinery. 78.33-5 Section 78.33-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 78.33-5 Accidents to machinery. (a) In the event of an...

  4. 46 CFR 122.208 - 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. 122.208 Section 122.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS CARRYING MORE THAN 150... Voyage Records § 122.208 Accidents to machinery. The owner, managing operator, or master shall...

  5. 46 CFR 122.208 - 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. 122.208 Section 122.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS CARRYING MORE THAN 150... Voyage Records § 122.208 Accidents to machinery. The owner, managing operator, or master shall...

  6. 46 CFR 122.208 - 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. 122.208 Section 122.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS CARRYING MORE THAN 150... Voyage Records § 122.208 Accidents to machinery. The owner, managing operator, or master shall...

  7. 46 CFR 78.33-5 - Accidents to machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 3 2012-10-01 2012-10-01 false Accidents to machinery. 78.33-5 Section 78.33-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PASSENGER VESSELS OPERATIONS Reports of Accidents, Repairs, and Unsafe Equipment § 78.33-5 Accidents to machinery. (a) In the event of an...

  8. 46 CFR 185.208 - 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. 185.208 Section 185.208 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) OPERATIONS Marine Casualties and Voyage Records § 185.208 Accidents to machinery. The owner,...

  9. 4. FIRST FLOOR INTERIOR, AMMONIA COMPRESSION DYNAMOS IN MACHINERY ROOM ...

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

    4. FIRST FLOOR INTERIOR, AMMONIA COMPRESSION DYNAMOS IN MACHINERY ROOM ALONG SOUTH SIDE OF WESTERN PORTION OF BUILDING, FROM EASTERN ENTRANCE TO MACHINERY ROOM, LOOKING WEST. - Oakland Naval Supply Center, Cold Storage Warehouse, South of C Street between First & Second Street, Oakland, Alameda County, CA

  10. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... refrigerating machinery. (a) Where refrigerating machines are installed in which anhydrous ammonia is used as a... a solution of aqua ammonia and machines using carbon dioxide are exempt from this requirement...) Machinery compartments containing equipment for ammonia shall be fitted with a sprinkler system providing...

  11. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... refrigerating machinery. (a) Where refrigerating machines are installed in which anhydrous ammonia is used as a... a solution of aqua ammonia and machines using carbon dioxide are exempt from this requirement...) Machinery compartments containing equipment for ammonia shall be fitted with a sprinkler system providing...

  12. 46 CFR 58.20-15 - Installation of refrigerating machinery.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... refrigerating machinery. (a) Where refrigerating machines are installed in which anhydrous ammonia is used as a... a solution of aqua ammonia and machines using carbon dioxide are exempt from this requirement...) Machinery compartments containing equipment for ammonia shall be fitted with a sprinkler system providing...

  13. Machinery Management. FMO: Fundamentals of Machine Operation. Third Edition.

    ERIC Educational Resources Information Center

    Bowers, Wendell

    This text is intended to provide a basic understanding of selecting, maintaining, and managing farm machinery. The following topics are covered in the individual chapters: dealing with typical problems in farm machinery management; measuring machine capacity; improving field efficiency; matching machine size and capacity; estimating power…

  14. Automated Recognition of Advanced Vibration Features for Machinery Fault Classification

    DTIC Science & Technology

    2001-04-05

    fault " using transitional failure data for commercial grade gearboxes . Features will be extracted from accelerometer data obtained on the Mechanical...Machinery Fault Classification DISTRIBUTION: Approved for public release, distribution unlimited This paper is part of the following report: TITLE: New...thru ADP013516 UNCLASSIFIED AUTOMATED RECOGNITION OF ADVANCED VIBRATION FEATURES FOR MACHINERY FAULT CLASSIFICATION Katherine McClintic, Robert Campbell

  15. Ketamine regulates the presynaptic release machinery in the hippocampus.

    PubMed

    Müller, Heidi Kaastrup; Wegener, Gregers; Liebenberg, Nico; Zarate, Carlos A; Popoli, Maurizio; Elfving, Betina

    2013-07-01

    In the search for new drug targets, that may help point the way to develop fast-acting treatments for mood disorders, we have explored molecular pathways regulated by ketamine, an NMDA receptor antagonist, which has consistently shown antidepressant response within a few hours of administration. Using Sprague-Dawley rats we investigated the effects of ketamine on the presynaptic release machinery responsible for neurotransmitter release at 1, 2 and 4 h as well as 7 days after administration of a single subanesthetic dose of ketamine (15 mg/kg). A large reduction in the accumulation of SNARE complexes was observed in hippocampal synaptic membranes after 1, 2 and 4 h of ketamine administration. In parallel, we found a selective reduction in the expression of the synaptic vesicle protein synaptotagmin I and an increase in the levels of synapsin I in hippocampal synaptosomes suggesting a mechanism by which ketamine reduces SNARE complex formation, in part, by regulating the number of synaptic vesicles in the nerve terminals. Moreover, ketamine reduced Thr(286)-phosphorylated αCaMKII and its interaction with syntaxin 1A, which identifies CaMKII as a potential target for second messenger-mediated actions of ketamine. In addition, despite previous reports of ketamine-induced inhibition of GSK-3, we were unable to detect regulation of its activity after ketamine administration. Our findings demonstrate that ketamine rapidly induces changes in the hippocampal presynaptic machinery similar to those that are obtained only with chronic treatments with traditional antidepressants. This suggests that reduction of neurotransmitter release in the hippocampus has possible relevance for the rapid antidepressant effect of ketamine.

  16. Ketamine regulates the presynaptic release machinery in the hippocampus

    PubMed Central

    Müller, Heidi Kaastrup; Wegener, Gregers; Liebenberg, Nico; Zarate, Carlos A.; Popoli, Maurizio; Elfving, Betina

    2013-01-01

    In the search for new drug targets, that may help point the way to develop fast-acting treatments for mood disorders, we have explored molecular pathways regulated by ketamine, an NMDA receptor antagonist, which has consistently shown antidepressant response within a few hours of administration. Using Sprague-Dawley rats we investigated the effects of ketamine on the presynaptic release machinery responsible for neurotransmitter release at 1, 2 and 4 h as well as 7 days after administration of a single subanesthetic dose of ketamine (15 mg/kg). A large reduction in the accumulation of SNARE complexes was observed in hippocampal synaptic membranes after 1, 2 and 4 h of ketamine administration. In parallel, we found a selective reduction in the expression of the synaptic vesicle protein synaptotagmin I and an increase in the levels of synapsin I in hippocampal synaptosomes suggesting a mechanism by which ketamine reduces SNARE complex formation, in part, by regulating the number of synaptic vesicles in the nerve terminals. Moreover, ketamine reduced Thr286-phosphorylated αCaMKII and its interaction with syntaxin 1A, which identifies CaMKII as a potential target for second messenger-mediated actions of ketamine. In addition, despite previous reports of ketamine-induced inhibition of GSK-3, we were unable to detect regulation of its activity after ketamine administration. Our findings demonstrate that ketamine rapidly induces changes in the hippocampal presynaptic machinery similar to those that are obtained only with chronic treatments with traditional antidepressants. This suggests that reduction of neurotransmitter release in the hippocampus has possible relevance for the rapid antidepressant effect of ketamine. PMID:23548331

  17. Cilostazol Modulates Autophagic Degradation of β-Amyloid Peptide via SIRT1-Coupled LKB1/AMPKα Signaling in Neuronal Cells

    PubMed Central

    Lee, Won Suk; Shin, Hwa Kyoung; Kim, Hye Young; Hong, Ki Whan; Kim, Chi Dae

    2016-01-01

    A neuroprotective role of autophagy mediates the degradation of β-amyloid peptide (Aβ) in Alzheimer’s disease (AD). The previous study showed cilostazol modulates autophagy by increasing beclin1, Atg5 and LC3-II expressions, and depletes intracellular Aβ accumulation. This study elucidated the mechanisms through which cilostazol modulates the autophagic degradation of Aβ in neurons. In N2a cells, cilostazol (10–30 μM), significantly increased the expression of P-AMPKα (Thr 172) and downstream P-ACC (acetyl-CoA carboxylase) (Ser 79) as did resveratrol (SIRT1 activator), or AICAR (AMPK activator), which were blocked by KT5720, compound C (AMPK inhibitor), or sirtinol. Furthermore, phosphorylated-mTOR (Ser 2448) and phosphorylated-P70S6K (Thr 389) expressions were suppressed, and LC3-II levels were elevated in association with decreased P62/Sqstm1 by cilostazol. Cilostazol increased cathepsin B activity and decreased p62/SQSTM 1, consequently decreased accumulation of Aβ1–42 in the activated N2aSwe cells, and these results were blocked by sirtinol, compound C and bafilomycin A1 (autophagosome blocker), suggesting enhanced autophagosome formation by cilostazol. In SIRT1 gene-silenced N2a cells, cilostazol failed to increase the expressions of P-LKB1 (Ser 428) and P-AMPKα, which contrasted with its effect in negative control cells transfected with scrambled siRNA duplex. Further, N2a cells transfected with expression vectors encoding pcDNA SIRT1 showed increased P-AMPKα expression, which mimicked the effect of cilostazol in N2a cells; suggesting cilostazol-stimulated expressions of P-LKB1 and P-AMPKα were SIRT1-dependent. Unlike their effects in N2a cells, in HeLa cells, which lack LKB1, cilostazol and resveratrol did not elevate SIRT1 or P-AMPKα expression, indicating cilostazol and resveratrol-stimulated expressions of SIRT1 and P-AMPKα are LKB1-dependent. In conclusion, cilostazol upregulates autophagy by activating SIRT1-coupled P-LKB1/P-AMPKα and

  18. SWI/SNF-dependent chromatin remodeling of RNR3 requires TAFIIs and the general transcription machinery

    PubMed Central

    Sharma, Vishva Mitra; Li, Bing; Reese, Joseph C.

    2003-01-01

    Gene expression requires the recruitment of chromatin remodeling activities and general transcription factors (GTFs) to promoters. Whereas the role of activators in recruiting chromatin remodeling activities has been clearly demonstrated, the contributions of the transcription machinery have not been firmly established. Here we demonstrate that the remodeling of the RNR3 promoter requires a number of GTFs, mediator and RNA polymerase II. We also show that remodeling is dependent upon the SWI/SNF complex, and that TFIID and RNA polymerase II are required for its recruitment to the promoter. In contrast, Gcn5p-dependent histone acetylation occurs independently of TFIID and RNA polymerase II function, and we provide evidence that acetylation increases the extent of nucleosome remodeling, but is not required for SWI/SNF recruitment. Thus, the general transcription machinery can contribute to nucleosome remodeling by mediating the association of SWI/SNF with promoters, thereby revealing a novel pathway for the recruitment of chromatin remodeling activities. PMID:12600943

  19. Activation of Peroxisome Proliferator-activated Receptor α Induces Lysosomal Biogenesis in Brain Cells

    PubMed Central

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J.; Sims, Katherine B.; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-01-01

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174

  20. Interactions of release factor RF3 with the translation machinery.

    PubMed

    O'Connor, Michael

    2015-08-01

    The bacterial release factor RF3 is a GTPase that has been implicated in multiple, incompletely understood steps of protein synthesis. This study explores the genetic interaction of RF3 with other components of the translation machinery. RF3 contributes to translation termination by recycling the class I release factors RF1 and RF2 off post-termination ribosomes. RF3 has also been implicated in dissociation of peptidyl-tRNAs from elongating ribosomes and in a post-peptidyltransferase quality control (post-PT QC) mechanism that selectively terminates ribosomes carrying erroneous peptides. A majority of the in vivo studies on RF3 have been carried out in K-12 strains of Escherichia coli which carry a partially defective RF2 protein with an Ala to Thr substitution at position 246. Here, the contribution of the K-12 specific RF2 variant to RF3 activities has been investigated. Strain reconstruction experiments in both E. coli and Salmonella enterica demonstrate that defects in termination and post-PT QC that are associated with RF3 loss, as well as phenotypes uncovered by phenotypic profiling, are all substantially ameliorated when the incompletely active K-12-specific RF2 protein is replaced by a fully active Ala246 RF2. These results indicate that RF3 loss is well tolerated in bacteria with fully active class I release factors, but that many of the previously reported phenotypes for RF3 deletion strains have been compromised by the presence of a partially defective RF2.

  1. Studying p53 family proteins in yeast: Induction of autophagic cell death and modulation by interactors and small molecules

    SciTech Connect

    Leão, Mariana; Gomes, Sara; Bessa, Cláudia; Soares, Joana; Raimundo, Liliana; Monti, Paola; Fronza, Gilberto; Pereira, Clara; Saraiva, Lucília

    2015-01-01

    In this work, the yeast Saccharomyces cerevisiae was used to individually study human p53, p63 (full length and truncated forms) and p73. Using this cell system, the effect of these proteins on cell proliferation and death, and the influence of MDM2 and MDMX on their activities were analyzed. When expressed in yeast, wild-type p53, TAp63, ΔNp63 and TAp73 induced growth inhibition associated with S-phase cell cycle arrest. This growth inhibition was accompanied by reactive oxygen species production and autophagic cell death. Furthermore, they stimulated rapamycin-induced autophagy. On the contrary, none of the tested p53 family members induced apoptosis either per se or after apoptotic stimuli. As previously reported for p53, also TAp63, ΔNp63 and TAp73 increased actin expression levels and its depolarization, suggesting that ACT1 is also a p63 and p73 putative yeast target gene. Additionally, MDM2 and MDMX inhibited the activity of all tested p53 family members in yeast, although the effect was weaker on TAp63. Moreover, Nutlin-3a and SJ-172550 were identified as potential inhibitors of the p73 interaction with MDM2 and MDMX, respectively. Altogether, the yeast-based assays herein developed can be envisaged as a simplified cell system to study the involvement of p53 family members in autophagy, the modulation of their activities by specific interactors (MDM2 and MDMX), and the potential of new small molecules to modulate these interactions. - Highlights: • p53, p63 and p73 are individually studied in the yeast S. cerevisiae. • p53 family members induce ROS production, cell cycle arrest and autophagy in yeast. • p53 family members increase actin depolarization and expression levels in yeast. • MDM2 and MDMX inhibit the activity of p53 family members in yeast. • Yeast can be a useful tool to study the biology and drugability of p53, p63 and p73.

  2. Occupational Accidents with Agricultural Machinery in Austria.

    PubMed

    Kogler, Robert; Quendler, Elisabeth; Boxberger, Josef

    2016-01-01

    The number of recognized accidents with fatalities during agricultural and forestry work, despite better technology and coordinated prevention and trainings, is still very high in Austria. The accident scenarios in which people are injured are very different on farms. The common causes of accidents in agriculture and forestry are the loss of control of machine, means of transport or handling equipment, hand-held tool, and object or animal, followed by slipping, stumbling and falling, breakage, bursting, splitting, slipping, fall, and collapse of material agent. In the literature, a number of studies of general (machine- and animal-related accidents) and specific (machine-related accidents) agricultural and forestry accident situations can be found that refer to different databases. From the database Data of the Austrian Workers Compensation Board (AUVA) about occupational accidents with different agricultural machinery over the period 2008-2010 in Austria, main characteristics of the accident, the victim, and the employer as well as variables on causes and circumstances by frequency and contexts of parameters were statistically analyzed by employing the chi-square test and odds ratio. The aim of the study was to determine the information content and quality of the European Statistics on Accidents at Work (ESAW) variables to evaluate safety gaps and risks as well as the accidental man-machine interaction.

  3. Global Design Optimization for Fluid Machinery Applications

    NASA Technical Reports Server (NTRS)

    Shyy, Wei; Papila, Nilay; Tucker, Kevin; Vaidyanathan, Raj; Griffin, Lisa

    2000-01-01

    Recent experiences in utilizing the global optimization methodology, based on polynomial and neural network techniques for fluid machinery design are summarized. Global optimization methods can utilize the information collected from various sources and by different tools. These methods offer multi-criterion optimization, handle the existence of multiple design points and trade-offs via insight into the entire design space can easily perform tasks in parallel, and are often effective in filtering the noise intrinsic to numerical and experimental data. Another advantage is that these methods do not need to calculate the sensitivity of each design variable locally. However, a successful application of the global optimization method needs to address issues related to data requirements with an increase in the number of design variables and methods for predicting the model performance. Examples of applications selected from rocket propulsion components including a supersonic turbine and an injector element and a turbulent flow diffuser are used to illustrate the usefulness of the global optimization method.

  4. A genome-wide siRNA screen for regulators of tumor suppressor p53 activity in human non-small lung cancer cells identifies components of the RNA splicing machinery as targets for anticancer treatment.

    PubMed

    Siebring-van Olst, Ellen; Blijlevens, Maxime; de Menezes, Renee X; van der Meulen-Muileman, Ida H; Smit, Egbert F; van Beusechem, Victor W

    2017-03-13

    Reinstating wild-type tumor suppressor p53 activity could be a valuable option for the treatment of cancer. To contribute to development of new treatment options for non-small cell lung cancer (NSCLC), we performed genome-wide siRNA screens for determinants of p53 activity in NSCLC cells. We identified many genes not previously known to be involved in regulating p53 activity. Silencing p53 pathway inhibitor genes was associated with loss of cell viability. The largest functional gene cluster influencing p53 activity was mRNA splicing. Prominent p53 activation was observed upon silencing of specific spliceosome components, rather than by general inhibition of the spliceosome. Ten genes were validated as inhibitors of p53 activity in multiple NSCLC cell lines: genes encoding the Ras-pathway activator SOS1, the zinc finger protein TSHZ3, the mitochondrial membrane protein COX16 and the spliceosome components SNRPD3, SF3A3, SF3B1, SF3B6, XAB2, CWC22 and HNRNPL. Silencing these genes generally increased p53 levels, with distinct effects on CDKN1A expression, induction of cell cycle arrest and cell death. Silencing spliceosome components was associated with alternative splicing of MDM4 mRNA, which could contribute to activation of p53. In addition, silencing splice factors was particularly effective in killing NSCLC cells, albeit in a p53-independent manner. Interestingly, silencing SNRPD3 and SF3A3 exerted much stronger cytotoxicity to NSCLC cells than to lung fibroblasts, suggesting that these genes could represent useful therapeutic targets. This article is protected by copyright. All rights reserved.

  5. Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain.

    PubMed

    Tabata, Keisuke; Matsunaga, Kohichi; Sakane, Ayuko; Sasaki, Takuya; Noda, Takeshi; Yoshimori, Tamotsu

    2010-12-01

    The endocytic and autophagic pathways are involved in the membrane trafficking of exogenous and endogenous materials to lysosomes. However, the mechanisms that regulate these pathways are largely unknown. We previously reported that Rubicon, a Beclin 1-binding protein, negatively regulates both the autophagic and endocytic pathways by unidentified mechanisms. In this study, we performed database searches to identify potential Rubicon homologues that share the common C-terminal domain, termed the RH domain. One of them, PLEKHM1, the causative gene of osteopetrosis, also suppresses endocytic transport but not autophagosome maturation. Rubicon and PLEKHM1 specifically and directly interact with Rab7 via their RH domain, and this interaction is critical for their function. Furthermore, we show that Rubicon but not PLEKHM1 uniquely regulates membrane trafficking via simultaneously binding both Rab7 and PI3-kinase.

  6. Mitochondria hyperfusion and elevated autophagic activity are key mechanisms for cellular bioenergetic preservation in centenarians

    PubMed Central

    Pinti, Marcello; Lanzarini, Catia; Ascione, Barbara; Gibellini, Lara; Dika, Emi; Patrizi, Annalisa; Tommasino, Chiara; Capri, Miriam; Cossarizza, Andrea; Baracca, Alessandra; Lenaz, Giorgio; Solaini, Giancarlo; Franceschi, Claudio; Malorni, Walter; Salvioli, Stefano

    2014-01-01

    Mitochondria have been considered for long time as important determinants of cell aging because of their role in the production of reactive oxygen species. In this study we investigated the impact of mitochondrial metabolism and biology as determinants of successful aging in primary cultures of fibroblasts isolated from the skin of long living individuals (LLI) (about 100 years old) compared with those from young (about 27 years old) and old (about 75 years old) subjects. We observed that fibroblasts from LLI displayed significantly lower complex I-driven ATP synthesis and higher production of H2O2 in comparison with old subjects. Despite these changes, bioenergetics of these cells appeared to operate normally. This lack of functional consequences was likely due to a compensatory phenomenon at the level of mitochondria, which displayed a maintained supercomplexes organization and an increased mass. This appears to be due to a decreased mitophagy, induced by hyperfused, elongated mitochondria. The overall data indicate that longevity is characterized by a preserved bioenergetic function likely attained by a successful mitochondria remodeling that can compensate for functional defects through an increase in mass, i.e. a sort of mitochondrial “hypertrophy”. PMID:24799450

  7. Protective Effect of Sevoflurane Postconditioning against Cardiac Ischemia/Reperfusion Injury via Ameliorating Mitochondrial Impairment, Oxidative Stress and Rescuing Autophagic Clearance

    PubMed Central

    Yu, Shuchun; Luo, Zhenzhong; Hua, Fuzhou; Yuan, Linhui; Zhou, Zhidong; Liu, Qin; Du, Xiaohong; Chen, Sisi; Zhang, Lieliang; Xu, Guohai

    2015-01-01

    Background and Purpose Myocardial infarction leads to heart failure. Autophagy is excessively activated in myocardial ischemia/reperfusion (I/R) in rats. The aim of this study is to investigate whether the protection of sevoflurane postconditioning (SPC) in myocardial I/R is through restored impaired autophagic flux. Methods Except for the sham control (SHAM) group, each rat underwent 30 min occlusion of the left anterior descending coronary (LAD) followed by 2 h reperfusion. Cardiac infarction was determined by 2,3,5-triphenyltetrazolium chloride triazole (TTC) staining. Cardiac function was examined by hemodynamics and echocardiography. The activation of autophagy was evaluated by autophagosome accumulation, LC3 conversion and p62 degradation. Potential molecular mechanisms were investigated by immunoblotting, real-time PCR and immunofluorescence staining. Results SPC improved the hemodynamic parameters, cardiac dysfunction, histopathological and ultrastructural damages, and decreased myocardial infarction size after myocardial I/R injury (P < 0.05 vs. I/R group). Compared with the cases in I/R group, myocardial ATP and NAD+ content, mitochondrial function related genes and proteins, and the expressions of SOD2 and HO-1 were increased, while the expressions of ROS and Vimentin were decreased in the SPC group (P < 0.05 vs. I/R group). SPC significantly activated Akt/mTOR signaling, and inhibited the formation of Vps34/Beclin1 complex via increasing expression of Bcl2 protein (P < 0.05 vs. I/R group). SPC suppressed elevated expressions of LC3 II/I ratio, Beclin1, Atg5 and Atg7 in I/R rat, which indicated that SPC inhibited over-activation of autophagy, and promoted autophagosome clearance. Meanwhile, SPC significantly suppressed the decline of Opa1 and increases of Drp1 and Parkin induced by I/R injury (P < 0.05 vs. I/R group). Moreover, SPC maintained the contents of ATP by reducing impaired mitochondria. Conclusion SPC protects rat hearts against I/R injury via

  8. Navigation of Construction and Agriculture Machinery

    NASA Astrophysics Data System (ADS)

    Stempfhuber, Werner

    2008-09-01

    Over the last two decades terrestrial and global 3D measurement sensors in the field of engineering geodesy have seen a significant upturn. With modern measurement techniques, a 3D trajectory of a moving object can be determined within a few centimetres (mostly with Global Navigation Satellite Systems, GNSS), under certain circumstances and with an overall understanding of the applied method accuracies of within 5 to 10 millimetres can be achieved (tracking total station). New application areas have been now created in the fields of construction, mining and agriculture. The guidance or control of heavy machinery demands a navigation sensor with an appropriate measurement rate and accuracy, as well as stable and reliable performance. The 3D position, together with the orientation as well as the long and cross inclination information is hereby just one part of the absolute machine guidance or control unit. Data collection, verification, management and interaction of the position information with the 6 degrees of freedom, together and the machine controller, are needed for the overall system. Rotation ring sensors for height control or height guidance are well-known amongst construction jobs and have been in use for more than 20 years. The first GPS-based guidance system for yield mapping was used 15 years ago (Auernhammer 1995). Optimization and improvements in efficiency are the principal reasons for the current developments in the area of 3D-based machine control and guidance. This paper will describe the state-of-the-art and general approaches as well as the real-time 3D measurement techniques in construction and agriculture environment.

  9. 18. INTERIOR VIEW OF THE GENERATOR HOUSE, SHOWING CONCRETE MACHINERY ...

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

    18. INTERIOR VIEW OF THE GENERATOR HOUSE, SHOWING CONCRETE MACHINERY FOOTINGS, LOOKING SOUTHWEST. - Tower Hill No. 2 Mine, Approximately 0.47 mile Southwest of intersection of Stone Church Road & Township Route 561, Hibbs, Fayette County, PA

  10. Therapeutic interventions to disrupt the protein synthetic machinery in melanoma.

    PubMed

    Kardos, Gregory R; Robertson, Gavin P

    2015-09-01

    Control of the protein synthetic machinery is deregulated in many cancers, including melanoma, to increase the protein production. Tumor suppressors and oncogenes play key roles in protein synthesis from the transcription of rRNA and ribosome biogenesis to mRNA translation initiation and protein synthesis. Major signaling pathways are altered in melanoma to modulate the protein synthetic machinery, thereby promoting tumor development. However, despite the importance of this process in melanoma development, involvement of the protein synthetic machinery in this cancer type is an underdeveloped area of study. Here, we review the coupling of melanoma development to deregulation of the protein synthetic machinery. We examine existing knowledge regarding RNA polymerase I inhibition and mRNA translation focusing on their inhibition for therapeutic applications in melanoma. Furthermore, the contribution of amino acid biosynthesis and involvement of ribosomal proteins are also reviewed as future therapeutic strategies to target deregulated protein production in melanoma.

  11. 87. DAM TAINTER GATE OPERATING MACHINERY TRAVELING HOIST ...

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

    87. DAM - TAINTER GATE OPERATING MACHINERY - TRAVELING HOIST - AMERICAN TYPE ASSEMBLY (ML-5-55/111-FS), February 1938 - Upper Mississippi River 9-Foot Channel Project, Lock & Dam No. 5, Minneiska, Winona County, MN

  12. 101. STARBOARD AIRPLANE ELEVATOR MACHINERY ROOM AFT LOOKING FORWARD ...

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

    101. STARBOARD AIRPLANE ELEVATOR MACHINERY ROOM - AFT LOOKING FORWARD PORT TO STARBOARD SHOWING ELEVATOR ENGINE, LIFTING WIRES, HYDRAULIC PIPING WITH REMOTE OPERATOR. - U.S.S. HORNET, Puget Sound Naval Shipyard, Sinclair Inlet, Bremerton, Kitsap County, WA

  13. 14. MACHINERY DETAILS: CENTER WHEEL FRAME AND AXEL, JACK WHEEL ...

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

    14. MACHINERY DETAILS: CENTER WHEEL FRAME AND AXEL, JACK WHEEL FRAME, JACK NUT HOUSING, JACK NUT, ETC. - Niantic River Swing Bridge, Spanning Niantic River between East Lyme & Waterford, Old Lyme, New London County, CT

  14. 62. VIEW SHOWING INSTALLATION TAINTER VALVE MACHINERY MONOLITH NO. 321, ...

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

    62. VIEW SHOWING INSTALLATION TAINTER VALVE MACHINERY MONOLITH NO. 32-1, LOOKING WEST Photograph No. 8571. October 24, 1949 - Upper Mississippi River 9-Foot Channel Project, Lock & Dam 27, Granite City, Madison County, IL

  15. 15. MACHINERY DETAILS: LATCH WHEEL BRACKET, LATCH POCKET, LOCK BAR, ...

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

    15. MACHINERY DETAILS: LATCH WHEEL BRACKET, LATCH POCKET, LOCK BAR, LATCH CRADLE, SPLIT COLLAR, ETC. - Niantic River Swing Bridge, Spanning Niantic River between East Lyme & Waterford, Old Lyme, New London County, CT

  16. 16. DETAIL VIEW OF AUXILIARY LOCK MITER GATE OPERATING MACHINERY ...

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

    16. DETAIL VIEW OF AUXILIARY LOCK MITER GATE OPERATING MACHINERY LOCATED IN INTERMEDIATE WALL, LOOKING NORTHWEST - Upper Mississippi River 9-Foot Channel Project, Lock & Dam 27, Granite City, Madison County, IL

  17. 51. FIRST FLOOR INTERIOR VIEW TO SOUTHEAST: Winding machinery to ...

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

    51. FIRST FLOOR INTERIOR VIEW TO SOUTHEAST: Winding machinery to right, and belt-driven machine tools to left. Museum mezzanine in upper portion of photo. - San Francisco Cable Railway, Washington & Mason Streets, San Francisco, San Francisco County, CA

  18. Building, roof, with machinery penthouses on left and harbor control ...

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

    Building, roof, with machinery penthouses on left and harbor control tower on right. Camera facing south - Naval Supply Center, Broadway Complex, Warehouse, 911 West Broadway, San Diego, San Diego County, CA

  19. 21. Detail of remains of machinery house viewed from below ...

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

    21. Detail of remains of machinery house viewed from below anchor-span deck, showing drawspan cable running back to the winding drum of the winch; view to northeast. - Summer Street Bridge, Spanning Reserved Channel, Boston, Suffolk County, MA

  20. 7. Detail, machinery shed atop east portal of Tunnel 28, ...

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

    7. Detail, machinery shed atop east portal of Tunnel 28, showing shaft and pulley system, 210mm lens with electronic flash fill. - Central Pacific Transcontinental Railroad, Tunnel No. 28, Milepost 134.75, Applegate, Placer County, CA

  1. 46. Interior detail of barge crane engine house with machinery. ...

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

    46. Interior detail of barge crane engine house with machinery. Lambert Hoisting Engine Company Winder Powered by A Gray Marine Diesel. - Barbour Boat Works, Tryon Palace Drive, New Bern, Craven County, NC

  2. 5. INTERIOR, FIRST FLOOR, WEST VIEW OF MACHINERY FRAME, SHOWING ...

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

    5. INTERIOR, FIRST FLOOR, WEST VIEW OF MACHINERY FRAME, SHOWING MAIN VERTICAL SHAFT WITH CAST-IRON WALLOWER GEAR AT LOWER END, AND THE GREAT SPUR WHEEL OF ALL WOODEN CONSTRUCTION - Robeson-Williams Grist Mill, Roslyn, Nassau County, NY

  3. 20. DETAIL VIEW OF MOVABLE SPAN MACHINERY UNDER GRID DECK ...

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

    20. DETAIL VIEW OF MOVABLE SPAN MACHINERY UNDER GRID DECK AT CENTRAL CONCRETE SUPPORT PEDESTAL, SHOWING DRIVE GEARS, ELECTRIC MOTOR AND STEEL BEAMS (taken in January 1984) - Sharptown Bridge, Spanning Nanticoke River, State Route 313, Sharptown, Wicomico County, MD

  4. 36. DETAILS FOR SCREENING MACHINERY, DEER ISLAND PUMPING STATION, METROPOLITAN ...

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

    36. DETAILS FOR SCREENING MACHINERY, DEER ISLAND PUMPING STATION, METROPOLITAN WATER AND SEWERAGE BOARD, METROPOLITAN SEWERAGE WORKS, DECEMBER 1909. Aperture card 6611-1 - Deer Island Pumping Station, Boston, Suffolk County, MA

  5. 14. Overview of bay without sluice gate machinery to northeast ...

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

    14. Overview of bay without sluice gate machinery to northeast - Mississippi River 9-Foot Channel, Lock & Dam No. 1, In Mississippi River at Mississippi Boulevard, below Ford Parkway Bridge, Saint Paul, Ramsey County, MN

  6. Activation of peroxisome proliferator-activated receptor α induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders.

    PubMed

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J; Sims, Katherine B; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-04-17

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role.

  7. Precision optical angular position marker system for rotating machinery

    NASA Technical Reports Server (NTRS)

    Barranger, J. P.

    1983-01-01

    An optical system is described which generates one or more markers of the angular shaft position of rotating machinery. The system consists of a light source, an optical cable, a machinery mounted lens assembly, a light detector, and a signal conditioner. Light reflected by targets on the rotor is converted to a digital output signal. The system is highly immune to extreme environments of vibration and temperature and achieved a 0.002 percent precision under operational test conditions.

  8. 65. CALIFORNIA STREET CABLE RAILWAY WINDING MACHINERY: Photocopy of ...

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

    65. CALIFORNIA STREET CABLE RAILWAY - WINDING MACHINERY: Photocopy of February 1955 photograph showing the winding machinery of the California Street Cable Railroad. The two suspended sheaves on the right of the photograph bore down on the cable as it left the winders, supplying tension to the cable and eliminating the need for a long tension run. - San Francisco Cable Railway, Washington & Mason Streets, San Francisco, San Francisco County, CA

  9. 8. MACHINERY SHED STORAGE ROOM ADDITION DETAIL SHOWING MATRIX OF ...

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

    8. MACHINERY SHED STORAGE ROOM ADDITION DETAIL SHOWING MATRIX OF NAILS USED TO ADHERE PORTLAND CEMENT PLASTER, SOUTH ADOBE WALL ADJACENT TO WINDOW Note: Photographs Nos. AZ-159-A-9 through AZ-159-A-10 are photocopies of photographs. The original prints and negatives are located in the SCS Tucson Plant Materials Center, Tucson, Arizona. Photographer Ted F. Spaller. - Tucson Plant Material Center, Machinery Shed, 3241 North Romero Road, Tucson, Pima County, AZ

  10. Cardiac arrest triggers hippocampal neuronal death through autophagic and apoptotic pathways

    PubMed Central

    Cui, Derong; Shang, Hanbing; Zhang, Xiaoli; Jiang, Wei; Jia, Xiaofeng

    2016-01-01

    The mechanism of neuronal death induced by ischemic injury remains unknown. We investigated whether autophagy and p53 signaling played a role in the apoptosis of hippocampal neurons following global cerebral ischemia-reperfusion (I/R) injury, in a rat model of 8-min asphyxial cardiac arrest (CA) and resuscitation. Increased autophagosome numbers, expression of lysosomal cathepsin B, cathepsin D, Beclin-1, and microtubule-associated protein light chain 3 (LC3) suggested autophagy in hippocampal cells. The expression of tumor suppressor protein 53 (p53) and its target genes: Bax, p53-upregulated modulator of apoptosis (PUMA), and damage-regulated autophagy modulator (DRAM) were upregulated following CA. The p53-specific inhibitor pifithrin-α (PFT-α) significantly reduced the expression of pro-apoptotic proteins (Bax and PUMA) and autophagic proteins (LC3-II and DRAM) that generally increase following CA. PFT-α also reduced hippocampal neuronal damage following CA. Similarly, 3-methyladenine (3-MA), which inhibits autophagy and bafilomycin A1 (BFA), which inhibits lysosomes, significantly inhibited hippocampal neuronal damage after CA. These results indicate that CA affects both autophagy and apoptosis, partially mediated by p53. Autophagy plays a significant role in hippocampal neuronal death induced by cerebral I/R following asphyxial-CA. PMID:27273382

  11. WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion

    PubMed Central

    King, Jason S.; Gueho, Aurélie; Hagedorn, Monica; Gopaldass, Navin; Leuba, Florence; Soldati, Thierry; Insall, Robert H.

    2013-01-01

    Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH-null lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that, in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage at which hydrolases can be retrieved and reused. PMID:23885127

  12. Altered MCM Protein Levels and Autophagic Flux in Aged and Systemic Sclerosis Dermal Fibroblasts

    PubMed Central

    Dumit, Verónica I.; Küttner, Victoria; Käppler, Jakob; Piera-Velazquez, Sonsoles; Jimenez, Sergio A.; Bruckner-Tuderman, Leena; Uitto, Jouni; Dengjel, Jörn

    2014-01-01

    Aging is a common risk factor of many disorders. With age, the level of insoluble extracellular matrix increases leading to increased stiffness of a number of tissues. Matrix accumulation can also be observed in fibrotic disorders, such as systemic sclerosis (SSc). Although the intrinsic aging process in skin is phenotypically distinct from SSc, here we demonstrate similar behavior of aged and SSc skin fibroblasts in culture. We have used quantitative proteomics to characterize the phenotype of dermal fibroblasts from healthy subjects of various ages and from patients with SSc. Our results demonstrate that proteins involved in DNA and RNA processing decrease with age and in SSc, while those involved in mitochondrial and other metabolic processes behave the opposite. Specifically, mini-chromosome maintenance (MCM) helicase proteins are less abundant with age and SSc, and they exhibit an altered subcellular distribution. We observed that lower levels of MCM7 correlate with reduced cell proliferation, lower autophagic capacity and higher intracellular protein expression phenotypes of aged and SSc cells. Additionally, we show that SSc fibroblasts exhibit higher levels of senescence than their healthy counterparts, suggesting further similarities between the fibrotic disorder and the aging process. Hence, at the molecular level, SSc fibroblasts exhibit intrinsic characteristics of fibroblasts from aged skin. PMID:24496236

  13. Hypoxia-mediated autophagic flux inhibits silver nanoparticle-triggered apoptosis in human lung cancer cells

    PubMed Central

    Jeong, Jae-Kyo; Gurunathan, Sangiliyandi; Kang, Min-Hee; Han, Jae Woong; Das, Joydeep; Choi, Yun-Jung; Kwon, Deug-Nam; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Song, Hyuk; Kim, Jin-Hoi

    2016-01-01

    Solid tumors are frequently associated with resistance to chemotherapy because the fraction of hypoxic tumor cells is substantial. To understand the underlying mechanism of hypoxia on silver nanoparticle (AgNPs)-induced apoptosis, the expression of hypoxia-inducible factor (HIF)-1α, a hallmark of hypoxia, was measured in the presence and absence of AgNPs. The results showed that HIF-1α expression was upregulated after AgNPs treatment under both hypoxic and normoxic conditions. Cell viability assays showed that AgNPs promoted cell death in cancer cells but not in non-cancer cells, as cancer cells are slightly more acidic than normal cells. However, reactive oxygen species generation induced by AgNPs in lung cancer cells caused high susceptibility to oxidative stress, whereas pre-exposure to hypoxia blocked AgNPs-induced oxidative stress. Notably, HIF-1α inhibited AgNPs-induced mitochondria-mediated apoptosis by regulating autophagic flux through the regulation of ATG5, LC3-II, and p62. Further, cell viability after treatment of cancer cells with AgNPs under hypoxic conditions was lower in HIF-1α siRNA-transfected cells than in control siRNA-transfected cells, indicating that HIF-1α knockdown enhances hypoxia induced decrease in cell viability. Our results suggest that hypoxia-mediated autophagy may be a mechanism for the resistance of AgNPs-induced apoptosis and that strategies targeting HIF-1α may be used for cancer therapy. PMID:26867977

  14. The Protective Effect of Gangliosides on Lead (Pb)-Induced Neurotoxicity Is Mediated by Autophagic Pathways.

    PubMed

    Meng, Hongtao; Wang, Lan; He, Junhong; Wang, Zhufeng

    2016-03-25

    Lead (Pb) is a ubiquitous environmental and industrial pollutant and can affect intelligence development and the learning ability and memory of children. Therefore, necessary measures should be taken to protect the central nervous system (CNS) from Pb toxicity. Gangliosides are sialic acid-containing glycosphingolipids that are constituents of mammalian cell membranes and are more abundantly expressed in the CNS. Studies have shown that gangliosides constitute a useful tool in the attempt to promote functional recovery of CNS and can reverse Pb-induced impairments of synaptic plasticity in rats. However, the detailed mechanisms have yet to be fully understood. In our present study, we tried to investigate the role of gangliosides in Pb-induced injury in hippocampus neurons and to further confirm the detailed mechanism. Our results show that Pb-induced injuries in the spatial reference memory were associated with a reduction of cell viability and cell apoptosis, and treatment with gangliosides markedly ameliorated the Pb-induced injury by inhibition of apoptosis action. Gangliosides further attenuated Pb-induced the abnormal autophagic process by regulation of mTOR pathways. In summary, our study establishes the efficacy of gangliosides as neuroprotective agents and provides a strong rationale for further studies on the underlying mechanisms of their neuroprotective functions.

  15. Cytoplasmic Lipid Droplets Are Sites of Convergence of Proteasomal and Autophagic Degradation of Apolipoprotein B

    PubMed Central

    Cheng, Jinglei; Fujita, Akikazu; Tokumoto, Toshinobu

    2006-01-01

    Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make “ApoB-crescents.” ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50–100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12–24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways. PMID:16597703

  16. E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells

    PubMed Central

    Hatchi, Elodie; Rodier, Geneviève; Sardet, Claude

    2011-01-01

    The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies. PMID:22024746

  17. Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus.

    PubMed

    Khositseth, Sookkasem; Uawithya, Panapat; Somparn, Poorichaya; Charngkaew, Komgrid; Thippamom, Nattakan; Hoffert, Jason D; Saeed, Fahad; Michael Payne, D; Chen, Shu-Hui; Fenton, Robert A; Pisitkun, Trairak

    2015-12-17

    Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI.

  18. Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus

    PubMed Central

    Khositseth, Sookkasem; Uawithya, Panapat; Somparn, Poorichaya; Charngkaew, Komgrid; Thippamom, Nattakan; Hoffert, Jason D.; Saeed, Fahad; Michael Payne, D.; Chen, Shu-Hui; Fenton, Robert A.; Pisitkun, Trairak

    2015-01-01

    Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown. We employed proteomic analysis of inner medullary collecting ducts (IMCD) from rats fed with a potassium-free diet for 1 day. IMCD protein quantification was performed by mass spectrometry using a label-free methodology. A total of 131 proteins, including the water channel AQP2, exhibited significant changes in abundance, most of which were decreased. Bioinformatic analysis revealed that many of the down-regulated proteins were associated with the biological processes of generation of precursor metabolites and energy, actin cytoskeleton organization, and cell-cell adhesion. Targeted LC-MS/MS and immunoblotting studies further confirmed the down regulation of 18 selected proteins. Electron microscopy showed autophagosomes/autophagolysosomes in the IMCD cells of rats deprived of potassium for only 1 day. An increased number of autophagosomes was also confirmed by immunofluorescence, demonstrating co-localization of LC3 and Lamp1 with AQP2 and several other down-regulated proteins in IMCD cells. AQP2 was also detected in autophagosomes in IMCD cells of potassium-deprived rats by immunogold electron microscopy. Thus, enhanced autophagic degradation of proteins, most notably including AQP2, is an early event in hypokalemia-induced NDI. PMID:26674602

  19. Hydrogen peroxide impairs autophagic flux in a cell model of nonalcoholic fatty liver disease.

    PubMed

    Jiang, Pengtao; Huang, Zhen; Zhao, Hong; Wei, Taotao

    2013-04-19

    Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, but the pathogenesis of NAFLD is not fully clear. The aim of this study was to determine whether autophagy plays a role in the pathogenesis of NAFLD. We found that the levels of autophagy were elevated in hepatoma cells upon exposure to free fatty acids, as confirmed by the increase in the number of autophagosomes. However, exposure of hepatoma cells to H2O2 and TNF-α, two typical "second hit" factors, increased the initiation of autophagy but inhibited the autophagic flux. The inhibition of autophagy sensitized cells to pro-apoptotic stimuli. Taken together, our results suggest that autophagy acts as a protective mechanism in the pathogenesis of NAFLD and that impairment of autophagy might induce more severe lesions of the liver. These findings will be a benefit to the understanding of the pathogenesis of NAFLD and might suggest a strategy for the prevention and cure of NAFLD.

  20. A179L, a viral Bcl-2 homologue, targets the core Bcl-2 apoptotic machinery and its upstream BH3 activators with selective binding restrictions for Bid and Noxa

    PubMed Central

    Galindo, Inmaculada; Hernaez, Bruno; Díaz-Gil, Gema; Escribano, Jose M.; Alonso, Covadonga

    2008-01-01

    Several large DNA viruses encode Bcl-2 protein homologues involved in the regulation of the cellular apoptosis cascade. This regulation often involves the interaction of these viral proteins with diverse cellular Bcl-2 family members. We have identified the specific interactions of A179L, an African swine fever virus (ASFV) Bcl-2 homologue, with the active forms of the porcine BH3-only Bid protein (truncated Bid p13 and p15). Transient expression of ASFV A179L gene in Vero cells prevented apoptosis induced by these active forms of Bid protein. Interestingly, A179L protein was able to interact, also with the main core Bcl-2 proapoptotic proteins Bax and Bak, and with several BH3-only proteins with selective binding restrictions for full length Bid and Noxa. These results suggest a fine regulation for A179L action in the suppression of apoptosis in infected cells which is essential for efficient virus replication. PMID:18329683