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Sample records for activated bax regulate

  1. An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway.

    Science.gov (United States)

    Garner, Thomas P; Reyna, Denis E; Priyadarshi, Amit; Chen, Hui-Chen; Li, Sheng; Wu, Yang; Ganesan, Yogesh Tengarai; Malashkevich, Vladimir N; Almo, Steve S; Cheng, Emily H; Gavathiotis, Evripidis

    2016-08-04

    Pro-apoptotic BAX is a cell fate regulator playing an important role in cellular homeostasis and pathological cell death. BAX is predominantly localized in the cytosol, where it has a quiescent monomer conformation. Following a pro-apoptotic trigger, cytosolic BAX is activated and translocates to the mitochondria to initiate mitochondrial dysfunction and apoptosis. Here, cellular, biochemical, and structural data unexpectedly demonstrate that cytosolic BAX also has an inactive dimer conformation that regulates its activation. The full-length crystal structure of the inactive BAX dimer revealed an asymmetric interaction consistent with inhibition of the N-terminal conformational change of one protomer and the displacement of the C-terminal helix α9 of the second protomer. This autoinhibited BAX dimer dissociates to BAX monomers before BAX can be activated. Our data support a model whereby the degree of apoptosis induction is regulated by the conformation of cytosolic BAX and identify an unprecedented mechanism of cytosolic BAX inhibition. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Allostery in BAX protein activation.

    Science.gov (United States)

    Jiang, Zhenyan; Zhang, Hansi; Böckmann, Rainer A

    2016-11-01

    BAX is a member of the proapoptotic BCL-2 family of proteins, which is involved in the regulation of the intrinsic pathway of apoptosis. In the process of apoptosis, BH3-only molecules activate cytosolic BAX. Activated BAX molecules insert into the mitochondrial outer membrane with their [Formula: see text]-helix and form oligomers that lead to membrane poration, resulting in the release of apoptogenic factors including cytochrome c. Recently, a novel interaction site for the binding of the BIM SAHB ligand to BAX was reported. BIM SAHB binding was shown to invoke the exposure of the 6A7 epitope (amino acids 13-19) and of the BH3 domain of BAX, followed by mobilization of the BAX [Formula: see text]-helix. However, the intramolecular pathway for signal transmission in BAX, from BIM SAHB binding to mobilization of the [Formula: see text]-helix largely remained elusive. For a molecular understanding of the activation of BAX, and thus the first steps in apoptosis, we performed microsecond atomistic molecular dynamics simulations both of the BAX protein and of the BAX:BIM SAHB complex in aqueous solution. In agreement with experiment, the 6A7 and BH3 domains adopt a more solvent-exposed conformation within the BAX:BIM SAHB complex. BIM SAHB binding was found to stabilize the secondary structure of the [Formula: see text]9-helix. A force distribution analysis revealed a force network of residue-residue interactions responsible for signal transmission from the BIM SAHB binding site predominantly via the [Formula: see text]4- and [Formula: see text]6-helices to the [Formula: see text]9-helix on the opposite site of the protein.

  3. Bax activation by Bim?

    National Research Council Canada - National Science Library

    Czabotar, P E; Colman, P M; Huang, D C S

    2009-01-01

    .... Although some data support a role for certain BH3-only proteins, such as Bim or tBid, to directly activate Bax, others have led to the conclusion that BH3-only proteins act indirectly by antagonizing...

  4. Quercetin induces tumor-selective apoptosis through down-regulation of Mcl-1 and activation of Bax

    Science.gov (United States)

    Cheng, Senping; Gao, Ning; Zhang, Zhuo; Chen, Gang; Budhraja, Amit; Ke, Zunji; Son, Young-ok; Wang, Xin; Luo, Jia; Shi, Xianglin

    2010-01-01

    Purpose To investigate the in vivo antitumor efficacy of querctin in U937 xenografts and the functional role of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia cells. Experimental Design Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercein, as well as Mcl-1 expression and Bax activation were investigated in xenografts of leukemia cells. Results Administration of quercetin caused pronounced apoptosis in both transformed and primary leukemia cells, but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was accompanied by Mcl-1 down-regulation and Bax conformational change and mitochondrial translocation which triggered cytochrome c release. Knockdown of Bax by siRNA reversed querctin-induced apoptosis. Knockout of Bax abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-mediated Bax activation, translocation and cell death. Conversely, interruption of Mcl-1 by siRNA enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence of Bax had no effect on quercetin-mediated Mcl-1 down-regulation. Furthermore, in vivo administration of quercetin attenuated tumor growth in U937 xenografts. The TUNEL positive apoptotic cells in tumor sections increased in quercetin-treated mice as compared with controls. Mcl-1 down-regulation and Bax activation were observed in xenografts. Conclusions These data suggest that quercetin may be useful for the treatment of leukemia by preferentially inducing apoptosis in leukemia versus normal hematopoietic cells, through a process involving Mcl-1 down-regulation, which in turn potentiates Bax activation and mitochondrial translocation, culminating in apoptosis. PMID:21138867

  5. Astilbic Acid Induced COLO 205 cell Apoptosis by Regulating Bcl-2 and Bax Expression and Activating Caspase-3

    Institute of Scientific and Technical Information of China (English)

    ZhengXiao-liang; SunHong-xiang; LiuXue-li; ChenYun-xiang; QianBo-chu

    2005-01-01

    To investigate the effect of astilbic acid (3β,6β-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis.Methods Proliferation of COLO 205 cells was measued by MTT assay. Content of DNA in COLO 205 cell was measued by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope.DNA fragmentation was visualized by agarose gel electrophoresis.Apoptosis rate and cell cycle distribution were deter-mined by flow cytometric analysis.Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis.The change of relative mitochondral transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. Results The IC50 (96h) of AA for inhibiting COLO 205 cell proliferation was 61.56±0.34 μmol/L.AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 μmol/L showed typical morphological changes of apoptosis and DNA “ladder” pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25% for COLO 205 cells treated with AA 64 μmol/L for 48h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO 1μmol/L could increase the viability of COLO 205 cells treated with AA for 48h.Conclusion AA showed potent inhibitory activity on COLO 205 cells proliferation,and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulating Bcl-2 expression, and up-regulating Bax expression, lowering relative MTP, and activating caspase-3 pathway.

  6. Small molecules reveal an alternative mechanism of Bax activation.

    Science.gov (United States)

    Brahmbhatt, Hetal; Uehling, David; Al-Awar, Rima; Leber, Brian; Andrews, David

    2016-04-15

    The pro-apoptotic protein Bax commits a cell to death by permeabilizing the mitochondrial outer membrane (MOM). To obtain small-molecule probes for elucidating the molecular mechanism(s) of Bax activation, we screened for compounds that induced Bax-mediated liposome permeabilization. We identified five structurally different small molecules that promoted both Bax targeting to and oligomerization at membranes. All five compounds initiated Bax oligomerization in the absence of membranes by a mechanism unlike Bax activation by Bcl-2 homology 3 domain (BH3) proteins. Some of the compounds induced Bax/Bak-dependent apoptosis in cells. Activation of Bax by the most active compound was poorly inhibited by the anti-apoptotic protein Bcl-XL and requires a cysteine residue at position 126 of Bax that is not required for activation by BH3 proteins. Our results reveal a novel pathway for Bax activation independent of pro-apoptotic BH3 proteins that may have important implications for the regulation of Bax activity in cells. © 2016 The Author(s).

  7. Astilbic acid induced COLO 205 cell apoptosis by regulating Bcl-2 and Bax expression and activating caspase-3

    Institute of Scientific and Technical Information of China (English)

    Xiao-liang ZHENG; Hong-xiang SUN; Xue-li LIU; Yun-xiang CHEN; Bo-chu QIAN

    2004-01-01

    AIM: To investigate the effect of astilbic acid (3β, 6β-dihydroxyolean-12-en-27-oic acid, AA) on human colorectal carcinoma COLO 205 cell proliferation and apoptosis. METHODS: Proliferation of COLO 205 cells was measued by MTT assay. Content of DNA in COLO 205 cell was measued by modified diphenylamine assay. AA-induced morphological changes was observed with fluorescence microscope and transmission electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptosis rate and cell cycle distribution were determined by flow cytometric analysis. Expressions of Bcl-2 and Bax proteins were visioned by immunohistochemical analysis. The change of relative mitochondral transmembrane potential (MTP) in COLO 205 cell was analyzed with FCM after rhodamine 123 staining. RESULTS: The ICs0 (96 h) of AA for inhibiting COLO 205 cell proliferation was 61.56±0.34 μmol/L. AA induced a marked concentration- and time-dependent inhibition of COLO 205 cell proliferation and reduced the DNA content in COLO 205 cell. Cells treated with AA 64 μmol/L showed typical morphological changes of apoptosis and DNA "ladder" pattern. The cell cycle was arrested in G0/G1 phase, and the apoptosis rate was 28.25 % for COLO 205 cells treated with AA 64 μmol/L for 48 h. Meanwhile the expression of Bcl-2 protein was decreased while that of Bax was increased and relative MTP was decreased as well. DEVD-CHO 1 μmol/L could increase the viability of COLO 205 cells treated with AA for 48 h. CONCLUSION: AA showed potent inhibitory activity on COLO 205 cells proliferation, and could induce COLO 205 cells apoptosis through disturbing DNA replication, down-regulatin Bcl-2 expression,and up-regulating Bax expression,lowering relative MTP, and activating caspase-3 pathway.

  8. Cytosolic Ku70 regulates Bax-mediated cell death.

    Science.gov (United States)

    Hada, Manila; Subramanian, Chitra; Andrews, Phillip C; Kwok, Roland P S

    2016-10-01

    The first known function of Ku70 is as a DNA repair factor in the nucleus. Using neuronal neuroblastoma cells as a model, we have established that cytosolic Ku70 binds to the pro-apoptotic protein Bax in the cytosol and blocks Bax's cell death activity. Ku70-Bax binding is regulated by Ku70 acetylation in that when Ku70 is acetylated Bax dissociates from Ku70, triggering cell death. We propose that Ku70 may act as a survival factor in these cells such that Ku70 depletion triggers Bax-dependent cell death. Here, we addressed two fundamental questions about this model: (1) Does all Bax, which is a cytosolic protein, bind to all cytosolic Ku70? and (2) Is Ku70 a survival factor in cells types other than neuronal neuroblastoma cells? We show here that, in neuronal neuroblastoma cells, only a small fraction of Ku70 binds to a small fraction of Bax; most Bax is monomeric. Interestingly, there is no free or monomeric Ku70 in the cytosol; most cytosolic Ku70 is in complex with other factors forming several high molecular weight complexes. A fraction of cytosolic Ku70 also binds to cytosolic Ku80, Ku70's binding partner in the nucleus. Ku70 may not be a survival factor in some cell types (Ku70-depletion less sensitive) because Ku70 depletion does not affect survival of these cells. These results indicate that, in addition to Ku70 acetylation, other factors may be involved in regulating Ku70-Bax binding in the Ku70-depletion less sensitive cells because Ku70 acetylation in these cells is not sufficient to dissociate Bax from Ku70 or to activate Bax.

  9. Direct Activation of Bax Protein for Cancer Therapy

    Science.gov (United States)

    Liu, Zhiqing; Ding, Ye; Ye, Na; Wild, Christopher; Chen, Haiying; Zhou, Jia

    2015-01-01

    Bax, a central cell death regulator, is an indispensable gateway to mitochondrial dysfunction and a major pro-apoptotic member of the Bcl-2 family proteins that control apoptosis in normal and cancer cells. Dysfunction of apoptosis renders the cancer cell resistant to treatment as well as promotes tumorigenesis. Bax activation induces mitochondrial membrane permeabilization, thereby leading to the release of apoptotic factor cytochrome c and consequently cancer cell death. A number of drugs in clinical use are known to indirectly activate Bax. Intriguingly, recent efforts demonstrate that Bax can serve as a promising direct target for small-molecule drug discovery. Several direct Bax activators have been identified to hold promise for cancer therapy with the advantages of specificity and the potential of overcoming chemo- and radioresistance. Further investigation of this new class of drug candidates will be needed to advance them into the clinic as a novel means to treat cancer. PMID:26395559

  10. Bax

    National Research Council Canada - National Science Library

    Cui, Qinghua; Valentin, Mayda; Janumyan, Yelena; Yang, Elizabeth

    2009-01-01

    .... We also discovered that the cell cycle function of Bcl-2 and Bcl-xL was dependent on Bax and Bak, and in bax -/- bak -/- double knockout cells, features of G0 quiesecence were already present and p27...

  11. DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX

    Science.gov (United States)

    Guan, J-J; Zhang, X-D; Sun, W; Qi, L; Wu, J-C; Qin, Z-H

    2015-01-01

    DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis. PMID:25633293

  12. Direct and selective small-molecule activation of proapoptotic BAX

    Science.gov (United States)

    Gavathiotis, Evripidis; Reyna, Denis E; Bellairs, Joseph A; Leshchiner, Elizaveta S; Walensky, Loren D

    2013-01-01

    BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX’s ‘on switch’. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis. PMID:22634637

  13. Connexin 43 enhances Bax activation via JNK activation in sunitinib-induced apoptosis in mesothelioma cells.

    Science.gov (United States)

    Uzu, Miaki; Sato, Hiromi; Shimizu, Ayaka; Shibata, Yukihiro; Ueno, Koichi; Hisaka, Akihiro

    2017-06-01

    The constituent protein of gap junctions, connexin (Cx), interacts with various proteins via its C-terminus region, including kinases, cell-adhesion proteins, and a pro-apoptotic protein, Bax. This molecular interaction may affect expression and functioning of the interacting proteins and modulate the cellular physiology. In our previous work, Cx43 was found to interact directly with Bax and in the presence of sunitinib, lead to the Bax-mediated apoptosis in mesothelioma cells. In this study, we investigated the mechanism of how Cx43 promotes Bax-mediated apoptosis using the same cell line. Treatment with sunitinib increased the expression of the active conformation of the Bax protein, which was predominantly localized at the mitochondria, only in Cx43-transfected cells. Bax oligomerization and decrease in the mitochondrial membrane potential were also observed. The involvement of c-Jun N-terminal kinase (JNK) in the interaction of Cx43 and Bax was further examined. Treatment with sunitinib increased the expression of phosphorylated (active) form of JNK only in the Cx43-transfected cells. Phosphorylated JNK and active Bax were co-localized, and the co-localization was suppressed by the knockdown of Cx43. Moreover, JNK inhibition clearly suppressed Bax activation. In conclusion, we identified a novel Cx43-JNK-Bax axis regulating the process of apoptosis for the first time. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  14. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression

    Science.gov (United States)

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC. PMID:27642320

  15. Matrine inhibits diethylnitrosamine-induced HCC proliferation in rats through inducing apoptosis via p53, Bax-dependent caspase-3 activation pathway and down-regulating MLCK overexpression.

    Science.gov (United States)

    Zhang, Xiaolin; Yu, Hao

    2016-01-01

    The proliferation of hepatocellular carcinoma (HCC) cells is one of the leading causes of liver cancer mortality in humans. The inhibiting effects of matrine on HCC cell proliferation have been studied, but the mechanism of that inhibition has not been fully elucidated. Since, apoptosis plays an important role in HCC cell proliferation. We examined the apoptosis-inducing effect of matrine on tumor cells. Western blot analysis of p53, Bax, cleaved caspase-3 and myosin light chain kinase (MLCK) revealed that matrine induced tumor cell apoptosis by controlling anoikis. It activated p53, Bax-dependent caspase-3 and blocked the ECM-integrin mediated cell survival pathway through down-regulating MLCK over-expression in the liver of rats with diethyl nitrosamine (DENA)-induced HCC. Our results suggest that matrine can inhibit the proliferation of HCC cells through inducing tumor cell apoptosis via activation of the p53 pathway and inhibition of MLCK overexpression. Matrine may thus be used as a potentially promising reagent to inhibit HCC cell proliferation and MLCK may be a novel target for the treatment of HCC.

  16. Ziyuglycoside II-induced apoptosis in human gastric carcinoma BGC-823 cells by regulating Bax/Bcl-2 expression and activating caspase-3 pathway

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    Zhu, A.K. [Department of General Surgery, Nanjing Medical University, Affiliated Hangzhou Hospital, Hangzhou (China); Zhou, H.; Xia, J.Z. [Department of General Surgery, Nanjing Medical University, Affiliated Wuxi Second Hospital, Wuxi (China); Jin, H.C. [Department of General Surgery, Nanjing Medical University, Affiliated Hangzhou Hospital, Hangzhou (China); Wang, K. [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu Province (China); Yan, J.; Zuo, J.B. [Department of General Surgery, Nanjing Medical University, Affiliated Wuxi Second Hospital, Wuxi (China); Zhu, X. [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu Province (China); Shan, T. [Department of General Surgery, Nanjing Medical University, Affiliated Wuxi Second Hospital, Wuxi (China)

    2013-08-13

    Ziyuglycoside II is an active compound of Sanguisorba officinalis L. that has anti-inflammation, antioxidation, antibiosis, and homeostasis properties. We report here on the anticancer effect of ziyuglycoside II on human gastric carcinoma BGC-823 cells. We investigated the effects of ziyuglycoside II on cell growth, cell cycle, and cell apoptosis of this cell line. Our results revealed that ziyuglycoside II could inhibit the proliferation of BGC-823 cells by inducing apoptosis but not cell cycle arrest, which was associated with regulation of Bax/Bcl-2 expression, and activation of the caspase-3 pathway. Our study is the first to report the antitumor potential of ziyuglycoside II in BGC-823 gastric cancer cells. Ziyuglycoside II may become a potential therapeutic agent against gastric cancer in the future.

  17. Activation of JNK/Bim/Bax pathway in UV-induced apoptosis

    Science.gov (United States)

    Liu, Lei; Hui, Li; Zhang, Zhen-zhen

    2011-03-01

    Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways are involved. JNK has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the molecular mechanism through which JNK regulates apoptosis, especially how JNK activates Bax in response to UV irradiation is still controversial. In this study, using real-time single-cell analysis, we studied the machinery of Bax activation during UV-induced apoptosis. UV treatment resulted in a series of events: phosphorylation of JNK, mitochondrial translocation of Bim, and subsequent activation of Bax. The activation of Bim and Bax could be inhibited in the presence of SP600125 (a specific inhibitor of JNK), suggesting that UV irradiation activated the JNK/Bim/Bax pathway.

  18. Conformational Heterogeneity in the Activation Mechanism of Bax.

    Science.gov (United States)

    Barnes, C Ashley; Mishra, Pushpa; Baber, James L; Strub, Marie-Paule; Tjandra, Nico

    2017-08-01

    Bax is known for its pro-apoptotic role within the mitochondrial pathway of apoptosis. However, the mechanism for transitioning Bax from cytosolic to membrane-bound oligomer remains elusive. Previous nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) studies defined monomeric Bax as conformationally homogeneous. Yet it has recently been proposed that monomeric Bax exists in equilibrium with a minor state that is distinctly different from its NMR structure. Here, we revisited the structural analysis of Bax using methods uniquely suited for unveiling "invisible" states of proteins, namely, NMR paramagnetic relaxation enhancements and EPR double electron-electron resonance (DEER). Additionally we examined the effect of glycerol, the co-solvent of choice in DEER studies, on the structure of Bax using NMR chemical-shift perturbations and residual dipolar couplings. Based on our combined NMR and EPR results, Bax is a conformationally homogeneous protein prior to its activation. Published by Elsevier Ltd.

  19. A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells

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    Claudia Fabiola Méndez-Catalá

    2013-08-01

    Full Text Available We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF, in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.

  20. Eucommia ulmoides cortex, geniposide and aucubin regulate lipotoxicity through the inhibition of lysosomal BAX.

    Science.gov (United States)

    Lee, Geum-Hwa; Lee, Mi-Rin; Lee, Hwa-Young; Kim, Seung Hyun; Kim, Hye-Kyung; Kim, Hyung-Ryong; Chae, Han-Jung

    2014-01-01

    In this study we examined the inhibition of hepatic dyslipidemia by Eucommia ulmoides extract (EUE). Using a screening assay for BAX inhibition we determined that EUE regulates BAX-induced cell death. Among various cell death stimuli tested EUE regulated palmitate-induced cell death, which involves lysosomal BAX translocation. EUE rescued palmitate-induced inhibition of lysosomal V-ATPase, α-galactosidase, α-mannosidase, and acid phosphatase, and this effect was reversed by bafilomycin, a lysosomal V-ATPase inhibitor. The active components of EUE, aucubin and geniposide, showed similar inhibition of palmitate-induced cell death to that of EUE through enhancement of lysosome activity. Consistent with these in vitro findings, EUE inhibited the dyslipidemic condition in a high-fat diet animal model by regulating the lysosomal localization of BAX. This study demonstrates that EUE regulates lipotoxicity through a novel mechanism of enhanced lysosomal activity leading to the regulation of lysosomal BAX activation and cell death. Our findings further indicate that geniposide and aucubin, active components of EUE, may be therapeutic candidates for non-alcoholic fatty liver disease.

  1. A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

    Science.gov (United States)

    Teijido, Oscar; Ganesan, Yogesh Tengarai; Llanos, Raul; Peton, Ashley; Urtecho, Jean-Baptiste; Soprani, Adauri; Villamayor, Aimee; Antonsson, Bruno; Manon, Stéphen; Dejean, Laurent

    2016-03-15

    Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

    Science.gov (United States)

    Kushnareva, Yulia; Andreyev, Alexander Y.; Kuwana, Tomomi; Newmeyer, Donald D.

    2012-01-01

    Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event. PMID:23049480

  3. Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation.

    Science.gov (United States)

    Düssmann, H; Rehm, M; Concannon, C G; Anguissola, S; Würstle, M; Kacmar, S; Völler, P; Huber, H J; Prehn, J H M

    2010-02-01

    Mitochondrial outer membrane permeabilisation (MOMP) during apoptosis is triggered by the activation and oligomerisation of Bax and Bak, but a quantification of these processes in individual cells has not yet been performed. Single-cell imaging of Bax translocation and oligomerisation in Bax-deficient DU-145 cells expressing CFP-Bax and YFP-Bax revealed that both processes started only minutes before or concomitantly with MOMP, with the majority of Bax translocation and oligomerisation occurring downstream of MOMP. Quantification of YFP-Bax concentrations at mitochondria revealed an increase of only 1.8 + or - 1.5% at MOMP onset. This was increased to 11.2 + or - 3.6% in bak-silenced cells. These data suggested that Bax activation exceeded by far the quantities required for MOMP induction, and that minimal Bax or Bak activation may be sufficient to trigger rapid pore formation. In a cellular automaton modelling approach that incorporated the quantities and movement probabilities of Bax and its inhibitors, activators and enablers in the mitochondrial membrane, we could re-model rapid pore formation kinetics at submaximal Bax activation.

  4. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

    Institute of Scientific and Technical Information of China (English)

    Bonsu Ku; Chengyu Liang; Jae U Jung; Byung-Ha Oh

    2011-01-01

    Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic Bc1-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague.Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, hut was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-XL, MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could he expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.

  5. Pro-apoptotic cBid and Bax exhibit distinct membrane remodeling activities: An AFM study.

    Science.gov (United States)

    Unsay, Joseph D; Cosentino, Katia; Sporbeck, Katharina; García-Sáez, Ana J

    2017-01-01

    Bcl-2 proteins are key regulators of the mitochondrial outer membrane (MOM) permeabilization that mediates apoptosis. During apoptosis, Bid is cleaved (cBid) and translocates to the MOM, where it activates Bax. Bax then oligomerizes and induces MOM permeabilization. However, little is known about how these proteins affect membrane organization aside from pore formation. In previous studies, we have shown that both cBid and Bax are able to remodel membranes and stabilize curvature. Here, we dissected the independent effects of Bax and cBid on supported lipid structures mimicking the mitochondrial composition by means of atomic force spectroscopy. We show that cBid did not permeabilize the membrane but lowered the membrane breakthrough force. On the other hand, Bax effects were dependent on its oligomeric state. Monomeric Bax did not affect the membrane properties. In contrast, oligomeric Bax lowered the breakthrough force of the membrane, which in the context of pore formation, implies a lowering of the line tension at the edge of the pore. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    Science.gov (United States)

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  7. Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

    Science.gov (United States)

    Zhao, Guoping; Zhu, Yanglong; Eno, Colins O.; Liu, Yanlong; DeLeeuw, Lynn; Burlison, Joseph A.; Chaires, Jonathan B.; Trent, John O.

    2014-01-01

    The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer. PMID:24421393

  8. Cytosolic Bax

    Science.gov (United States)

    Vogel, Sandra; Raulf, Nina; Bregenhorn, Stephanie; Biniossek, Martin L.; Maurer, Ulrich; Czabotar, Peter; Borner, Christoph

    2012-01-01

    Bax is kept inactive in the cytosol by refolding its C-terminal transmembrane domain into the hydrophobic binding pocket. Although energetic calculations predicted this conformation to be stable, numerous Bax binding proteins were reported and suggested to further stabilize inactive Bax. Unfortunately, most of them have not been validated in a physiological context on the endogenous level. Here we use gel filtration analysis of the cytosol of primary and established cells to show that endogenous, inactive Bax runs 20–30 kDa higher than recombinant Bax, suggesting Bax dimerization or the binding of a small protein. Dimerization was excluded by a lack of interaction of differentially tagged Bax proteins and by comparing the sizes of dimerized recombinant Bax with cytosolic Bax on blue native gels. Surprisingly, when analyzing cytosolic Bax complexes by high sensitivity mass spectrometry after anti-Bax immunoprecipitation or consecutive purification by gel filtration and blue native gel electrophoresis, we detected only one protein, called p23 hsp90 co-chaperone, which consistently and specifically co-purified with Bax. However, this protein could not be validated as a crucial inhibitory Bax binding partner as its over- or underexpression did not show any apoptosis defects. By contrast, cytosolic Bax exhibits a slight molecular mass shift on SDS-PAGE as compared with recombinant Bax, which suggests a posttranslational modification and/or a structural difference between the two proteins. We propose that in most healthy cells, cytosolic endogenous Bax is a monomeric protein that does not necessarily need a binding partner to keep its pro-apoptotic activity in check. PMID:22277657

  9. BH3-Triggered Structural Reorganization Drives the Activation of Pro-apoptotic BAX

    Science.gov (United States)

    Gavathiotis, Evripidis; Reyna, Denis E.; Davis, Marguerite L.; Bird, Gregory H.; Walensky, Loren D.

    2010-01-01

    Summary BAX is a pro-apoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α-helices 1 and 2 is displaced, the carboxy terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an auto-activating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals new pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway. PMID:21070973

  10. cBid, Bax and Bcl-xL exhibit opposite membrane remodeling activities

    Science.gov (United States)

    Bleicken, S; Hofhaus, G; Ugarte-Uribe, B; Schröder, R; García-Sáez, A J

    2016-01-01

    The proteins of the Bcl-2 family have a crucial role in mitochondrial outer membrane permeabilization during apoptosis and in the regulation of mitochondrial dynamics. Current models consider that Bax forms toroidal pores at mitochondria that are responsible for the release of cytochrome c, whereas Bcl-xL inhibits pore formation. However, how Bcl-2 proteins regulate mitochondrial fission and fusion remains poorly understood. By using a systematic analysis at the single vesicle level, we found that cBid, Bax and Bcl-xL are able to remodel membranes in different ways. cBid and Bax induced a reduction in vesicle size likely related to membrane tethering, budding and fission, besides membrane permeabilization. Moreover, they are preferentially located at highly curved membranes. In contrast, Bcl-xL not only counterbalanced pore formation but also membrane budding and fission. Our findings support a mechanism of action by which cBid and Bax induce or stabilize highly curved membranes including non-lamellar structures. This molecular activity reduces the energy for membrane remodeling, which is a necessary step in toroidal pore formation, as well as membrane fission and fusion, and provides a common mechanism that links the two main functions of Bcl-2 proteins. PMID:26913610

  11. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.

    Science.gov (United States)

    Kuwana, Tomomi; Bouchier-Hayes, Lisa; Chipuk, Jerry E; Bonzon, Christine; Sullivan, Barbara A; Green, Douglas R; Newmeyer, Donald D

    2005-02-18

    Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.

  12. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    Science.gov (United States)

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

  13. S-palmitoylation represents a novel mechanism regulating the mitochondrial targeting of BAX and initiation of apoptosis

    Science.gov (United States)

    Fröhlich, M; Dejanovic, B; Kashkar, H; Schwarz, G; Nussberger, S

    2014-01-01

    The intrinsic pathway of apoptotic cell death is mainly mediated by the BCL-2-associated X (BAX) protein through permeabilization of the mitochondrial outer membrane (MOM) and the concomitant release of cytochrome c into the cytosol. In healthy, non-apoptotic cells, BAX is predominantly localized in the cytosol and exhibits a dynamic shuttle cycle between the cytosol and the mitochondria. Thus, the initial association with mitochondria represents a critical regulatory step enabling BAX to insert into MOMs, promoting the release of cytochrome c and ultimately resulting in apoptosis. However, the molecular mode of how BAX associates with MOMs and whether a cellular regulatory mechanism governs this process is poorly understood. Here we show that in both primary tissues and cultured cells, the association with MOMs and the proapoptotic action of BAX is controlled by its S-palmitoylation at Cys-126. A lack of BAX palmitoylation reduced BAX mitochondrial translocation, BAX oligomerization, caspase activity and apoptosis. Furthermore, ectopic expression of specific palmitoyl transferases in cultured healthy cells increases BAX S-palmitoylation and accelerates apoptosis, whereas malignant tumor cells show reduced BAX S-palmitoylation consistent with their reduced BAX-mediated proapoptotic activity. Our findings suggest that S-palmitoylation of BAX at Cys126 is a key regulatory process of BAX-mediated apoptosis. PMID:24525733

  14. DMFC (3,5-dimethyl-(7)H-furo[3,2-g]chromen-7-one) regulates Bim to trigger Bax and Bak activation to suppress drug-resistant human hepatoma.

    Science.gov (United States)

    Xiang, Jun; Wang, Zhe; Liu, Qianqian; Li, Xia; Sun, Jianguo; Fung, Kwok-Pui; Liu, Feiyan

    2017-03-01

    3,5-Dimethyl-(7)H-furo[3,2-g]chromen-7-one (DMFC) is a coumarin derivative with anti-cancer activity against human hepatoma cells, but the mechanisms underlying DMFC function in cancer suppression is unknown. In this study, we aimed at elucidating the molecular mechanisms underlying DMFC anti-cancer activity and determining whether DMFC is effective in suppression of drug-resistant human hepatocellular carcinoma. We show here that DMFC effectively suppresses both the parent and the multidrug-resistant hepatoma cell growth in vitro and DMFC suppresses hepatoma cell growth at least in part through inducing tumor cell apoptosis. In the molecular level, we observed that DMFC treatment decreases Bcl-2 level by a post-transcriptional mechanism and activates Bim transcription to increase Bim mRNA and protein level in hepatoma cells. Furthermore, co-immunoprecipitation studies revealed that DMFC-induced Bim interrupts interactions between Bcl-2 and Bax and between Mcl-1 and Bak, resulting in dissociation of Bax from Bcl-2 and Bak from Mcl-1 and subsequent activation of both Bax and Bak. Activation of Bax and Bak leads to mitochondrial outer membrane permeabilization and cytochrome c release. Consistent with its potent apoptosis-inducing activity, DMFC exhibited potent activity against the multidrug-resistant hepatoma xenograft growth in vivo. Therefore, we determine that DMFC suppresses hepatoma growth through decreasing Bcl-2 and increasing Bim to induce tumor cell apoptosis and hold great promise for further development as a therapeutic agent to treat chemoresistant hepatoma.

  15. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis

    Science.gov (United States)

    Westphal, D; Kluck, R M; Dewson, G

    2014-01-01

    The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different ‘modes' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane. PMID:24162660

  16. Targeting Bax interaction sites reveals that only homo-oligomerization sites are essential for its activation

    Science.gov (United States)

    Peng, R; Tong, J-S; Li, H; Yue, B; Zou, F; Yu, J; Zhang, L

    2013-01-01

    Bax is a proapoptotic Bcl-2 family member that has a central role in the initiation of mitochondria-dependent apoptosis. However, the mechanism of Bax activation during apoptosis remains unsettled. It is believed that the activation of Bax is mediated by either dissociation from prosurvival Bcl-2 family members, or direct association with BH3-only members. Several interaction sites on Bax that mediate its interactions with other Bcl-2 family members, as well as its proapoptotic activity, have been identified in previous studies by other groups. To rigorously investigate the functional role of these interaction sites, we knocked in their respective mutants using HCT116 colon cancer cells, in which apoptosis induced by several stimuli is strictly Bax-dependent. Bax-mediated apoptosis was intact upon knock-in (KI) of K21E and D33A, which were shown to block the interaction of Bax with BH3-only activators. Apoptosis was partially reduced by KI of D68R, which impairs the interaction of Bax with prosurvival members, and S184V, a constitutively mitochondria-targeting mutant. In contrast, apoptosis was largely suppressed by KI of L70A/D71A, which blocks homo-oligomerization of Bax and its binding to prosurvival Bcl-2 family proteins. Collectively, our results suggest that the activation of endogenous Bax in HCT116 cells is dependent on its homo-oligomerization sites, but not those previously shown to interact with BH3-only activators or prosurvival proteins only. We therefore postulate that critical interaction sites yet to be identified, or mechanisms other than protein-protein interactions, need to be pursued to delineate the mechanism of Bax activation during apoptosis. PMID:23392123

  17. Bax/Bak activation in the absence of Bid, Bim, Puma, and p53.

    Science.gov (United States)

    Zhang, J; Huang, K; O'Neill, K L; Pang, X; Luo, X

    2016-06-16

    How BH3-only proteins activate Bax/Bak, the two gateway proteins of the mitochondria-dependent apoptotic pathway, remains incompletely understood. Although all pro-apoptotic BH3-only proteins are known to bind/neutralize the anti-apoptotic Bcl-2 proteins, the three most potent ones, Bid (tBid), Bim, and Puma, possess an additional activity of directly activating Bax/Bak in vitro. This latter activity has been proposed to be responsible for triggering Bax/Bak activation following apoptotic stimulation. To test this hypothesis, we generated Bid(-/)(-)Bim(-/)(-)Puma(-/)(-) (TKO), TKO/Bax(-/)(-)/Bak(-/)(-) (PentaKO), and PentaKO/Mcl-1(-/-) (HexaKO) HCT116 cells through gene editing. Surprisingly, although the TKO cells were resistant to several apoptotic stimuli, robust apoptosis was induced upon the simultaneous inactivation of Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins known to suppress Bax/Bak activation and activity. Importantly, such apoptotic activity was completely abolished in the PentaKO cells. In addition, ABT-737, a BH3 mimetic that inhibits Bcl-xL/Bcl-w/Bcl-2, induced Bax activation in HexaKO cells reconstituted with endogenous level of GFP-Bax. Further, by generating TKO/p53(-/-) (QKO) cells, we demonstrated that p53, a tumor suppressor postulated to directly activate Bax, is not required for Bid/Bim/Puma-independent Bax/Bak activation. Together, these results strongly suggest that the direct activation activities of Bid (tBid), Bim, Puma, and p53 are not essential for activating Bax/Bak once the anti-apoptotic Bcl-2 proteins are neutralized.

  18. Pin1-Induced Proline Isomerization in Cytosolic p53 Mediates BAX Activation and Apoptosis.

    Science.gov (United States)

    Follis, Ariele Viacava; Llambi, Fabien; Merritt, Parker; Chipuk, Jerry E; Green, Douglas R; Kriwacki, Richard W

    2015-08-20

    The cytosolic fraction of the tumor suppressor p53 activates the apoptotic effector protein BAX to trigger apoptosis. Here we report that p53 activates BAX through a mechanism different from that associated with activation by BH3 only proteins (BIM and BID). We observed that cis-trans isomerization of proline 47 (Pro47) within p53, an inherently rare molecular event, was required for BAX activation. The prolyl isomerase Pin1 enhanced p53-dependent BAX activation by catalyzing cis-trans interconversion of p53 Pro47. Our results reveal a signaling mechanism whereby proline cis-trans isomerization in one protein triggers conformational and functional changes in a downstream signaling partner. Activation of BAX through the concerted action of cytosolic p53 and Pin1 may integrate cell stress signals to induce a direct apoptotic response. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Influence of Apoptin on Up-regulation of the Expression of Bad and Bax

    Institute of Scientific and Technical Information of China (English)

    GUO Tai; YANG Qian

    2005-01-01

    The chicken anemia virus protein, apoptin, which manifests selectivity and specificity to tumor cells, induces a p53-independent and Bcl-2-insensitive type of apoptosis in various human tumor cells. In this study, the apoptin gene was cloned from the total DNA of chicken anemia virus, and the recombinant vector was constructed. We used oligonucleotide microarray to study the changes of four genes, including Bcl-2, Bcl-xL, Bad and Bax. The post-transfection with the recombinant was also studied. The pro-apoptotic genes(Bad and Bax) and anti-apoptosis genes(Bcl-2 and Bcl-xL) were up-regulated in contrast to the controls. According to the published data, either Bcl-2 or Bcl-xL can form non-functional heterodimers by Bad and Bax binding together, resulting in blocking partly the release of cytochrome c from mitochondria. However, apoptosis could be inhibited by neither the endogenous Bcl-xL nor Bcl-2 over-expression. The experiments show that the apoptin-induced apoptotic pathway is related to the up-regulation of Bad and Bax. Bad was up-regulated by apoptin; then this up-regulated product of Bad was in favor of displacing Bax from binding to Bcl-xL or Bcl-2. Consequently, Bax exerted a pro-apoptotic dysfunction to mitochondria, thereby inducing the release of cytochrome c. Finally, apoptin induced the apoptosis of HHCC cells. These results indicate that the oligonucleotide microarray can reveal the genes related to the apoptosis induced by apoptin in HHCC cells.

  20. Bax Inhibitor-1 down-regulation in the progression of chronic liver diseases

    Directory of Open Access Journals (Sweden)

    Burra Patrizia

    2010-04-01

    Full Text Available Abstract Background Bax inhibitor-1 (BI-1 is an evolutionary conserved endoplasmic reticulum protein that, when overexpressed in mammalian cells, suppresses the apoptosis induced by Bax, a pro-apoptotic member of the Bcl-2 family. The aims of this study were: (1 to clarify the role of intrinsic anti- and pro-apoptotic mediators, evaluating Bax and BI-1 mRNA and protein expressions in liver tissues from patients with different degrees of liver damage; (2 to determine whether HCV and HBV infections modulate said expression. Methods We examined 62 patients: 39 with chronic hepatitis (CH (31 HCV-related and 8 HBV-related; 7 with cirrhosis (6 HCV-related and 1 HBV-related; 13 with hepatocellular carcinoma (HCC [7 in viral cirrhosis (6 HCV- and 1 HBV-related, 6 in non-viral cirrhosis]; and 3 controls. Bax and BI-1 mRNAs were quantified by real-time PCR, and BI-1 protein expression by Western blot. Results CH tissues expressed significantly higher BI-1 mRNA levels than cirrhotic tissues surrounding HCC (P Conclusions BI-1 expression is down-regulated as liver damage progresses. The high BI-1 mRNAs levels observed in early liver disease may protect virus-infected cells against apoptosis, while their progressive downregulation may facilitate hepatocellular carcinogenesis. HCV genotype seems to have a relevant role in Bax transcript expression.

  1. Quercetin induces tumor-selective apoptosis through downregulation of Mcl-1 and activation of Bax.

    Science.gov (United States)

    Cheng, Senping; Gao, Ning; Zhang, Zhuo; Chen, Gang; Budhraja, Amit; Ke, Zunji; Son, Young-ok; Wang, Xin; Luo, Jia; Shi, Xianglin

    2010-12-01

    To investigate the in vivo antitumor efficacy of quercetin in U937 xenografts and the functional roles of Mcl-1 and Bax in quercetin-induced apoptosis in human leukemia. Leukemia cells were treated with quercetin, after which apoptosis, Mcl-1 expression, and Bax activation and translocation were evaluated. The efficacy of quercetin as well as Mcl-1 expression and Bax activation were investigated in xenografts of U937 cells. Administration of quercetin caused pronounced apoptosis in both transformed and primary leukemia cells but not in normal blood peripheral mononuclear cells. Quercetin-induced apoptosis was accompanied by Mcl-1 downregulation and Bax conformational change and mitochondrial translocation that triggered cytochrome c release. Knockdown of Bax by siRNA reversed quercetin-induced apoptosis and abrogated the activation of caspase and apoptosis. Ectopic expression of Mcl-1 attenuated quercetin-mediated Bax activation, translocation, and cell death. Conversely, interruption of Mcl-1 by siRNA enhanced Bax activation and translocation, as well as lethality induced by quercetin. However, the absence of Bax had no effect on quercetin-mediated Mcl-1 downregulation. Furthermore, in vivo administration of quercetin attenuated tumor growth in U937 xenografts. The TUNEL-positive apoptotic cells in tumor sections increased in quercetin-treated mice as compared with controls. Mcl-1 downregulation and Bax activation were also observed in xenografts. These data suggest that quercetin may be useful for the treatment of leukemia by preferentially inducing apoptosis in leukemia versus normal hematopoietic cells through a process involving Mcl-1 downregulation, which, in turn, potentiates Bax activation and mitochondrial translocation, culminating in apoptosis. ©2010 AACR.

  2. ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

    Science.gov (United States)

    Song, Shanshan; Jacobson, Krista N; McDermott, Kimberly M; Reddy, Sekhar P; Cress, Anne E; Tang, Haiyang; Dudek, Steven M; Black, Stephen M; Garcia, Joe G N; Makino, Ayako; Yuan, Jason X-J

    2016-01-15

    Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. Copyright

  3. Allosteric sensitization of proapoptotic BAX.

    Science.gov (United States)

    Pritz, Jonathan R; Wachter, Franziska; Lee, Susan; Luccarelli, James; Wales, Thomas E; Cohen, Daniel T; Coote, Paul; Heffron, Gregory J; Engen, John R; Massefski, Walter; Walensky, Loren D

    2017-09-01

    BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.

  4. Calpain and reactive oxygen species targets Bax for mitochondrial permeabilisation and caspase activation in zerumbone induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Praveen K Sobhan

    Full Text Available Fluorescent protein based signaling probes are emerging as valuable tools to study cell signaling because of their ability to provide spatio- temporal information in non invasive live cell mode. Previously, multiple fluorescent protein probes were employed to characterize key events of apoptosis in diverse experimental systems. We have employed a live cell image based approach to visualize the key events of apoptosis signaling induced by zerumbone, the active principle from ginger Zingiber zerumbet, in cancer cells that enabled us to analyze prominent apoptotic changes in a hierarchical manner with temporal resolution. Our studies substantiate that mitochondrial permeabilisation and cytochrome c dependent caspase activation dominate in zerumbone induced cell death. Bax activation, the essential and early event of cell death, is independently activated by reactive oxygen species as well as calpains. Zerumbone failed to induce apoptosis or mitochondrial permeabilisation in Bax knockout cells and over-expression of Bax enhanced cell death induced by zerumbone confirming the essential role of Bax for mitochondrial permeabilsation. Simultaneous inhibition of reactive oxygen species and calpain is required for preventing Bax activation and cell death. However, apoptosis induced by zerumbone was prevented in Bcl 2 and Bcl-XL over-expressing cells, whereas more protection was afforded by Bcl 2 specifically targeted to endoplasmic reticulum. Even though zerumbone treatment down-regulated survival proteins such as XIAP, Survivin and Akt, it failed to affect the pro-apoptotic proteins such as PUMA and BIM. Multiple normal diploid cell lines were employed to address cytotoxic activity of zerumbone and, in general, mammary epithelial cells, endothelial progenitor cells and smooth muscle cells were relatively resistant to zerumbone induced cell death with lesser ROS accumulation than cancer cells.

  5. Driving p53 Response to Bax Activation Greatly Enhances Sensitivity to Taxol by Inducing Massive Apoptosis

    Directory of Open Access Journals (Sweden)

    Paola De Feudis

    2000-05-01

    Full Text Available The proapoptotic gene bax is one of the downstream effectors of p53. The p53 binding site in the bax promoter is less responsive to p53 than the one in the growth arrest mediating gene p21. We introduced the bax gene under the control of 13 copies of a strong p53 responsive element into two ovarian cancer cell lines. The clones expressing bax under the control of p53 obtained from the wild-type (wt p53-expressing cell line A2780 were much more sensitive (500- to 1000-fold to the anticancer agent taxol than the parent cell line, with a higher percentage of cells undergoing apoptosis after drug treatment that was clearly p53-dependent and bax-mediated. Xenografts established in nude mice from one selected clone (A2780/C3 were more responsive to taxol than the parental line and the apoptotic response of A2780/C3 tumors was also increased after treatment. Introduction of the same plasmid into the p53 null SKOV3 cell line did not alter the sensitivity to taxol or the induction of apoptosis. In conclusion, driving the p53 response (after taxol treatment by activating the bax gene rather than the p21 gene results in induction of massive apoptosis, in vitro and in vivo, and greatly enhances sensitivity to the drug.

  6. Bax Activation Blocks Self-Renewal and Induces Apoptosis of Human Glioblastoma Stem Cells.

    Science.gov (United States)

    Daniele, Simona; Pietrobono, Deborah; Costa, Barbara; Giustiniano, Mariateresa; La Pietra, Valeria; Giacomelli, Chiara; La Regina, Giuseppe; Silvestri, Romano; Taliani, Sabrina; Trincavelli, Maria Letizia; Da Settimo, Federico; Novellino, Ettore; Martini, Claudia; Marinelli, Luciana

    2017-04-11

    Glioblastoma (GBM) is characterized by a poor response to conventional chemotherapeutic agents, attributed to the insurgence of drug resistance mechanisms and to the presence of a subpopulation of glioma stem cells (GSCs). GBM cells and GSCs present, among others, an overexpression of antiapoptotic proteins and an inhibition of pro-apoptotic ones, which help to escape apoptosis. Among pro-apoptotic inducers, the Bcl-2 family protein Bax has recently emerged as a promising new target in cancer therapy along with first BAX activators (BAM7, Compound 106, and SMBA1). Herein, a derivative of BAM-7, named BTC-8, was employed to explore the effects of Bax activation in different human GBM cells and in their stem cell subpopulation. BTC-8 inhibited GBM cell proliferation, arrested the cell cycle, and induced apoptosis through the induction of mitochondrial membrane permeabilization. Most importantly, BTC-8 blocked proliferation and self-renewal of GSCs and induced their apoptosis. Notably, BTC-8 was demonstrated to sensitize both GBM cells and GSCs to the alkylating agent Temozolomide. Overall, our findings shed light on the effects and the relative molecular mechanisms related to Bax activation in GBM, and they suggest Bax-targeting compounds as promising therapeutic tools against the GSC reservoir.

  7. Gynostemma pentaphyllum protects mouse male germ cells against apoptosis caused by zearalenone via Bax and Bcl-2 regulation.

    Science.gov (United States)

    Yuan, Hui; Deng, Youtian; Yuan, Liyun; Wu, Jing; Yuan, Zhihang; Yi, Jine; Zhang, Ming; Guo, Chengzhi; Wen, Lixin; Li, Rongfang; Zhu, Li; He, Zuping

    2010-03-01

    The objective of this study was to explore the effects of Gynostemma pentaphyllum on Zearalenone-induced apoptosis in mouse male germ cells. Fifty Kunming male mice at 25-days-old were classified into five groups: group A was the control (10% ethanol, 0.5 ml/day); group B with 10 microg Zearalenone/day; group C with 10 microg Zearalenone and 50 mg/kg/day Gynostemma pentaphyllum; group D with 10 microg Zearalenone and 100 mg/kg/day Gynostemma pentaphyllum; and group E with 10 microg Zearalenone and 200 mg/kg/day Gynostemma pentaphyllum. It was found that Gynostemma pentaphyllum has a marked effect on protecting male germ cells against Zearalenone-induced apoptosis, as evidenced by a reduced apoptosis rate of male germ cells and Bax expression as well as an enhancement of Bcl-2 expression in Gynostemma pentaphyllum-treated groups compared to the control. In addition, Gynostemma pentaphyllum remarkably improved pathologic changes of testicular tissue, reduced the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD) caused by Zearalenone. Taken together, these results suggest that Gynostemma pentaphyllum protects against toxicity caused by Zearalenone through anti-oxidation and anti-apoptosis via the regulation of Bax and Bcl-2 expression.

  8. Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation.

    Science.gov (United States)

    Tessner, Teresa G; Muhale, Filipe; Riehl, Terrence E; Anant, Shrikant; Stenson, William F

    2004-12-01

    Prostaglandin E2 (PGE2) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE2 (dmPGE2) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax(-/-) mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE2 to attenuate apoptosis was lost in Bax(-/-) mice. Positional analysis revealed that apoptosis in the Bax(-/-) mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax(-/-) HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE2. Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE2. Treatment with dmPGE2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE2. The in vitro studies indicate that dmPGE2, most likely by signaling through the E prostaglandin receptor EP2, reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.

  9. Neohesperidin induces cellular apoptosis in human breast adenocarcinoma MDA-MB-231 cells via activating the Bcl-2/Bax-mediated signaling pathway.

    Science.gov (United States)

    Xu, Fei; Zang, Jia; Chen, Daozhen; Zhang, Ting; Zhan, Huiying; Lu, Mudan; Zhuge, Hongxiang

    2012-11-01

    Neohesperidin, a flavonoid compound found in high amounts in Poncirus trifoliata, has free radical scavenging activity. For the first time, our study indicated that neohesperidin also induces cell apoptosis in human breast adenocarcinoma MDA-MB-231 cells, which was possibly mediated by regulating the P53/Bcl-2/Bax pathway. MDA-MB-231 cells were subjected to treatment with neohesperidin. MTT and Trypan blue exclusion assays were applied to assess the cell viability. The morphological changes of cells were observed using an inverted microscope, and cell apoptosis was detected by flow cytometric analysis. Immunoblot analysis was conducted to evaluate the protein expressions of apoptosis-related genes, including P53, Bcl-2 and Bax. Our results indicated that the proliferation of MDA-MB-231 cells was inhibited by the treatment with neohesperidin in a time- and dose-dependent manner. The IC50 values of neohesperidin at 24 and 48 h were 47.4 +/- 2.6 microM and 32.5 +/- 1.8 microM, respectively. The expressions of P53 and Bax in the neohesperidin-treated cells were significantly up-regulated, while that of Bcl-2 was down-regulated. Our study suggested that neohesperidin could induce apoptosis of MDA-MB-231 cells, a process which was associated with the activation of the Bcl-2/Bax-mediated signaling pathway.

  10. Mangiferin attenuates contusive spinal cord injury in rats through the regulation of oxidative stress, inflammation and the Bcl‑2 and Bax pathway.

    Science.gov (United States)

    Luo, Yang; Fu, Changfeng; Wang, Zhenyu; Zhang, Zhuo; Wang, Hongxia; Liu, Yi

    2015-11-01

    Mangiferin has antioxidant, antiviral, apoptosis regulating, anti‑inflammatory, antitumor and antidiabetic effects, which can also inhibit osteoclast formation and bone resorption. However, whether mangiferin ameliorates the neurological pain of spinal cord injury (SCI) in ratS remains to be elucidated. The present study investigated the therapeutic effects of mangiferin on neurological function, the water content of spinal cord, oxidative stress, the expression of inflammatory cytokines and the protein expression of Bcl‑2/Bax in a SCI rat model. In the present study, the Basso, Beattie and Bresnahan scores, and the water content of the spinal cord were used to analyze the therapeutic effects of mangiferin on neurological pain in the SCI rat. The concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and the serum levels of glutathione peroxidase (GSH‑PX), nuclear factor‑κB p65 unit, tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6 and caspase‑3/9 were detected using commercial kits. The expression levels of Bcl‑2 and Bax were measured using western blot analysis. The results demonstrated that administrating mangiferin began to ameliorate neurological function and the water content of the spinal cord in the SCI rat. The mangiferin‑treated group were found to have lower oxidative stress activity and lower expression levels of inflammatory cytokines, compared with the SCI rat. In addition, mangiferin significantly reduced the protein expression of Bax and promoted the protein expression of Bcl-2 in the SCI rat model. Finally, mangiferin markedly suppressed the expression of caspase‑3/9, indicating that the protective action of mangiferin may be associated with anti‑apoptosis activation. In conclusion, mangiferin attenuated contusive SCI in the rats through regulating oxidative stress, inflammation and the Bcl‑2 and Bax pathway.

  11. Oleanolic acid from Prunella Vulgaris L. induces SPC-A-1 cell line apoptosis via regulation of Bax, Bad and Bcl-2 expression.

    Science.gov (United States)

    Feng, Liang; Au-Yeung, Wai; Xu, You-Hua; Wang, Shan-Shan; Zhu, Quan; Xiang, Ping

    2011-01-01

    Prunella vulgaris L. (PV) has been used as a herb for chemoprevention of lung cancer. In this study, the main active compound, oleanolic acid (OA) was isolated from an ethanol extract and its chemical structure was identified according to the results of high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC) and liquid chromatography-mass spectrography (LC-MS). Results for cell viability indictated no notable differences between OA and ethanol extract of PV in lung adenocarcinoma SPC-A-1 cells measured by MTT assay. Consistent concentration-response curves. Fluorescence detection with acridine orange-ethidium bromide was used to evaluate apoptosis of SPC-A-1 cells. OA at 16 and 8 microM group increased significantly the apoptosis rate compared with normal and 1% DMSO groups (p<0.05). In addition, immunocytochemistry assays showed increase in Bax and Bad protein expression while Bcl-2 decreased. Moreover, the ratio of Bax/Bcl-2 was heightened by OA treatment. The results suggest OA induced apoptosis of lung adenocarcinoma cells through down-regulating Bcl-2 expression, and up-regulating Bax and Bad expression.

  12. 光动力学调控miR143激活Bcl-2/Bax的信号路径对人宫颈癌的治疗机制%Regulation Effect of Photodynamic Therapy on Bcl-2/Bax Signal Path Activated by miR143 in Human Cervical Cancer

    Institute of Scientific and Technical Information of China (English)

    兰艳丽; 刘韵

    2016-01-01

    Objective To investigate the regulation effect of photodynamic therapy on Bcl-2/Bax signal path activated by miR143 in human cervical cancer ,and lay foundation for cervical cancer treated with photodynamic therapy along with miR 143. Methods Human HeLa cells received miR 143 interference ( miR143 group ) , photodynamic irradiation treatment ( PDT group ) and miR143 interference combined with photodynamic irradiation (PDT +miR143 group).The rate of inhibition,apoptosis and invasion were detected by CCK8,flow cytometry and Transwell model ,respectively.Meanwhile the expression levels of miR143, Bcl-2 and Bax before and after different treatments were detected by fluorescence quantitative PCR .Results The inhibition rates and apoptosis rates among the 3 groups were significantly different (P0.05).Conclusion In cervical cancer,photodynamic therapy upregulated the Bcl-2/Bax signal path activated by miR 143,and lay foundation for cervical cancer treated with photodynamic therapy along with miR 143.%目的 探讨在宫颈癌中光动力学调控对miR143激活Bcl-2/Bax的信号路径的影响,为光动力学联合miR143应用于宫颈癌的治疗机制奠定基础.方法 人宫颈癌HeLa细胞分别进行miR143干扰处理(miR143组)、光动力照射处理(PDT组)及miR143干扰联合光动力照射处理(PDT+miR143组),采用CCK8法、流式细胞术及Transwell小室侵袭模型对各组处理后HeLa细胞的抑制率、凋亡率及侵袭能力进行分析,同时采用荧光定量PCR检测不同处理前后各组细胞的miR143、Bcl-2和Bax的mRNA表达水平.结果 3组之间的细胞抑制率、凋亡率差异有统计学意义(P<0.05),PDT+miR143组细胞抑制率和凋亡率分别高于PDT组和miR143组,差异具有统计学意义(P<0.05),而PDT+miR143组穿膜细胞数显著高于miR143组和PDT组(P<0.05).处理后,3组miR143和Bcl-2 mRNA表达水平较处理前均显著升高(P<0.0001),且处理后PDT+miR143组miR143和Bcl-2 mRNA表达

  13. Enterovirus 71 2B Induces Cell Apoptosis by Directly Inducing the Conformational Activation of the Proapoptotic Protein Bax.

    Science.gov (United States)

    Cong, Haolong; Du, Ning; Yang, Yang; Song, Lei; Zhang, Wenliang; Tien, Po

    2016-11-01

    To survive and replicate within a host, many viruses have evolved strategies that target crucial components within the apoptotic cascade, leading to either inhibition or induction of cell apoptosis. Enterovirus 71 (EV71) infections have been demonstrated to impact the mitochondrial apoptotic pathway and induce apoptosis in many cell lines. However, the detailed mechanism of EV71-induced apoptosis remains to be elucidated. In this study, we report that EV71 2B protein (2B) localized to the mitochondria and induced cell apoptosis by interacting directly with and activating the proapoptotic protein Bax. 2B recruited Bax to the mitochondria and induced Bax conformational activation. In addition, mitochondria isolated from 2B-expressing cells that were treated with a recombinant Bax showed increased Bax interaction and cytochrome c (Cyt c) release. Importantly, apoptosis in cells with either EV71 infection or 2B expression was dramatically reduced in Bax knockdown cells but not in Bak knockdown cells, suggesting that Bax played a pivotal role in EV71- or 2B-induced apoptosis. Further studies indicate that a hydrophobic region of 18 amino acids (aa) in the C-terminal region of 2B (aa 63 to 80) was responsible for the location of 2B in the mitochondria. A hydrophilic region of 14 aa in the N-terminal region of 2B was functional in Bax interaction and its subsequent activation. Moreover, overexpression of the antiapoptotic protein Bcl-XL abrogates 2B-induced release of Cyt c and caspase activation. Therefore, this study provides direct evidence that EV71 2B induces cell apoptosis and impacts the mitochondrial apoptotic pathway by directly modulating the redistribution and activation of proapoptotic protein Bax. EV71 infections are usually accompanied by severe neurological complications. It has also been postulated that the induction of cell apoptosis resulting from tissue damage is a possible process of EV71-related pathogenesis. In this study, we report that EV71 2B

  14. Robotic assistance for performing vocational rehabilitation activities using BaxBot.

    Science.gov (United States)

    Ashley, Kyle; Alqasemi, Redwan; Dubey, Rajiv

    2017-07-01

    Activities of Daily Living (ADL's) refer to tasks that people do on a daily basis, such as self-feeding, cleaning the house, or bathing. These activities often require a degree of functional mobility that may be outside the ability of a person suffering from cognitive or physical impairment. This work describes methods of performing ADL's with a mobile robotic system. We examined the needs of potential users and caregivers through surveys to determine the most needed applications for robotic assistance. Using this information, we extended the functionality of our BaxBot mobile robotic system to provide meaningful, autonomous assistance in performing three specific ADL's with minimal user interaction.

  15. Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax.

    Science.gov (United States)

    Blatt, Neal B; Boitano, Anthony E; Lyssiotis, Costas A; Opipari, Anthony W; Glick, Gary D

    2008-11-01

    Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.

  16. Bax, Bak and beyond - mitochondrial performance in apoptosis.

    Science.gov (United States)

    Peña-Blanco, Aida; García-Sáez, Ana J

    2017-07-29

    Bax and Bak are members of the Bcl-2 family and core regulators of the intrinsic pathway of apoptosis. Upon apoptotic stimuli, they are activated and oligomerize at the mitochondrial outer membrane (MOM) to mediate its permeabilization, which is considered a key step in apoptosis. However, the molecular mechanism underlying Bax and Bak function has remained a key question in the field. Here, we review recent structural and biophysical evidence that has changed our understanding of how Bax and Bak promote MOM permeabilization. We also discuss how the spatial regulation of Bcl-2 family preference for binding partners contributes to regulate Bax and Bak activation. Finally, we consider the contribution of mitochondrial composition, dynamics and interaction with other organelles to apoptosis commitment. A new perspective is emerging, in which the control of apoptosis by Bax and Bak goes beyond them and is highly influenced by additional mitochondrial components. © 2017 Federation of European Biochemical Societies.

  17. A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2.

    Science.gov (United States)

    Renault, Thibaud T; Dejean, Laurent M; Manon, Stéphen

    2017-01-01

    Bcl-2 family members form a network of protein-protein interactions that regulate apoptosis through permeabilization of the mitochondrial outer membrane. Deciphering this intricate network requires streamlined experimental models, including the heterologous expression in yeast. This approach had previously enabled researchers to identify domains and residues that underlie the conformational changes driving the translocation, the insertion and the oligomerization of the pro-apoptotic protein Bax at the level of the mitochondrial outer membrane. Recent studies that combine experiments in yeast and in mammalian cells have shown the unexpected effect of the anti-apoptotic protein Bcl-xL on the priming of Bax. As demonstrated with the BH3-mimetic molecule ABT-737, this property of Bcl-xL, and of Bcl-2, is crucial to elaborate about how apoptosis could be reactivated in tumoral cells.

  18. Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes.

    Science.gov (United States)

    Andreu-Fernández, Vicente; Sancho, Mónica; Genovés, Ainhoa; Lucendo, Estefanía; Todt, Franziska; Lauterwasser, Joachim; Funk, Kathrin; Jahreis, Günther; Pérez-Payá, Enrique; Mingarro, Ismael; Edlich, Frank; Orzáez, Mar

    2017-01-10

    The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

  19. Emodin inhibits LOVO colorectal cancer cell proliferation via the regulation of the Bcl-2/Bax ratio and cytochrome c.

    Science.gov (United States)

    Ma, Liang; Li, Wusheng

    2014-10-01

    In this study, the effect of emodin and its mechanism of action were investigated in LOVO colorectal cancer cells. Cell growth was determined using a Cell Counting kit-8 assay, and the results demonstrated that emodin significantly inhibited the growth of LOVO cells in a concentration-dependent manner. In order to investigate the anticancer mechanism of emodin, reverse transcription polymerase chain reaction assays were performed to determine the B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax) expression ratio in LOVO colorectal cancer cells following treatment with emodin. The results showed that emodin induced a significant increase in the Bax expression level and a marked reduction of the Bcl-2 expression level in LOVO cells. In addition, emodin was found to have an inhibitory effect on the mitochondrial membrane potential and the results from the western blot analysis revealed that cytochrome c was released from the mitochondria to the cytoplasm. In combination, these results suggest that emodin inhibits cancer cell growth via the regulation of the Bcl-2/Bax ratio and by its effect on the mitochondrial apoptosis pathway.

  20. Hyperactivity and depression-like traits in Bax KO mice.

    Science.gov (United States)

    Krahe, Thomas E; Medina, Alexandre E; Lantz, Crystal L; Filgueiras, Cláudio C

    2015-11-02

    The Bax gene is a member of the Bcl-2 gene family and its pro-apoptotic Bcl-associated X (Bax) protein is believed to be crucial in regulating apoptosis during neuronal development as well as following injury. With the advent of mouse genomics, mice lacking the pro-apoptotic Bax gene (Bax KO) have been extensively used to study how cell death helps to determine synaptic circuitry formation during neurodevelopment and disease. Surprisingly, in spite of its wide use and the association of programmed neuronal death with motor dysfunctions and depression, the effects of Bax deletion on mice spontaneous locomotor activity and depression-like traits are unknown. Here we examine the behavioral characteristics of Bax KO male mice using classical paradigms to evaluate spontaneous locomotor activity and depressive-like responses. In the open field, Bax KO animals exhibited greater locomotor activity than their control littermates. In the forced swimming test, Bax KO mice displayed greater immobility times, a behavior despair state, when compared to controls. Collectively, our findings corroborate the notion that a fine balance between cell survival and death early during development is critical for normal brain function later in life. Furthermore, it points out the importance of considering depressive-like and hyperactivity behavioral phenotypes when conducting neurodevelopmental and other studies using the Bax KO strain. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Quercetin inhibits human breast cancer cell proliferation and induces apoptosis via Bcl-2 and Bax regulation.

    Science.gov (United States)

    Duo, Jian; Ying, Guo-Guang; Wang, Guo-Wen; Zhang, Li

    2012-06-01

    Breast cancer is a disease in which cancer cells form in the tissues of the breast. The present study aimed to explore the effect of the flavonoid compound quercetin on the growth and apoptosis of human breast cancer cells. Varying concentrations (12.5, 25, 50, 100, 200 µM) of quercetin were applied to cultured MCF-7 human breast cancer cells for defined lengths of time. At 50 to 200 µM doses, quercetin significantly inhibited the proliferation of MCF-7 cells assessed by MTT colorimetry, in both dose- and time-dependent manners (Papoptosis after 48 h of exposure (Pquercetin treatment Bcl-2 expression decreased significantly while Bax expression increased significantly (Pquercetin inhibits cell growth and induces apoptosis in MCF-7 human breast cancer cells. The mechanisms behind these effects may stem from the downregulation of Bcl-2 protein expression and upregulation of Bax expression.

  2. Oridonin induces apoptosis of HeLa cells via altering expres sion of Bcl-2/Bax and activating caspase-3/ICAD pathway

    Institute of Scientific and Technical Information of China (English)

    Chun-ling ZHANG; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2004-01-01

    AIM: To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro. METHODS: Viability of oridonin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis.Caspase activity was assayed using fiuorometric protease assay. ICAD, Bcl-2, and Bax proteins expression were detected by Western blot analysis. RESULTS: Oridonin induced oligonucleosomal fragmentation of DNA and increased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase (ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activation and down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-AspGlu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro5-[4-(1-piperidinyl)butoxy]-1 (2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. In addition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. CONCLUSION:Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation of caspase-3/ICAD pathway.

  3. Amygdalin induces apoptosis through regulation of Bax and Bcl-2 expressions in human DU145 and LNCaP prostate cancer cells.

    Science.gov (United States)

    Chang, Hyun-Kyung; Shin, Mal-Soon; Yang, Hye-Young; Lee, Jin-Woo; Kim, Young-Sick; Lee, Myoung-Hwa; Kim, Jullia; Kim, Khae-Hawn; Kim, Chang-Ju

    2006-08-01

    Prostate cancer is one of the most common non-skin cancers in men. Amygdalin is one of the nitrilosides, natural cyanide-containing substances abundant in the seeds of plants of the prunasin family that have been used to treat cancers and relieve pain. In particular, D-amygdalin (D-mandelonitrile-beta-D-gentiobioside) is known to exhibit selective killing effect on cancer cells. Apoptosis, programmed cell death, is an important mechanism in cancer treatment. In the present study, we prepared the aqueous extract of the amygdalin from Armeniacae semen and investigated whether this extract induces apoptotic cell death in human DU145 and LNCaP prostate cancer cells. In the present results, DU145 and LNCaP cells treated with amygdalin exhibited several morphological characteristics of apoptosis. Treatment with amygdalin increased expression of Bax, a pro-apoptotic protein, decreased expression of Bcl-2, an anti-apoptotic protein, and increased caspase-3 enzyme activity in DU145 and LNCaP prostate cancer cells. Here, we have shown that amygdalin induces apoptotic cell death in human DU145 and LNCaP prostate cancer cells by caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. The present study reveals that amygdalin may offer a valuable option for the treatment of prostate cancers.

  4. SIRT1 regulates in vitro apoptosis of chondrocytes via p53/bax and NF-κB/peroxisome proliferator-activated receptor-γ coactivator-1α pathways%SIRT1经p53和NF-κB的相关蛋白途径调控软骨细胞凋亡的体外研究

    Institute of Scientific and Technical Information of China (English)

    刘弼; 肖德明; 雷鸣; 王大平; 熊建义; 马经野; 许蕴; 史敦云; 张晓丽

    2012-01-01

    Objective To investigate the role and mechanism of SIRT1 in regulating the apoptosis of chondrocytes in vitro. Methods The articular chondrocytes from New Zealand rabbits were routinely cultured and divided into 3 even groups( n =10):SIRT1 activator (resveratrol) group,control group and SIRT1 inhibitor (nicotinamide) group.The apoptosis of chondrocytes was induced by sodium nitroprusside (SNP).MTT assay was used to detect the activity of cartilage cells in each group.DAPI staining and flow cytometry were applied to detect the apoptosis of chondrocytes.Western Blot was used to detect the expressions of SIRT1,p53,NF-κB,bax,PGC-1α in the cells in each group.RT-PCR method was applied to detect the mRNA expressions of SIRT1,type Ⅱ collagen and aggrecan in the cells in each group. Results The average OD value was 0.139 ±0.016 in the SIRT1 activator group,0.098 ±0.006 in the control group and 0.079 ± 0.002 in the SIRT1 inhibitor group.There was a significant difference among the 3 groups ( F =51.273,P =0.000) and between groups as well ( P < 0.05).In terms of cellular apoptosis,the SIRT1 inhibitor group is the highest,the control group lower and the SIRT1 activator group the lowest.Compared with the control group,the expressions of SIRT1 and PGC-1α were increased in the SIRT1 activator group but decreased in the SIRT1 inhibitor group.The expressions of p53,NF-κB and bax were lower in the SIRT1 activator group than in the control group while those were higher in the SIRT1 inhibitor group.The mRNA expressions of SIRT1,type Ⅱ collagen and aggrecan were the highest in the SIRT1 activator group,lower in the control group and the lowest in the SIRT1 inhibitor group. Conclusion SIRT1 may regulate the apoptosis of chondrocytes by regulating the expressions of p53 and NF-κB which in turn changes the expressions of downstream bax and PGC-1α.%目的 探讨SIRT1经p53/bax和NF-κB/过氧化物酶体增殖物受体γ共激活因子-1α(PGC-1α)途径调控软

  5. Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax.

    Science.gov (United States)

    Starkov, Anatoly A; Polster, Brian M; Fiskum, Gary

    2002-10-01

    Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.

  6. Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species

    Science.gov (United States)

    Subburaj, Yamunadevi; Cosentino, Katia; Axmann, Markus; Pedrueza-Villalmanzo, Esteban; Hermann, Eduard; Bleicken, Stephanie; Spatz, Joachim; García-Sáez, Ana J.

    2015-08-01

    Bax is a key regulator of apoptosis that mediates the release of cytochrome c to the cytosol via oligomerization in the outer mitochondrial membrane before pore formation. However, the molecular mechanism of Bax assembly and regulation by other Bcl-2 members remains obscure. Here, by analysing the stoichiometry of Bax oligomers at the single-molecule level, we find that Bax binds to the membrane in a monomeric state and then self-assembles in Bax does not exist in a unique oligomeric state, but as several different species based on dimer units. Moreover, we show that cBid activates Bax without affecting its assembly, while Bcl-xL induces the dissociation of Bax oligomers. On the basis of our experimental data and theoretical modelling, we propose a new mechanism for the molecular pathway of Bax assembly to form the apoptotic pore.

  7. Hyperhomocysteinemia induces cardiac injury by up-regulation of p53-dependent Noxa and Bax expression through the p53 DNA methylation in ApoE-/-mice

    Institute of Scientific and Technical Information of China (English)

    Shengchao Ma; Huiping Zhang; Weiwei Sun; HuiHui Gong; Yanhua Wang; Changjian Ma; Ju Wang

    2013-01-01

    Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease and has a strong correlation with heart failure.However,the effects of HHcy on cardiac tissue remain less well understood.To elucidate the role of p53-dependent apoptosis in HHcy-induced cardiac injury,we fed ApoE-/-mice with high methionine diet to establish HHcy model.Serum Hcy,cardiac enzymes,and lipids were measured.The protein levels of Noxa,DNMT1,caspases-3/9,and p53 were determined by enzyme-linked immunosorbent assay.Bcl-2 and Bax proteins were detected by immunohistochemistry staining.S-adenosyl methionine and S-adenosyl homocysteine concentrations were determined by high-performance liquid chromatography.The mRNA levels of p53 and DNMT1 were analyzed by real-time polymerase chain reaction (PCR) and the methylation levels of p53 were analyzed by nested methylation-specific-PCR.Our data showed that the concentrations of serum Hey and lipids were increased in Meth group compared with the N-control group,which indicated that the model was established successfully.The expression levels of p53 and Noxa were increased in Meth group,while the methylation status of p53 was hypomethylation.The activities of caspase-3/9 were increased in Meth group compared with the N-control group.In addition,immunohistochemistry staining showed that the expression of Bax was significantly increased in Meth and Meth-F group compared with the N-control group.In summary,HHcy induces cardiac injury by up-regulation of p53-dependent pro-apoptotic related genes Noxa and Bax,while p53 DNA hypomethylation is a key molecular mechanism in pathological process induced by HHcy.

  8. Nano-SiO2 induces apoptosis via activation of p53 and Bax mediated by oxidative stress in human hepatic cell line.

    Science.gov (United States)

    Ye, Yiyi; Liu, Jianwen; Xu, Jianhe; Sun, Lijuan; Chen, Mingcang; Lan, Minbo

    2010-04-01

    Nanoparticles such as nano-SiO(2) are increasingly used in food, cosmetics, diagnosis, imaging and drug delivery. However, toxicological data of nano-SiO(2) on hepatic cells in vitro and their detailed molecular mechanisms still remain unclear. In order to assess toxicity of nano-SiO(2), L-02 cells were exposed to 0.2, 0.4 and 0.6 mg/ml of SiO(2) colloids (21, 48 and 86 nm) for 12, 24, 36 and 48h. Lactate dehydrogenase released from damaged cells were quantified, cellular ultrastructural organization was observed, and the levels of reactive oxygen species (ROS), lipid peroxidation and glutathione were measured. Apoptosis induced by 21 nm SiO(2) was characterized by annexin V-FITC/PI staining and DNA ladder assay. Furthermore, apoptosis related proteins such as p53, Bax and Bcl-2 were analyzed by using western blot analysis. Our data indicated that nano-SiO(2) caused cytotoxicity in size, dose and time dependent manners. Oxidative stress and apoptosis were induced by exposure to 21 nm SiO(2). Moreover, the expression of p53 and Bax was increased in time and dose dependent patterns, whereas the expression of Bcl-2 was not significantly changed. In conclusion, ROS-mediated oxidative stress, the activation of p53 and up-regulation of Bax/Bcl-2 ratio are involved in mechanistic pathways of 21 nm SiO(2) induced apoptosis in L-02 cells.

  9. Inhibition of benzopyrene-diol-epoxide (BPDE)-induced bax and caspase-9 by cadmium: Role of mitogen activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Jagat J.; Gupta, Suresh K. [State University of New York College at Buffalo, Environ. Toxicol. and Chem., Great Lakes Center, 1300 Elmwood Avenue, Buffalo, NY 14222 (United States); Kumar, Subodh [State University of New York College at Buffalo, Environ. Toxicol. and Chem., Great Lakes Center, 1300 Elmwood Avenue, Buffalo, NY 14222 (United States)], E-mail: kumars@buffalostate.edu

    2009-02-10

    Cadmium, a major metal constituent of tobacco smoke, elicits synergistic enhancement of cell transformation when combined with benzo[a]pyrene (BP) or other polynuclear aromatic hydrocarbons (PAHs). The mechanism underlying this synergism is not clearly understood. Present study demonstrates that (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen of BP, induces apoptosis in human leukemic HL-60 cells and others, and cadmium at non-cytotoxic concentration inhibits BPDE-induced apoptosis. We observed that BPDE treatment also activates all three MAP kinases e.g. ERK1/2, p38 and JNK in HL-60 cells, and inhibition of BPDE-induced apoptosis by cadmium is associated with down-regulation of pro-apoptotic bax induction/caspase-9 activation and up-regulation of ERK phosphorylation, whereas p38 MAP kinase and c-Jun phosphorylation (indicative of JNK activation) remain unaffected. Inhibition of ERKs by prior treatment of cells with 10 {mu}M U0126 relieves cadmium-mediated inhibition of apoptosis/bax induction/caspase-9 activation. Our results suggest that cadmium inhibits BPDE-induced apoptosis by modulating apoptotic signaling through up-regulation of ERK, which is known to promote cell survival.

  10. DHT inhibits the Aβ25-35-induced apoptosis by regulation of seladin-1, survivin, XIAP, bax, and bcl-xl expression through a rapid PI3-K/Akt signaling in C6 glial cell lines.

    Science.gov (United States)

    Bing, Lelin; Wu, Junfeng; Zhang, Jianfeng; Chen, Yinghui; Hong, Zhen; Zu, Hengbing

    2015-01-01

    Previous evidences indicate that androgen is neuroprotective in the brain. However, the underling mechanisms remain to be fully elucidated. Moreover, it is controversial whether dihydrotestosterone (DHT) modulates the expression of apoptosis-related effectors, such as survivin, XIAP, bax, and bcl-xl proteins mediated by the PI3-K/Akt pathway, which contributes to androgen neuroprotection. In this study using a C6 glial cell model, apoptotic cells were detected by flow cytometry. Akt, seladin-1, survivin, XIAP, bcl-xl, and bax protein expression is investigated by Western blot. After amyloid β-protein fragment (Aβ25-35) treatment, apoptotic cells at early (annexin V+, PI-) and late (annexin V+, PI+) stages were significantly increased. Apoptosis at early and late was obviously inhibited in the presence of DHT. The effect of DHT was markedly blocked by PI3-K inhibitor LY294002.To elicit the mechanism of DHT protection, the expression of seladin-1, survivin, XIAP, bax, and bcl-xl protein was determined in C6 cells treated with Aβ25-35, DHT, or LY294002. Aβ25-35 significantly downregulated the expression of seladin-1, survivin, XIAP, bcl-xl protein and upregulated the expression of bax protein. DHT significantly inhibited the expression of bax, seladin-1, survivin, XIAP, and bcl-xl protein induced by Aβ25-35. Further, we found the effect of DHT was significantly inhibited by LY294002. Collectively, in a C6 glial cell model, we firstly found that DHT inhibits Aβ25-35-induced apoptosis by a rapid nongenic PI-3K/Akt activation as well as regulation of seladin-1, survivin, XIAP, bcl-xl, and bax proteins.

  11. Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores.

    Science.gov (United States)

    Salvador-Gallego, Raquel; Mund, Markus; Cosentino, Katia; Schneider, Jale; Unsay, Joseph; Schraermeyer, Ulrich; Engelhardt, Johann; Ries, Jonas; García-Sáez, Ana J

    2016-02-15

    Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane. © 2016 The Authors.

  12. BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax.

    Science.gov (United States)

    Wilfling, F; Weber, A; Potthoff, S; Vögtle, F-N; Meisinger, C; Paschen, S A; Häcker, G

    2012-08-01

    During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.

  13. BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation

    Science.gov (United States)

    Brustovetsky, Tatiana; Li, Tsyregma; Yang, Youyun; Zhang, Jiang-Ting; Antonsson, Bruno; Brustovetsky, Nickolay

    2010-01-01

    BAX cooperates with truncated BID (tBID) and Ca2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state. PMID:20655869

  14. Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

    Directory of Open Access Journals (Sweden)

    Yoon TH

    2012-03-01

    Full Text Available Ki-Chun Yoo1, Chang-Hwan Yoon1, Dongwook Kwon2, Kyung-Hwan Hyun1, Soo Jung Woo1, Rae-Kwon Kim1, Eun-Jung Lim1, Yongjoon Suh1, Min-Jung Kim1, Tae Hyun Yoon2, Su-Jae Lee11Laboratory of Molecular Biochemistry, 2Laboratory of Nanoscale Characterization and Environmental Chemistry, Department of Chemistry, Hanyang University, Seoul, Republic of KoreaBackground: Titanium dioxide (TiO2 has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined.Methods and results: In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70 together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation.Conclusion: These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein

  15. Methylglyoxal reduces mitochondrial potential and activates Bax and caspase-3 in neurons: Implications for Alzheimer's disease.

    Science.gov (United States)

    Tajes, Marta; Eraso-Pichot, Abel; Rubio-Moscardó, Fanny; Guivernau, Biuse; Bosch-Morató, Mònica; Valls-Comamala, Victòria; Muñoz, Francisco J

    2014-09-19

    Alzheimer's disease (AD) is characterized by the oxidative stress generated from amyloid β-peptide (Aβ) aggregates. It produces protein nitrotyrosination, after the reaction with nitric oxide to form peroxynitrite, being triosephosphate isomerase (TPI) one of the most affected proteins. TPI is a glycolytic enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Methylglyoxal (MG) is a by-product of TPI activity whose production is triggered when TPI is nitrotyrosinated. MG is harmful to cells because it glycates proteins. Here we found protein glycation when human neuroblastoma cells were treated with Aβ. Moreover glycation was also observed when neuroblastoma cells overexpressed mutated TPI where Tyr165 or Tyr209, the two tyrosines close to the catalytic center, were changed by Phe in order to mimic the effect of nitrotyrosination. The pathological relevance of these findings was studied by challenging cells with Aβ oligomers and MG. A significant decrease in mitochondrial transmembrane potential, one of the first apoptotic events, was obtained. Therefore, increasing concentrations of MG were assayed searching for MG effect in neuronal apoptosis. We found a decrease of the protective Bcl2 and an increase of the proapoptotic caspase-3 and Bax levels. Our results suggest that MG is triggering apoptosis in neurons and it would play a key role in AD neurodegeneration.

  16. Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance

    DEFF Research Database (Denmark)

    Erler, Janine Terra; Cawthorne, Christopher J; Williams, Kaye J;

    2004-01-01

    of the Bcl-2 protein family. Oxygen deprivation of human colon cancer cells in vitro provoked decreased mRNA and protein levels of proapoptotic Bid and Bad. Hypoxia-inducible factor 1 (HIF-1) was dispensable for the down-regulation of Bad but required for that of Bid, consistent with the binding of HIF-1......alpha to a hypoxia-responsive element (positions -8484 to -8475) in the bid promoter. Oxygen deprivation resulted in proteosome-independent decreased expression of Bax in vitro, consistent with a reduction in global translation efficiency. The physiological relevance of Bid and Bax down...

  17. The Impact of Adenosine Fast Induction of Myocardial Arrest during CABG on Myocardial Expression of Apoptosis-Regulating Genes Bax and Bcl-2

    Directory of Open Access Journals (Sweden)

    Ahmed Shalaby

    2009-01-01

    Full Text Available Background. We studied the effect of fast induction of cardiac arrest with denosine on myocardial bax and bcl-2 expression. Methods and Results. 40 elective CABG patients were allocated into two groups. The adenosine group (n=20 received 250 μg/kg adenosine into the aortic root followed by blood potassium cardioplegia. The control group received potassium cardioplegia in blood. Bcl-2 and bax were measured. Bax was reduced in the postoperative biopsies (1.38 versus 0.47, P=.002 in the control group. Bcl-2 showed a reducing tendency (0.14 versus 0.085, P=.07. After the adenosine treatment, the expression of both bax (0.52 versus 0.59, P=.4 and bcl-2 (0.104 versus 0.107, P=.4 remained unaltered after the operation. Conclusion. Open heart surgery is associated with rapid reduction in the expression of apoptosis regulating genes bax and bcl-2. Fast Adenosine induction abolished changes in their expression.

  18. Benzo(a)pyrene-7,8-diol-9,10-epoxide induced p53-independent necrosis via the mitochondria-associated pathway involving Bax and Bak activation.

    Science.gov (United States)

    Zhang, W; Liu, N; Wang, X; Jin, X; Du, H; Peng, G; Xue, J

    2015-02-01

    Benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is a highly reactive DNA damage agent and can induce cell death through both p53-independent and -dependent pathways. However, little is known about the molecular mechanisms of p53-independent pathways in BPDE-induced cell death. To understand the p53-independent mechanisms, we have now examined BPDE-induced cytotoxicity in p53-deficient baby mouse kidney (BMK) cells. The results showed that BPDE could induce Bax and Bak activation, cytochrome c release, caspases activation, and necrotic cell death in the BMK cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore, were interdependently activated by BPDE, with Bax and Bak translocation to and Bax/Bak homo-oligomerization in mitochondria, release of cytochrome c was induced. Importantly, cytochrome c release and necrotic cell death were diminished in BMK cells (Bax(-/-)), BMK cells (Bak(-/-)), and BMK cells (Bax(-/-)/Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate BPDE-induced cytochrome c release and necrosis. Together the findings suggested that BPDE-induced necrosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases. © The Author(s) 2015.

  19. Hypoxia-Mediated Down-Regulation of Bid and Bax in Tumors Occurs via Hypoxia-Inducible Factor 1-Dependent and -Independent Mechanisms and Contributes to Drug Resistance

    Science.gov (United States)

    Erler, Janine T.; Cawthorne, Christopher J.; Williams, Kaye J.; Koritzinsky, Marianne; Wouters, Bradley G.; Wilson, Clare; Miller, Crispin; Demonacos, Costas; Stratford, Ian J.; Dive, Caroline

    2004-01-01

    Solid tumors with disorganized, insufficient blood supply contain hypoxic cells that are resistant to radiotherapy and chemotherapy. Drug resistance, an obstacle to curative treatment of solid tumors, can occur via suppression of apoptosis, a process controlled by pro- and antiapoptotic members of the Bcl-2 protein family. Oxygen deprivation of human colon cancer cells in vitro provoked decreased mRNA and protein levels of proapoptotic Bid and Bad. Hypoxia-inducible factor 1 (HIF-1) was dispensable for the down-regulation of Bad but required for that of Bid, consistent with the binding of HIF-1α to a hypoxia-responsive element (positions −8484 to −8475) in the bid promoter. Oxygen deprivation resulted in proteosome-independent decreased expression of Bax in vitro, consistent with a reduction in global translation efficiency. The physiological relevance of Bid and Bax down-regulation was confirmed in tumors in vivo. Oxygen deprivation resulted in decreased drug-induced apoptosis and clonogenic resistance to agents with different mechanisms of action. The contribution of Bid and/or Bax down-regulation to drug responsiveness was demonstrated by the relative resistance of normoxic cells that had no or reduced expression of Bid and/or Bax and by the finding that forced expression of Bid in hypoxic cells resulted in increased sensitivity to the topoisomerase II inhibitor etoposide. PMID:15024076

  20. Effect of bax, bcl-2 and bcl-xL on regulating apoptosis in tissues of normal liver and hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Xiao-Zhong Guo; Xiao-Dong Shao; Min-Pei Liu; Jian-Hua Xu; Li-Nan Ren; Jia-Jun Zhao; Hong-Yu Li; Di Wang

    2002-01-01

    AIM: To investigate the expression of bax, bcl-2 and bcl-xL mRNA in the tissues of normal liver and hepatocellular carcinoma (HCC), and analyze the relationship between the expression of bax, bcl-2 and bcl-xL mRNA and clinical parameters of HCC patients.METHODS: The expression of bax, bcl-2 and bcl-xL mRNA of normal liver and HCC was measured by Northern blot. Statistical analyses were made by t test and correlation analysis.RESULTS: A very low mRNA level was indicated at bax,bcl-2 and bcl-xL in the HCC tissues in contrast to the tissues of normal liver by Northern blot analysis. The analyses of mRNA level revealed that HCC tissues exhibited a mean 7.6-fold decrease in bax, 4.2-fold in bcl-2 and 3.5-fold in bcl-xL in comparison with normal control tissues, respectively. Positive correlation was found between bax and bcl-xL (r=0.7061,P<0.01). There was no significance between the mRNA expression of these three genes and age, gender, tumor differentiation and tumor stage of HCC patients. CONCLUSION: The results are consistent with the fact that apoptosis rarely occurs in normal livers but increases in HCC, indicating that bcl-2 and bcl-xL may play a very important role in regulating the apoptosis of normal liver and HCC.

  1. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice

    Science.gov (United States)

    Karch, Jason; Kwong, Jennifer Q; Burr, Adam R; Sargent, Michelle A; Elrod, John W; Peixoto, Pablo M; Martinez-Caballero, Sonia; Osinska, Hanna; Cheng, Emily H-Y; Robbins, Jeffrey; Kinnally, Kathleen W; Molkentin, Jeffery D

    2013-01-01

    A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI: http://dx.doi.org/10.7554/eLife.00772.001 PMID:23991283

  2. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice.

    Science.gov (United States)

    Karch, Jason; Kwong, Jennifer Q; Burr, Adam R; Sargent, Michelle A; Elrod, John W; Peixoto, Pablo M; Martinez-Caballero, Sonia; Osinska, Hanna; Cheng, Emily H-Y; Robbins, Jeffrey; Kinnally, Kathleen W; Molkentin, Jeffery D

    2013-08-27

    A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI:http://dx.doi.org/10.7554/eLife.00772.001.

  3. Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL.

    Science.gov (United States)

    Garenne, David; Renault, Thibaud T; Manon, Stéphen

    2016-12-05

    The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.

  4. Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

    Science.gov (United States)

    Faião-Flores, Fernanda; Coelho, Paulo Rogério Pinto; Toledo Arruda-Neto, João Dias; Maria-Engler, Silvya Stuchi; Tiago, Manoela; Capelozzi, Vera Luiza; Giorgi, Ricardo Rodrigues; Maria, Durvanei Augusto

    2013-01-01

    Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and 7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT. PMID:23527236

  5. Apoptosis through Bcl-2/Bax and cleaved caspase up-regulation in melanoma treated by boron neutron capture therapy.

    Directory of Open Access Journals (Sweden)

    Fernanda Faião-Flores

    Full Text Available Boron neutron capture therapy (BNCT is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET particles, alpha and (7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.

  6. Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

    Science.gov (United States)

    Chura-Chambi, Rosa Maria; Arcuri, Helen Andrade; Lino, Felipe; Versati, Natan; Palma, Mario Sergio; Favaro, Denize C; Morganti, Ligia

    2017-05-01

    Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. (15) N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  7. Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38 MAPK, NF-κB/IκBα, and Bcl-2/Bax signaling

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    Wang YY

    2015-12-01

    Full Text Available Yuanyuan Wang, Rong Wang, Yujie Wang, Ruqin Peng, Yan Wu, Yongfang Yuan Department of Pharmacy, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China Background: Liver fibrosis is the consequence of diverse liver injuries and can eventually develop into liver cirrhosis. Ginkgo biloba extract (GBE is an extract from dried ginkgo leaves that has many pharmacological effects because of its various ingredients and has been shown to be hepatoprotective. Purpose and methods: Aimed to investigate the underlying protective mechanisms of GBE on carbon tetrachloride (CCl4-induced liver fibrosis in rats. Male Sprague Dawley rats were randomly divided into four groups: control group (C, model group (M, low-dose group (L, and high-dose group (H. Liver fibrosis was induced by CCl4 groups M, L, and H: group C was administered saline. In addition, GBE at different doses was used to treat groups L and H. Results: The results of hematoxylin and eosin staining, Masson’s trichrome staining, a liver function index, and a liver fibrosis index showed that GBE application noticeably mitigated fibrosis and improved the function of the liver. The western blotting and immunohistochemistry analyses indicated that GBE reduced liver fibrosis not only by inhibiting p38 MAPK and NF-κBp65 via inhibition of IκBα degradation but also by inhibiting hepatocyte apoptosis via downregulation of Bax, upregulation of Bcl-2, and subsequent inhibition of caspase-3 activation. Inflammation-associated factors and hepatic stellate cell (HSC-activation markers further demonstrated that GBE could effectively inhibit HSC activation and inflammation as a result of its regulation of p38 MAPK and nuclear factor-kappa B/IκBα signaling. Conclusion: Our findings indicated a novel role for GBE in the treatment of liver fibrosis. The potential mechanisms may be associated with the following signaling pathways: 1 the p38 MAPK

  8. Differential retrotranslocation of mitochondrial Bax and Bak

    Science.gov (United States)

    Todt, Franziska; Cakir, Zeynep; Reichenbach, Frank; Emschermann, Frederic; Lauterwasser, Joachim; Kaiser, Andrea; Ichim, Gabriel; Tait, Stephen WG; Frank, Stephan; Langer, Harald F; Edlich, Frank

    2015-01-01

    The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death. PMID:25378477

  9. Curcuma purpurascens BI. rhizome accelerates rat excisional wound healing: involvement of Hsp70/Bax proteins, antioxidant defense, and angiogenesis activity

    Science.gov (United States)

    Rouhollahi, Elham; Moghadamtousi, Soheil Zorofchian; Hajiaghaalipour, Fatemeh; Zahedifard, Maryam; Tayeby, Faezeh; Awang, Khalijah; Abdulla, Mahmood Ameen; Mohamed, Zahurin

    2015-01-01

    Purpose Curcuma purpurascens BI. is a member of Zingiberaceae family. The purpose of this study is to investigate the wound healing properties of hexane extract of C. purpurascens rhizome (HECP) against excisional wound healing in rats. Materials and methods Twenty four rats were randomly divided into 4 groups: A) negative control (blank placebo, acacia gum), B) low dose of HECP, C) high dose of HECP, and D) positive control, with 6 rats in each group. Full-thickness incisions (approximately 2.00 cm) were made on the neck area of each rat. Groups 1–4 were treated two-times a day for 20 days with blank placebo, HECP (100 mg/kg), HECP (200 mg/kg), and intrasite gel as a positive control, respectively. After 20 days, hematoxylin and eosin and Masson’s trichrome stainings were employed to investigate the histopathological alterations. Protein expressions of Bax and Hsp70 were examined in the wound tissues using immunohistochemistry analysis. In addition, levels of enzymatic antioxidants and malondialdehyde representing lipid peroxidation were measured in wound tissue homogenates. Results Macroscopic evaluation of wounds showed conspicuous elevation in wound contraction after topical administration of HECP at both doses. Moreover, histopathological analysis revealed noteworthy reduction in the scar width correlated with the enhanced collagen content and fibroblast cells, accompanied by a reduction of inflammatory cells in the granulation tissues. At the molecular level, HECP facilitates wound-healing process by downregulating Bax and upregulating Hsp70 protein at the wound site. The formation of new blood vessel was observed in Masson’s trichrome staining of wounds treated with HECP (100 and 200 mg/kg). In addition, HECP administration caused a significant surge in enzymatic antioxidant activities and a decline in lipid peroxidation. Conclusion These findings suggested that HECP accelerated wound-healing process in rats via antioxidant activity, angiogenesis

  10. Formononetin Induces Apoptosis of Human Osteosarcoma Cell Line U2OS by Regulating the Expression of Bcl-2, Bax and MiR-375 In Vitro and In Vivo

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    Wei Hu

    2015-09-01

    Full Text Available Background/Aims: Phytoestrogens are known to prevent tumor progression by inhibiting proliferation and inducing apoptosis in cancer cells. Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study investigates formononetin induction of apoptosis of human osteosarcoma cell line U2OS by regulating Bcl-2 and Bax expression in vitro and in vivo. Methods: U2OS cells were treated with different concentrations of formononetin and the proliferation of the cells was measured using an MTT assay. Cell apoptosis was examined by flow cytometry. The levels of miR-375, Bax and Bcl-2 protein expression in treated cells were determined by Western blot and RT-PCR. The antitumor activity of formononetin was also evaluated in vivo in nude mice bearing orthotopic tumor implants. Results: High concentrations of formononetin significantly suppress the proliferation of U2OS cells and induce cell apoptosis. Moreover, compared to control group the expression of Bcl-2 and miR-375 decreases with formononetin in the U2OS cells, while Bax increases. Conclusion: Formononetin has inhibitory effects on the proliferation of U2SO cells, both in vitro and in vivo. This antitumor effect is directly correlated with formononetin concentration.

  11. Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner.

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    Guo, W; Zhang, Y; Ling, Z; Liu, X; Zhao, X; Yuan, Z; Nie, C; Wei, Y

    2015-10-15

    Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.

  12. Parkin promotes proteasomal degradation of misregulated BAX.

    Science.gov (United States)

    Cakir, Zeynep; Funk, Kathrin; Lauterwasser, Joachim; Todt, Franziska; Zerbes, Ralf M; Oelgeklaus, Aline; Tanaka, Atsushi; van der Laan, Martin; Edlich, Frank

    2017-09-01

    The pro-apoptotic BCL-2 protein BAX commits human cells to apoptosis by permeabilizing the outer mitochondrial membrane. BAX activation has been suggested to require the separation of helix α5 from α6 - the 'latch' from the 'core' domain - among other conformational changes. Here, we show that conformational changes in this region impair BAX translocation to the mitochondria and retrotranslocation back into the cytosol, and therefore BAX inhibition, but not activation. Redirecting misregulated BAX to the mitochondria revealed an alternative mechanism of BAX inhibition. The E3 ligase parkin, which is known to trigger mitochondria-specific autophagy, ubiquitylates BAX K128 and targets the pro-apoptotic BCL-2 protein for proteasomal degradation. Retrotranslocation-deficient BAX is completely degraded in a parkin-dependent manner. Although only a minor pool of endogenous BAX escapes retrotranslocation into the cytosol, parkin-dependent targeting of misregulated BAX on the mitochondria provides substantial protection against BAX apoptotic activity. © 2017. Published by The Company of Biologists Ltd.

  13. MiR-467a is upregulated in radiation-induced mouse thymic lymphomas and regulates apoptosis by targeting Fas and Bax.

    Science.gov (United States)

    Gao, Fu; Chen, Song; Sun, Mingjuan; Mitchel, Ronald E J; Li, Bailong; Chu, Zhiyong; Cai, Jianming; Liu, Cong

    2015-01-01

    It has been reported dysregulation of certain microRNAs (miRNAs / miRs) is involved in tumorigenesis. However, the miRNAs associated with radiocarcinogenesis remain undefined. In this study, we validated the upregulation of miR-467a in radiation-induced mouse thymic lymphoma tissues. Then, we investigated whether miR-467a functions as an oncogenic miRNA in thymic lymphoma cells. For this purpose, we assessed the biological effect of miR-467a on thymic lymphoma cells. Using miRNA microarray, we found four miRNAs (miR-467a, miR-762, miR-455 and miR-714) were among the most upregulated (>4-fold) miRNAs in tumor tissues. Bioinformatics prediction suggests miR-467a may potentially regulate apoptosis pathway via targeting Fas and Bax. Consistently, in miR-467a-transfected cells, both proliferation and colony formation ability were significantly increased with decrease of apoptosis rate, while, in miR-467a-knockdown cells, proliferation was suppressed with increase of apoptosis rate, indicating that miR-467a may be involved in the regulation of apoptosis. Furthermore, miR-467a-knockdown resulted in smaller tumors and better prognosis in an in vivo tumor-transplanted model. To explain the mechanism of apoptosis suppression by miR-467a, we explore the expression of candidate target genes (Fas and Bax) in miR-467a-transfected relative to negative control transfected cells using flow cytometry and immunoblotting. Fas and Bax were commonly downregulated in miR-467a-transfected EL4 and NIH3T3 cells, and all of the genes harbored miR-467a target sequences in the 3'UTR of their mRNA. Fas and Bax were actually downregulated in radiation-induced thymic lymphoma tissues, and therefore both were identified as possible targets of miR-467a in thymic lymphoma. To ascertain whether downregulation of Fas and / or Bax is involved in apoptosis suppression by miR-467a, we transfected vectors expressing Fas and Bax into miR-467a-upregulated EL4 cells. Then we found that both Fas- and Bax

  14. Anticancer Activities and Effects of Bufalin on Apoptosis-regulate Gene Bcl-xL, Bax in Orthotopic Transplantation Model of Human Colorectal Cancer in Nude Mice%蟾毒灵对裸鼠大肠癌原位移植瘤的抗肿瘤作用及其对凋亡相关基因Bcl-xL、Bax表达的影响

    Institute of Scientific and Technical Information of China (English)

    王杰; 奉典旭; 陈超; 倪振华; 左青松; 陈亚峰; 王旭; 张勇; 陈腾

    2011-01-01

    Objective To investigate the anticancer activity and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation model of human colorectal cancer in nude mice. Methods HCT-116 cells of human colorectal cancer were implanted into the colon to establish orthotopic transplantation tumor models of human colorectal cancer in nude mice. Sixty mice were randomly divided into five groups: NS group,5-Fu group,Low dose bufalin group(BL), Medium dose bufalin group(BM) and High dose bufalin group(BH), with 12 mice in each group. Each group was injected intraperitoneally for 7days,respectively. Six mice in each group were killed at d24 and survival time in each group was calculated. The volume of the tumor were measured and the tumor inhibiting rate were calculated. The apoptotic rate measured by TUNEL staining method, and the expression of apoptosis regulated genes Bcl-xl and Bax were detected by real-time fluorogenic quantitative polymerase chain reaction (RFQ-PCR) and Western blot in tumor tissues. Result The tumor volume of 5-Fu group and each Bufalin group were reduced significantly compared with NS group (P<0. 01),and the tumor inhibiting rate were 69. 6%,45. 6%,56. 2% and 58. 5% for group of 5-Fu,BL,BM and BH respectively. The survival time were prolonged in group BL and BM (P<0. 05). The apoptotic rate in each group of bufalin were increased significantly compared with NS group(P<0. 05). The result of RFQ-PCR and Western blot staining showed that the expression of BcI-xl was decreased both in mRNA and protein level, while the expression of Bax was increased. Conclusion Bufalin has significant anticancer activity in the orthotopic transnplantation model of human colorectal cancer in nude mice and it can induce apoptosis of transplanted tumor cells. The mechanism may be associated with the down-regulation of Bcl-xt, and up-regulation of Bax in tumor cells.%目的 探讨蟾毒灵对裸鼠大肠癌原位移植瘤的抗肿瘤作用及其可能

  15. The correlation between telomerase activity and Bax/Bcl-2 ratio in valproic acid-treated MCF-7 breast cancer cell line

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    Zahra Vafaiyan

    2015-07-01

    Conclusion: Our study demonstrated that cell viability of MCF-7 cells was decreased after treatment with VPA, probably through a reduction of telomerase activity and an increase in Bax/bcl-2 ratio. Therefore, it could be concluded that VPA is a potent anti-cancer agent for breast cancer cells through inhibition of telomerase activity and induction of apoptosis.

  16. Study of Ganoderma lucidum spores on pentylenetetrazol activation of hippocampal neurons bax expression%灵芝孢子粉对戊四氮活化海马神经细胞bax表达的研究

    Institute of Scientific and Technical Information of China (English)

    张金波; 王淑秋; 张淑红; 金岳雷; 朱金玲; 刘爽

    2012-01-01

    (P <0. 01 ) , the difference was statistically significant. Conclusion; The results of this study confirmed that model group and the treatment group of bax expression were significantly higher than the normal control group after PTZ kindled, it show that bax genes play a catalytic role in the regulation of apoptosis process. Bax expression were significantly lower than model group after given Ganoderma lucidum spores treatment. It point active ingredients of Ganoderma spore powder can fully act on the brain tissue and regulate the expression of bax in order to play the role of anti - apoptotic neuroprotection.

  17. Cancer-selective death of human breast cancer cells by leelamine is mediated by bax and bak activation.

    Science.gov (United States)

    Sehrawat, Anuradha; Kim, Su-Hyeong; Hahm, Eun-Ryeong; Arlotti, Julie A; Eiseman, Julie; Shiva, Sruti S; Rigatti, Lora H; Singh, Shivendra V

    2017-02-01

    The present study is the first to report inhibition of breast cancer cell growth in vitro and in vivo and suppression of self-renewal of breast cancer stem cells (bCSC) by a pine bark component (leelamine). Except for a few recent publications in melanoma, anticancer pharmacology of this interesting phytochemical is largely elusive. Leelamine (LLM) dose-dependently inhibited viability of MDA-MB-231 (triple-negative), MCF-7 (estrogen receptor-positive), and SUM159 (triple-negative) human breast cancer cells in association with apoptotic cell death induction. To the contrary, a normal mammary epithelial cell line derived from fibrocystic breast disease and spontaneously immortalized (MCF-10A) was fully resistant to LLM-mediated cell growth inhibition and apoptosis induction. LLM also inhibited self-renewal of breast cancer stem cells. Apoptosis induction by LLM in breast cancer cells was accompanied by a modest increase in reactive oxygen species production, which was not due to inhibition of mitochondrial electron transport chain complexes. Nevertheless, ectopic expression of manganese superoxide dismutase conferred partial protection against LLM-induced cell death but only at a lower yet pharmacologically relevant concentration. Exposure of breast cancer cells to LLM resulted in (a) induction and/or activation of multidomain proapoptotic proteins Bax and Bak, (b) caspase-9 activation, and (c) cytosolic release of cytochrome c. Bax and Bak deficiency in immortalized fibroblasts conferred significant protection against cell death by LLM. Intraperitoneal administration of LLM (7.5 mg/kg; 5 times/wk) suppressed the growth of orthotopic SUM159 xenografts in mice without any toxicity. In conclusion, the present study provides critical preclinical data to warrant further investigation of LLM. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia

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    Bitar Fadi F

    2006-07-01

    Full Text Available Abstract Background The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1β and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2. Methods Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1β expression. Results TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1β expression was significantly increased at 14 days. Conclusion Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.

  19. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl

  20. Anti-apoptotic mechanism of Bacoside rich extract against reactive nitrogen species induced activation of iNOS/Bax/caspase 3 mediated apoptosis in L132 cell line.

    Science.gov (United States)

    Anand, T; Pandareesh, M D; Bhat, Pratiksha V; Venkataramana, M

    2014-10-01

    Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

  1. Imbalanced expression of Bcl-xL and Bax in platelets treated with plasma from immune thrombocytopenia.

    Science.gov (United States)

    Qiao, Jianlin; Liu, Yun; Li, Depeng; Wu, Yulu; Li, Xiaoqian; Yao, Yao; Niu, Mingshan; Fu, Chunling; Li, Hongchun; Ma, Ping; Li, Zhenyu; Xu, Kailin; Zeng, Lingyu

    2016-04-01

    Immune thrombocytopenia is a heterogeneous autoimmune disease, characterized by accelerated platelet destruction and impaired platelet production. Bcl-xL and Bax play an opposite role in the regulation of apoptotic process with Bcl-xL for cell survival and Bax for cell apoptosis. Given the critical roles in the regulation of platelet apoptosis, whether Bcl-xL or Bax was involved in the pathogenesis of ITP remains unknown. The aim of this study is to evaluate the expression profile of Bcl-xL and Bax in platelets treated with ITP plasma. Normal washed platelets were treated with plasma from 20 active ITP patients or 10 age and gender-matched control to mimic the ITP in vivo environment. Mitochondrial depolarization, platelet apoptosis and activation were measured by flow cytometry. Expression of Bcl-xL, Bax and caspase-3 were also measured by quantitative real-time PCR and western blot. Our results demonstrated increased mitochondrial depolarization, platelet apoptosis and activation in platelets after treated with ITP plasma in comparison to control. In addition, decreased expression of Bcl-xL, increased expression of Bax and activity of caspase-3 were also observed. Furthermore, a negative correlation of Bcl-xL with Bax was found in platelets treated with ITP plasma. In conclusion, imbalanced expression of Bcl-xL and Bax might be associated with platelet apoptosis in ITP and therapeutically targeting them might be a novel approach in the treatment of ITP.

  2. Anti-tumour activity of a novel coumarin-chalcone hybrid is mediated through intrinsic apoptotic pathway by inducing PUMA and altering Bax/Bcl-2 ratio.

    Science.gov (United States)

    Singh, Neetu; Sarkar, Jayanta; Sashidhara, Koneni V; Ali, Shakir; Sinha, Sudhir

    2014-06-01

    Coumarins and chalcones are secondary plant metabolites which have shown an array of pharmacological properties including anti-tumour activity. We have previously reported on the synthesis and anti-proliferative activity of a series of novel coumarin-chalcone hybrids. Now we report on the in vivo efficacy as well as mechanism of action of the most potent molecule of the series, S009-131. Oral administration of this molecule resulted in regression of tumours induced by HeLa cell xenografts in nod SCID mice. The molecule inhibited proliferation of cervical cancer cells (HeLa and C33A) by inducing apoptosis and arresting cell cycle at G2/M phase. Apoptosis was induced through induction of caspase-dependent intrinsic pathway and alterations in the cellular levels of Bcl-2 family proteins. The mitochondrial transmembrane potential got highly depleted in S009-131 treated cells due to an increase in Bax/Bcl-2 ratio and intracellular ROS. The molecule induced release of cytochrome c into the cytosol and activation of initiator caspase-9 and executioner caspases-3/7. Tumour suppressor protein p53 and its transcriptional target PUMA were up regulated, suggesting their role in mediating the cell death. These results suggest that S009-131 is a potent candidate for the chemotherapy of cervical carcinoma.

  3. Bone Marrow Stromal Cells Promote Neuronal Restoration in Rats with Traumatic Brain Injury: Involvement of GDNF Regulating BAD and BAX Signaling

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    Qin Shen

    2016-02-01

    Full Text Available Background/Aims: To investigate the effects of bone marrow stromal cells (BMSCs and underlying mechanisms in traumatic brain injury (TBI. Methods: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43 and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. Results: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF. The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. Conclusion: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

  4. Bcl-xL retrotranslocates Bax from the mitochondria into the cytosol

    Science.gov (United States)

    Edlich, Frank; Banerjee, Soojay; Suzuki, Motoshi; Cleland, Megan M.; Arnoult, Damien; Wang, Chunxin; Neutzner, Albert; Tjandra, Nico; Youle, Richard J.

    2011-01-01

    Summary The Bcl-2 family member Bax translocates from the cytosol to mitochondria where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by pro-survival Bcl-2 proteins, but how they inhibit Bax remains controversial, because they neither co-localize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-xL in detergents and cell free MOMP activity, but unexpectedly induce Bax accumulation on mitochondria. Fluorescence Loss in Photobleaching (FLIP) reveals constant retrotranslocation of wt Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on pro-survival Bcl-2 family proteins and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-xL inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax. PMID:21458670

  5. Bax, Bcl2, and p53 differentially regulate neomycin- and gentamicin-induced hair cell death in the zebrafish lateral line.

    Science.gov (United States)

    Coffin, Allison B; Rubel, Edwin W; Raible, David W

    2013-10-01

    Sensorineural hearing loss is a normal consequence of aging and results from a variety of extrinsic challenges such as excessive noise exposure and certain therapeutic drugs, including the aminoglycoside antibiotics. The proximal cause of hearing loss is often death of inner ear hair cells. The signaling pathways necessary for hair cell death are not fully understood and may be specific for each type of insult. In the lateral line, the closely related aminoglycoside antibiotics neomycin and gentamicin appear to kill hair cells by activating a partially overlapping suite of cell death pathways. The lateral line is a system of hair cell-containing sense organs found on the head and body of aquatic vertebrates. In the present study, we use a combination of pharmacologic and genetic manipulations to assess the contributions of p53, Bax, and Bcl2 in the death of zebrafish lateral line hair cells. Bax inhibition significantly protects hair cells from neomycin but not from gentamicin toxicity. Conversely, transgenic overexpression of Bcl2 attenuates hair cell death due to gentamicin but not neomycin, suggesting a complex interplay of pro-death and pro-survival proteins in drug-treated hair cells. p53 inhibition protects hair cells from damage due to either aminoglycoside, with more robust protection seen against gentamicin. Further experiments evaluating p53 suggest that inhibition of mitochondrial-specific p53 activity confers significant hair cell protection from either aminoglycoside. These results suggest a role for mitochondrial p53 activity in promoting hair cell death due to aminoglycosides, likely upstream of Bax and Bcl2.

  6. Identification of Bax-Interacting Proteins in Oligodendrocyte Progenitors during Glutamate Excitotoxicity and Perinatal Hypoxia–Ischemia

    Directory of Open Access Journals (Sweden)

    Sopio Simonishvili

    2013-11-01

    Full Text Available OPC (oligodendrocyte progenitor cell death contributes significantly to the pathology and functional deficits following hypoxic-ischemic injury in the immature brain and to deficits resulting from demyelinating diseases, trauma and degenerative disorders in the adult CNS. Glutamate toxicity is a major cause of oligodendroglial death in diverse CNS disorders, and previous studies have demonstrated that AMPA/kainate receptors require the pro-apoptotic protein Bax in OPCs undergoing apoptosis. The goal of the present study was to define the pro-apoptotic and anti-apoptotic effectors that regulate Bax in healthy OPCs and after exposure to excess glutamate in vitro and following H–I (hypoxia–ischemia in the immature rat brain. We show that Bax associates with a truncated form of Bid, a BH3-only domain protein, subsequent to glutamate treatment. Furthermore, glutamate exposure reduces Bax association with the anti-apoptotic Bcl family member, Bcl-xL. Cell fractionation studies demonstrated that both Bax and Bid translocate from the cytoplasm to mitochondria during the early stages of cell death consistent with a role for Bid as an activator, whereas Bcl-xL, which normally complexes with both Bax and Bid, disassociates from these complexes when OPCs are exposed to excess glutamate. Bax remained unactivated in the presence of insulin-like growth factor-1, and the Bcl-xL complexes were protected. Our data similarly demonstrate loss of Bcl-xL–Bax association in white matter following H–I and implicate active Bad in Bax-mediated OPC death. To identify other Bax-binding partners, we used proteomics and identified cofilin as a Bax-associated protein in OPCs. Cofilin and Bax associated in healthy OPCs, whereas the Bax–cofilin association was disrupted during glutamate-induced OPC apoptosis.

  7. Identification of Bax-interacting proteins in oligodendrocyte progenitors during glutamate excitotoxicity and perinatal hypoxia–ischemia

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    Sopio Simonishvili

    2013-12-01

    Full Text Available OPC (oligodendrocyte progenitor cell death contributes significantly to the pathology and functional deficits following hypoxic-ischemic injury in the immature brain and to deficits resulting from demyelinating diseases, trauma and degenerative disorders in the adult CNS. Glutamate toxicity is a major cause of oligodendroglial death in diverse CNS disorders, and previous studies have demonstrated that AMPA/kainate receptors require the pro-apoptotic protein Bax in OPCs undergoing apoptosis. The goal of the present study was to define the pro-apoptotic and anti-apoptotic effectors that regulate Bax in healthy OPCs and after exposure to excess glutamate in vitro and following H–I (hypoxia–ischemia in the immature rat brain. We show that Bax associates with a truncated form of Bid, a BH3-only domain protein, subsequent to glutamate treatment. Furthermore, glutamate exposure reduces Bax association with the anti-apoptotic Bcl family member, Bcl-xL. Cell fractionation studies demonstrated that both Bax and Bid translocate from the cytoplasm to mitochondria during the early stages of cell death consistent with a role for Bid as an activator, whereas Bcl-xL, which normally complexes with both Bax and Bid, disassociates from these complexes when OPCs are exposed to excess glutamate. Bax remained unactivated in the presence of insulin-like growth factor-1, and the Bcl-xL complexes were protected. Our data similarly demonstrate loss of Bcl-xL–Bax association in white matter following H–I and implicate active Bad in Bax-mediated OPC death. To identify other Bax-binding partners, we used proteomics and identified cofilin as a Bax-associated protein in OPCs. Cofilin and Bax associated in healthy OPCs, whereas the Bax–cofilin association was disrupted during glutamate-induced OPC apoptosis.

  8. Lack of ligand-selective binding of the aryl hydrocarbon receptor to putative DNA binding sites regulating expression of Bax and paraoxonase 1 genes.

    Science.gov (United States)

    DeGroot, Danica E; Hayashi, Ai; Denison, Michael S

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals through its ability to bind specific DNA recognition sites (dioxin responsive elements (DREs)), and activate transcription of adjacent genes. While the DRE has a highly conserved consensus sequence, it has been suggested that the nucleotide specificity of AhR DNA binding may be ligand-dependent. The upstream regulatory regions of the murine Bax and human paraoxonase 1 (PON1) genes reportedly contain unique DRE-like sequences that respond to AhRs activated by some ligands but not others. Given the significant implications of this observation to understanding the diversity in AhR responses and that of other ligand-dependent nuclear receptors, a combination of DNA binding, nuclear translocation and gene expression analysis was used to investigate the molecular mechanisms underlying these ligand-selective responses. Although known AhR agonists stimulated AhR nuclear translocation, DRE binding and gene expression, the ligand-selective DRE-like DNA elements identified in the Bax and PON1 upstream regulatory regions failed to bind ligand-activated AhR or confer AhR-responsiveness upon a reporter gene. These results argue against the reported ligand-selectivity of AhR DNA binding and suggest DNA binding by ligand activated AhR involves DRE-containing DNA.

  9. USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

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    Aman Wang

    2017-05-01

    Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

  10. Transient activation of microglia following acute alcohol exposure in developing mouse neocortex is primarily driven by BAX-dependent neurodegeneration.

    Science.gov (United States)

    Ahlers, Katelin E; Karaçay, Bahri; Fuller, Leah; Bonthius, Daniel J; Dailey, Michael E

    2015-10-01

    Fetal alcohol exposure is the most common known cause of preventable mental retardation, yet we know little about how microglia respond to, or are affected by, alcohol in the developing brain in vivo. Using an acute (single day) model of moderate (3 g/kg) to severe (5 g/kg) alcohol exposure in postnatal day (P) 7 or P8 mice, we found that alcohol-induced neuroapoptosis in the neocortex is closely correlated in space and time with the appearance of activated microglia near dead cells. The timing and molecular pattern of microglial activation varied with the level of cell death. Although microglia rapidly mobilized to contact and engulf late-stage apoptotic neurons, apoptotic bodies temporarily accumulated in neocortex, suggesting that in severe cases of alcohol toxicity the neurodegeneration rate exceeds the clearance capacity of endogenous microglia. Nevertheless, most dead cells were cleared and microglia began to deactivate within 1-2 days of the initial insult. Coincident with microglial activation and deactivation, there was a transient increase in expression of pro-inflammatory factors, TNFα and IL-1β, after severe (5 g/kg) but not moderate (3 g/kg) EtOH levels. Alcohol-induced microglial activation and pro-inflammatory factor expression were largely abolished in BAX null mice lacking neuroapoptosis, indicating that microglial activation is primarily triggered by apoptosis rather than the alcohol. Therefore, acute alcohol exposure in the developing neocortex causes transient microglial activation and mobilization, promoting clearance of dead cells and tissue recovery. Moreover, cortical microglia show a remarkable capacity to rapidly deactivate following even severe neurodegenerative insults in the developing brain.

  11. Inhibition of Pro-Apoptotic BAX by a Noncanonical Interaction Mechanism

    Science.gov (United States)

    Barclay, Lauren A.; Wales, Thomas E.; Garner, Thomas P.; Wachter, Franziska; Lee, Susan; Guerra, Rachel M.; Stewart, Michelle L.; Braun, Craig R.; Bird, Gregory H.; Gavathiotis, Evripidis; Engen, John R.; Walensky, Loren D.

    2015-01-01

    SUMMARY BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1–α2 loop, and α2–α3 and α5–α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism. PMID:25684204

  12. Gastroprotective activity of Annona muricata leaves against ethanol-induced gastric injury in rats via Hsp70/Bax involvement.

    Science.gov (United States)

    Moghadamtousi, Soheil Zorofchian; Rouhollahi, Elham; Karimian, Hamed; Fadaeinasab, Mehran; Abdulla, Mahmood Ameen; Kadir, Habsah Abdul

    2014-01-01

    The popular fruit tree of Annona muricata L. (Annonaceae), known as soursop and graviola, is a widely distributed plant in Central and South America and tropical countries. Leaves of A. muricata have been reported to possess antioxidant and anti-inflammatory activities. In this study, the gastroprotective effects of ethyl acetate extract of A. muricata leaves (EEAM) were investigated against ethanol-induced gastric injury models in rats. The acute toxicity test of EEAM in rats, carried out in two doses of 1 g/kg and 2 g/kg, showed the safety of this plant, even at the highest dose of 2 g/kg. The antiulcer study in rats (five groups, n=6) was performed with two doses of EEAM (200 mg/kg and 400 mg/kg) and with omeprazole (20 mg/kg), as a standard antiulcer drug. Gross and histological features showed the antiulcerogenic characterizations of EEAM. There was significant suppression on the ulcer lesion index of rats pretreated with EEAM, which was comparable to the omeprazole effect in the omeprazole control group. Oral administration of EEAM to rats caused a significant increase in the level of nitric oxide and antioxidant activities, including catalase, glutathione, and superoxide dismutase associated with attenuation in gastric acidity, and compensatory effect on the loss of gastric wall mucus. In addition, pretreatment of rats with EEAM caused significant reduction in the level of malondialdehyde, as a marker for oxidative stress, associated with an increase in prostaglandin E2 activity. Immunohistochemical staining also demonstrated that EEAM induced the downregulation of Bax and upregulation of Hsp70 proteins after pretreatment. Collectively, the present results suggest that EEAM has a promising antiulcer potential, which could be attributed to its suppressive effect against oxidative damage and preservative effect toward gastric wall mucus.

  13. Asiaticoside: attenuation of neurotoxicity induced by MPTP in a rat model of Parkinsonism via maintaining redox balance and up-regulating the ratio of Bcl-2/Bax.

    Science.gov (United States)

    Xu, Chang-Liang; Wang, Qi-Zhi; Sun, Ling-Mei; Li, Xiu-Min; Deng, Ji-Min; Li, Lu-Fan; Zhang, Jin; Xu, Rong; Ma, Shi-Ping

    2012-01-01

    In this study, we investigated the neuroprotective effects of asiaticoside, a triterpenoid saponin isolated from the Chinese medicinal herb Centella asiatica, in the rats model of Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Rats were first injected with MPTP. One day after surgery, asiaticoside was administered and the behavioral tests were assessed. On 14th day, the rats were sacrificed, substantia nigra (SN) and striatum were dissected, and then dopamine (DA) and its metabolites in striatum and malonyldialdehyde (MDA) contents, reduced glutathione (GSH) level and gene expression level in SN were estimated. Treatment with asiaticoside was found to protect dopaminergic neuron by antagonizing MPTP induced neurotoxicity and to improve locomotor dysfunction. Asiaticoside significantly attenuated the MPTP-induced reduction of dopamine in the striatum. The content of MDA was significantly decreased while the GSH level was significantly increased in asiaticoside-treated groups. In addition, asiaticoside increased the Bcl-2/Bax ratio. These results indicated that asiaticoside was effective in reversing MPTP induced Parkinsonism via its neuroprotective effects including antioxidant activity, maintaining the metabolic balance of DA, and increasing ratio of Bcl-2/Bax.

  14. BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

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    Boohaker, Rebecca J.; Zhang, Ge; Carlson, Adina Loosley; Nemec, Kathleen N.

    2011-01-01

    The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration. PMID:21289292

  15. Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins.

    Science.gov (United States)

    Goodall, Katharine J; Finch-Edmondson, Megan L; van Vuuren, Joanne; Yeoh, George C; Gentle, Ian E; Vince, James E; Ekert, Paul G; Vaux, David L; Callus, Bernard A

    2016-01-01

    Apoptosis mediated by Bax or Bak is usually thought to be triggered by BH3-only members of the Bcl-2 protein family. BH3-only proteins can directly bind to and activate Bax or Bak, or indirectly activate them by binding to anti-apoptotic Bcl-2 family members, thereby relieving their inhibition of Bax and Bak. Here we describe a third way of activation of Bax/Bak dependent apoptosis that does not require triggering by multiple BH3-only proteins. In factor dependent myeloid (FDM) cell lines, cycloheximide induced apoptosis by a Bax/Bak dependent mechanism, because Bax-/-Bak-/- lines were profoundly resistant, whereas FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Addition of cycloheximide led to the rapid loss of Mcl-1 but did not affect the expression of other Bcl-2 family proteins. In support of these findings, similar results were observed by treating FDM cells with the CDK inhibitor, roscovitine. Roscovitine reduced Mcl-1 abundance and caused Bax/Bak dependent cell death, yet FDM lines lacking one or more genes for BH3-only proteins remained highly sensitive. Therefore Bax/Bak dependent apoptosis can be regulated by the abundance of anti-apoptotic Bcl-2 family members such as Mcl-1, independently of several known BH3-only proteins.

  16. Identification and expression analysis of alternatively spliced new transcript isoform of Bax gene in mouse.

    Science.gov (United States)

    Husain, Mohammed Amir; Ishqi, Hassan Mubarak; Sarwar, Tarique; Rehman, Sayeed Ur; Tabish, Mohammad

    2017-07-20

    Bax, a pro-apoptotic member of Bcl-2 family regulates apoptosis through homodimerization/heterodimerization with Bcl-2. Bax-α is the only product of the Bax gene that has been extensively studied. Bax-α exists in inactive form and several conformational changes are required during apoptosis to activate it. Here, we have identified a novel transcript variant of Bax gene in mouse which contains alternatively spliced new first exon that is different from the first exon of previously reported transcript. Conceptual translation of new transcript encodes a protein (Bax-α1), having different N-terminus. The existence of the new transcript variant was confirmed by reverse transcriptase-PCR, semi-nested PCR using primers designed for the newly identified transcript variant. The identity of PCR product obtained after semi-nested PCR was confirmed by DNA sequencing. Relative expression of new transcript variant with respect to reported transcript was also studied with the help of real time PCR. The existence of new transcript variant was further supported by the presence of clusters of overlapping ESTs from the database. Bax-α1 possibly displays heterogeneous properties as predicted by post-translational modification analysis tools. The differences in post-translational modifications might play important roles in divergent function of the new isoform. The three dimensional structure was generated by homology modelling to visualize the differences at N termini of known and newly identified variant. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Apopotic gene Bax expression in carotid plaque

    Institute of Scientific and Technical Information of China (English)

    Bao-Zhong MEN; Ding-Biao ZHOU; Huai-Yin SHI; Xiao-Ming ZHANG

    2006-01-01

    The expression of BAX in carotid atherosclerosis and its regulation is far from defined. Objectives To investigate BAX expression in stable/fibrous and instable/vulnerable carotid plaque and its clinical significance. Methods 25 cases of carotid plaque specimens obtained from endarterectomy were divided into two groups, stable/fibrous 14 cases, vulnerable/instable 11 cases; aortic artery and its branches from hepatic transplantation donors 6 case as control. The expression of proapoptotic BAX was detected by immunohistochemistry(IHC), in situ hybridization(ISH) and in situ TdT dUTP nick end labeling (TUNEL). Results 5 cases of BAX ( + ) were detected by ICH and ISH, 4 case of TUNEL ( + ) were detected by TUNEL in stable/fibrous carotid plaque , while 10 cases were BAX ( + )by IHC(P < 0.05) , 11case by ISH and 9 case by TUNEL were detected in instable/vulnerable carotid plaque ( P < 0.01 ), respectively. The intensity of BAX ( + ) cells by IHC and ISH was 8.63 ± 2.62 and 10.32 ± 3.12 in fibrous plaques, whereas 122 ± 21.64and 152 ± 23.35 in vulnerable plaques, respectively. No expression of BAX was found in controlled group. Conclusion The higher expression of Bax in vulnerable carotid plaque may be one mechanisms in molecular pathogenesis of carotid atherosclerosis which affect plaque stability and be the cause of higher incidence of stroke than fibrous carotid plaques, the regulation of BAX expression in different stage of atherosclerosis may provide targets in gene therapy for carotid atherosclerosis.

  18. Antrodia camphorata Potentiates Neuroprotection against Cerebral Ischemia in Rats via Downregulation of iNOS/HO-1/Bax and Activated Caspase-3 and Inhibition of Hydroxyl Radical Formation

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    Po-Sheng Yang

    2015-01-01

    Full Text Available Antrodia camphorata (A. camphorata is a fungus generally used in Chinese folk medicine for treatment of viral hepatitis and cancer. Our previous study found A. camphorata has neuroprotective properties and could reduce stroke injury in cerebral ischemia animal models. In this study, we sought to investigate the molecular mechanisms of neuroprotective effects of A. camphorata in middle cerebral artery occlusion (MCAO rats. A selective occlusion of the middle cerebral artery (MCA with whole blood clots was used to induce ischemic stroke in rats and they were orally treated with A. camphorata (0.25 and 0.75 g/kg/day alone or combined with aspirin (5 mg/kg/day. To provide insight into the functions of A. camphorata mediated neuroprotection, the expression of Bax, inducible nitric oxide synthase (iNOS, haem oxygenase-1 (HO-1, and activated caspase-3 was determined by Western blot assay. Treatment of aspirin alone significantly reduced the expressions of HO-1 (P<0.001, iNOS (P<0.001, and Bax (P<0.01 in ischemic regions. The reduction of these expressions was more potentiated when rats treated by aspirin combined with A. camphorata (0.75 g/kg/day. Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared to MCAO group (P<0.01. Moreover, treatment of A. camphorata significantly (P<0.05 reduced fenton reaction-induced hydroxyl radical (OH• formation at a dose of 40 mg/mL. Taken together, A. camphorata has shown neuroprotective effects in embolic rats, and the molecular mechanisms may correlate with the downregulation of Bax, iNOS, HO-1, and activated caspase-3 and the inhibition of OH• signals.

  19. Synergistic Effect of Subtoxic-dose Cisplatin and TRAIL to Mediate Apoptosis by Down-regulating Decoy Receptor 2 and Up-regulating Caspase-8, Caspase-9 and Bax Expression on NCI-H460 and A549 Cells

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    2013-05-01

    Full Text Available Objective(s: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL can selectively induce apoptosis in tumor cells, more than half of tumors including non-small cell lung cancer (NSCLC exhibit TRAIL-resistance. The purpose of this study was to determine whether subtoxic-dose cisplatin and TRAIL could synergistically enhance apoptosis on NSCLC cells and investigate its underlying mechanisms. Materials and Methods:NCI-H460 and A549 cells were treated with TRAIL alone, cisplatin alone or combination treatment in this study. The cytotoxicity was evaluated according to Sulforhodamine B assay, and apoptosis was examined using Hoechst 33342 staining and flow cytometry. The mRNA and protein levels of TRAIL receptors and apoptotic proteins including caspase-8, caspase-9, Bcl-2 and Bax were determined by RT-PCR and Western blotting, respectively. Results:Our results showed that NCI-H460 cells were sensitive to TRAIL, whereas A549 cells were resistant. However, subtoxic-dose cisplatin could enhance the both cells to TRAIL-mediated cell proliferation inhibition and apoptosis. The underlying mechanisms might be associated with the down-regulation of DcR2 and up-regulation of Caspase-8, Caspase-9 and Bax. Conclusion:Subtoxic-dose cisplatin could enhance both TRAIL- sensitive and TRAIL- resistant NSCLC cells to TRAIL-mediated apoptosis. These findings motivated further studies to evaluate such a combinatory therapeutic strategy against NSCLC in the animal models.

  20. Interaction of caveolin-1 with Ku70 inhibits Bax-mediated apoptosis.

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    Huafei Zou

    Full Text Available Caveolin-1, the structural protein component of caveolae, acts as a scaffolding protein that functionally regulates signaling molecules. We show that knockdown of caveolin-1 protein expression enhances chemotherapeutic drug-induced apoptosis and inhibits long-term survival of colon cancer cells. In vitro studies demonstrate that caveolin-1 is a novel Ku70-binding protein, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82-101 to the caveolin-binding domain (CBD of Ku70 (amino acids 471-478. Cell culture data show that caveolin-1 binds Ku70 after treatment with chemotherapeutic drugs. Mechanistically, we found that binding of caveolin-1 to Ku70 inhibits the chemotherapeutic drug-induced release of Bax from Ku70, activation of Bax, translocation of Bax to mitochondria and apoptosis. Potentiation of apoptosis by knockdown of caveolin-1 protein expression is greatly reduced in the absence of Bax expression. Finally, we found that overexpression of wild type Ku70, but not a mutant form of Ku70 that cannot bind to caveolin-1 (Ku70 Φ→A, limits the chemotherapeutic drug-induced Ku70/Bax dissociation and apoptosis. Thus, caveolin-1 acts as an anti-apoptotic protein in colon cancer cells by binding to Ku70 and inhibiting Bax-dependent cell death.

  1. Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2.

    Science.gov (United States)

    Zhang, Li-Min; Zhao, Xiao-Chun; Sun, Wen-Bo; Li, Rui; Jiang, Xiao-Jing

    2015-10-15

    Temporal post-conditioning helps provide neuroprotection against brain injury secondary to ischemia-reperfusion and is considered an effective intervention, but the exact mechanism of sevoflurane post-conditioning is unclear. The essential axis involves activator Bid, Bim, Puma (BH3s), Bax, and Bak; activates the mitochondrial death program; and might be involved in a cell death signal. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in cell growth and proliferation. We hypothesized that sevoflurane post-conditioning might inhibit Bid, Bim, Puma, Bax, and Bak expression and is activated by phosphor-Erk1/2 to decrease neuronal death. To test this hypothesis, we exposed primary cortical neuron cultures to oxygen-glucose deprivation for 1h, along with resuscitation for 24h (OGD/R). MTT assays, propidium iodide uptake (PI), JC-1 fluorescence, and Western blot indicated the following: decreased cell viability (PPuma, Bax, and Bak expression with OGD/R exposure. Inhibition of Erk1/2 phosphorylation could attenuate sevoflurane post-conditioning that mediated an increase in neuronal viability and mitochondrial membrane potential, as well as a decrease in cell death and Bid, Bim, Puma, Bax, and Bak expression after OGD/R treatment. The results demonstrated that sevoflurane post-conditioning caused a marked decrease in cortical neuronal death secondary to OGD/R exposure through the downregulation of the mitochondrial apoptosis axis involving Bid, Bim, Puma, Bax, and Bak that was mediated by the phosphorylation/activation of Erk1/2.

  2. Sulforaphene promotes Bax/Bcl2, MAPK-dependent human gastric cancer AGS cells apoptosis and inhibits migration via EGFR, p-ERK1/2 down-regulation.

    Science.gov (United States)

    Mondal, Arindam; Biswas, Raktim; Rhee, Yun-Hee; Kim, Jongkee; Ahn, Jin-Chul

    2016-01-01

    Gastric cancer migration and invasion considered as main causes of this cancer-related death around the world. Sulforaphene (4-isothiocyanato-4R-(methylsulfinyl)-1-butene), a structural analog of sulforaphane, has been found to exhibit anticancer potential against different cancers. Our aim was to investigate whether dietary isothiocyanate sulforaphene (SFE) can promote human gastric cancer (AGS) cells apoptosis and inhibit migration. Cells were treated with various concentrations of SFE and cell viability, morphology, intracellular ROS, migration and different signaling protein expressions were investigated. The results indicate that SFE decreases AGS cell viability and induces apoptosis in a dose-dependent manner. Intracellular ROS generation, dose- and time-dependent Bax/Bcl2 alteration and signaling proteins like cytochrome c, Casp-3, Casp-8 and PARP-1 higher expression demonstrated the SFE-induced apoptotic pathway in AGS cells. Again, SFE induced apoptosis also accompanied by the phosphorylation of mitogen-activated protein kinases (MAPKs) like JNK and P-38. Moreover, dose-dependent EGFR, p-ERK1/2 down-regulation and cell migration inhibition at non-toxic concentration confirms SFE activity in AGS cell migration inhibition. Thus, this study demonstrated effective chemotherapeutic potential of SFE by inducing apoptisis as well as inhibiting migration and their preliminary mechanism for human gastric cancer management.

  3. Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2 associated X protein) Bax in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge.

    Science.gov (United States)

    Luo, Sheng-Wei; Wang, Wei-Na; Sun, Zuo-Ming; Xie, Fu-Xing; Kong, Jing-Rong; Liu, Yuan; Cheng, Chang-Hong

    2016-07-01

    Bax is a pro-apoptotic member of Bcl-2 like superfamily, playing an important role in regulating the apoptosis. In this study, the full-length Bax (EcBax) was obtained, containing a 5'UTR of 64 bp, an ORF of 579 bp and a 3'UTR of 1021 bp. The EcBax gene encoded a polypeptide of 192 amino acids with an estimated molecular mass of 21.55 KDa and a predicted isoelectric point (pI) of 6.75. The deduced amino acid sequence analysis showed that EcBax comprised the conserved residues and the characteristic domains known to the critical function of Bax. qRT-PCR analysis revealed that EcBax mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in blood, followed by the expression in liver, gill, spleen, kidney, heart, muscle and intestine. A sharp increase of EcBax expression was observed in the vibrio challenge group by comparing with those in the control. Subcellular localization analysis revealed that EcBax was predominantly localized in the cytoplasm. EcBax exerted a regulatory role in modulating the mitochondrial membrane potential, promoting the cytochrome c release, and then activating the downstream caspase signaling. Moreover, the overexpression of EcBax can decrease the cell viability and antagonize NF-kB, AP-1, Stat3 promoter activity in Hela cells. These results indicate that EcBax containing the conserved domain of pro-apoptotic member of Bcl-2 family may disrupt the mammalian signaling and play a regulative role in the apoptotic process.

  4. EGR-1/Bax pathway plays a role in vitamin E δ-tocotrienol-induced apoptosis in pancreatic cancer cells.

    Science.gov (United States)

    Wang, Chen; Husain, Kazim; Zhang, Anying; Centeno, Barbara A; Chen, Dung-Tsa; Tong, Zhongsheng; Sebti, Säid M; Malafa, Mokenge P

    2015-08-01

    The anticancer activity of δ-tocotrienol, a bioactive vitamin E present in whole grain cereals, annatto beans and palm fruit, is strongly dependent on its effect on the induction of apoptosis. δ-Tocotrienol-induced apoptosis is associated with consistent induction in the expression of the proapoptotic protein Bcl-2-associated X protein (Bax). The molecular mechanism by which δ-tocotrienol regulates Bax expression is unknown. We carried out a DNA microarray study that identified δ-tocotrienol induction of the zinc finger transcription factor EGR-1 in pancreatic cancer cells. Here, we provide evidence linking δ-tocotrienol-induced apoptosis in pancreatic cancer cells to EGR-1 regulation of Bax expression. Forced expression of EGR-1 induces Bax expression and apoptosis in pancreatic cancer cells. In contrast, knockdown of δ-tocotrienol-induced EGR-1 by small interfering RNA attenuated δ-tocotrienol-induced Bax expression and reduced δ-tocotrienol-induced apoptosis. Further analyses showed that de novo protein synthesis was not required for δ-tocotrienol-induced EGR-1 expression, suggesting a direct effect of δ-tocotrienol on EGR-1 expression. Furthermore, a chromatin immunoprecipitation assay demonstrated that EGR-1 binds to the Bax gene promoter. Finally, δ-tocotrienol treatment induced Bax expression and activated EGR-1 in the pancreatic neoplastic cells of the PDX-Cre Kras genetically engineered model of pancreatic cancer. Our study provides the first evidence for EGR-1 as a direct target of vitamin E δ-tocotrienol, suggesting that EGR-1 may act as a proapoptotic factor in pancreatic cancer cells via induction of Bax. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Vucicevic, Ksenija; Jakovljevic, Vladimir; Colovic, Natasa; Tosic, Natasa; Kostic, Tatjana; Glumac, Irena; Pavlovic, Sonja; Karan-Djurasevic, Teodora; Colovic, Milica

    2016-04-01

    In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p=Bax ratio showed a negative correlation to lymphocyte doubling time (r=-0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.

  6. Pro-apoptotic Bax molecules densely populate the edges of membrane pores.

    Science.gov (United States)

    Kuwana, Tomomi; Olson, Norman H; Kiosses, William B; Peters, Bjoern; Newmeyer, Donald D

    2016-06-03

    How the pro-apoptotic Bax protein permeabilizes the mitochondrial outer membrane is not fully understood. Previously, using cryo-electron microscopy (cryo-EM), we showed that activated Bax forms large, growing pores. Whether formed in liposomes or in mitochondrial outer membranes, Bax-induced pores exhibit the same morphology, with negative curvature flanking the edges and with no visible protein structure protruding from the membranes. Here we used cryo-EM to show that gold-labeled Bax molecules, after activation by Bid, became localized strictly at pore edges. This argues that Bax acts at short range to deform the membrane. Also, Bax molecules populated the walls of both small and large pores at the same density, implying that Bax is continuously recruited to the pores as they widen. Moreover, because all Bax molecules became oligomerized after membrane insertion, we infer that Bax oligomers are present at pore edges. We suggest that oligomerization may promote pore enlargement.

  7. Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells

    DEFF Research Database (Denmark)

    Nielsen, M; Kaestel, C G; Eriksen, K W

    1999-01-01

    promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic...

  8. The porin VDAC2 is the mitochondrial platform for Bax retrotranslocation.

    Science.gov (United States)

    Lauterwasser, Joachim; Todt, Franziska; Zerbes, Ralf M; Nguyen, Thanh Ngoc; Craigen, William; Lazarou, Michael; van der Laan, Martin; Edlich, Frank

    2016-09-13

    The pro-apoptotic Bcl-2 protein Bax can permeabilize the outer mitochondrial membrane and therefore commit human cells to apoptosis. Bax is regulated by constant translocation to the mitochondria and retrotranslocation back into the cytosol. Bax retrotranslocation depends on pro-survival Bcl-2 proteins and stabilizes inactive Bax. Here we show that Bax retrotranslocation shuttles membrane-associated and membrane-integral Bax from isolated mitochondria. We further discover the mitochondrial porin voltage-dependent anion channel 2 (VDAC2) as essential component and platform for Bax retrotranslocation. VDAC2 ensures mitochondria-specific membrane association of Bax and in the absence of VDAC2 Bax localizes towards other cell compartments. Bax retrotranslocation is also regulated by nucleotides and calcium ions, suggesting a potential role of the transport of these ions through VDAC2 in Bax retrotranslocation. Together, our results reveal the unanticipated bifunctional role of VDAC2 to target Bax specifically to the mitochondria and ensure Bax inhibition by retrotranslocation into the cytosol.

  9. The C-terminal helix of Bcl-xL mediates Bax retrotranslocation from the mitochondria

    Science.gov (United States)

    Todt, F; Cakir, Z; Reichenbach, F; Youle, R J; Edlich, F

    2013-01-01

    The proapoptotic Bcl-2 protein Bax can commit a cell to apoptosis by translocation from the cytosol to the mitochondria and permeabilization of the outer mitochondrial membrane. Prosurvival Bcl-2 family members, such as Bcl-xL, control Bax activity. Bcl-xL recognizes Bax after a conformational change in the N-terminal segment of Bax on the mitochondria and retrotranslocates it back into the cytoplasm, stabilizing the inactive form of Bax. Here we show that Bax retrotranslocation depends on the C-terminal helix of Bcl-xL. Deletion or substitution of this segment reduces Bax retrotranslocation and correlates with the accumulation of GFP-tagged or endogenous Bax on the mitochondria of non-apoptotic cells. Unexpectedly, the substitution of the Bcl-xL membrane anchor by the corresponding Bax segment reverses the Bax retrotranslocation activity of Bcl-xL, but not that of Bcl-xL shuttling. Bax retrotranslocation depends on interaction to the Bcl-xL membrane anchor and interaction between the Bax BH3 domain and the Bcl-xL hydrophobic cleft. Interference with either interaction increases mitochondrial levels of endogenous Bax. In healthy cells, mitochondrial Bax does not permeabilize the outer mitochondrial membrane, but increases cell death after apoptosis induction. PMID:23079612

  10. Bax and Bak Pores: Are We Closing the Circle?

    Science.gov (United States)

    Cosentino, Katia; García-Sáez, Ana J

    2017-04-01

    Bax and its homolog Bak are key regulators of the mitochondrial pathway of apoptosis. On cell stress Bax and Bak accumulate at distinct foci on the mitochondrial surface where they undergo a conformational change, oligomerize, and mediate cytochrome c release, leading to cell death. The molecular mechanisms of Bax and Bak assembly and mitochondrial permeabilization have remained a longstanding question in the field. Recent structural and biophysical studies at several length scales have shed light on key aspects of Bax and Bak function that have shifted how we think this process occurs. These discoveries reveal an unexpected molecular mechanism in which Bax (and likely Bak) dimers assemble into oligomers with an even number of molecules that fully or partially delineate pores of different sizes to permeabilize the mitochondrial outer membrane (MOM) during apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. A nonapoptotic role for BAX and BAK in eicosanoid metabolism.

    Science.gov (United States)

    Zhang, Tejia; Walensky, Loren D; Saghatelian, Alan

    2015-06-19

    BCL-2 proteins are key regulators of programmed cell death. The interplay between pro and antiapoptotic BCL-2 members has important roles in many cancers. In addition to their apoptotic function, recent evidence supports key nonapoptotic roles for several BCL-2 proteins. We used an unbiased lipidomics strategy to reveal that the proapoptotic proteins BAX, and to a lesser extent BAK, regulate the cellular inflammatory response by mediating COX-2 expression and prostaglandin biosynthesis. COX-2 upregulation in response to the bacterial endotoxin lipopolysaccharide is blunted in the absence of BAX, and Bax(-/-) mouse embryonic fibroblasts display altered kinetics of NFκB and MAPK signaling following endotoxin treatment. Our approach uncovers a novel, nonapoptotic function for BAX in regulation of the cellular inflammatory response and suggests that inflammation and apoptosis are more tightly connected than previously anticipated.

  12. Gastroprotective activity of Annona muricata leaves against ethanol-induced gastric injury in rats via Hsp70/Bax involvement

    Directory of Open Access Journals (Sweden)

    Moghadamtousi SZ

    2014-10-01

    Full Text Available Soheil Zorofchian Moghadamtousi,1 Elham Rouhollahi,2 Hamed Karimian,2 Mehran Fadaeinasab,3 Mahmood Ameen Abdulla,2 Habsah Abdul Kadir1 1Biomolecular Research Group, Biochemistry Program, Institute of Biological Sciences, Faculty of Science, 2Department of Biomedical Science, Faculty of Medicine, 3Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia Abstract: The popular fruit tree of Annona muricata L. (Annonaceae, known as soursop and graviola, is a widely distributed plant in Central and South America and tropical countries. Leaves of A. muricata have been reported to possess antioxidant and anti-inflammatory activities. In this study, the gastroprotective effects of ethyl acetate extract of A. muricata leaves (EEAM were investigated against ethanol-induced gastric injury models in rats. The acute toxicity test of EEAM in rats, carried out in two doses of 1 g/kg and 2 g/kg, showed the safety of this plant, even at the highest dose of 2 g/kg. The antiulcer study in rats (five groups, n=6 was performed with two doses of EEAM (200 mg/kg and 400 mg/kg and with omeprazole (20 mg/kg, as a standard antiulcer drug. Gross and histological features showed the antiulcerogenic characterizations of EEAM. There was significant suppression on the ulcer lesion index of rats pretreated with EEAM, which was comparable to the omeprazole effect in the omeprazole control group. Oral administration of EEAM to rats caused a significant increase in the level of nitric oxide and antioxidant activities, including catalase, glutathione, and superoxide dismutase associated with attenuation in gastric acidity, and compensatory effect on the loss of gastric wall mucus. In addition, pretreatment of rats with EEAM caused significant reduction in the level of malondialdehyde, as a marker for oxidative stress, associated with an increase in prostaglandin E2 activity. Immunohistochemical staining also demonstrated that EEAM induced the

  13. A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway.

    Directory of Open Access Journals (Sweden)

    Sebastián Zamorano

    Full Text Available Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

  14. All-trans retinoic acid inhibits KIT activity and induces apoptosis in gastrointestinal stromal tumor GIST-T1 cell line by affecting on the expression of survivin and Bax protein

    Directory of Open Access Journals (Sweden)

    Taguchi Takahiro

    2010-12-01

    Full Text Available Abstract Background Imatinib, a selective tyrosine kinase inhibitor, has been used as a standard first-line therapy for irresectable and metastasized gastrointestinal stromal tumor (GIST patients. Unfortunately, most patients responding to imatinib will eventually exhibit imatinib-resistance, the cause of which is not fully understood. The serious clinical problem of imatinib-resistance demands alternative therapeutic strategy. This study was conducted to investigate the effect of all-trans retinoic acid (ATRA on GIST cell lines. Methods Cell proliferation was determined by trypan blue dye exclusion test. Western blot analysis was performed to test the expression of activated KIT, its downstream proteins, and apoptosis associated proteins. The cytotoxic interactions of imatinib with ATRA were evaluated using the isobologram of Steel and Peckham. Results and conclusion In this work, for the first time we have demonstrated that ATRA affected on cell proliferation of GIST-T1 and GIST-882 cell line through inhibition of cell growth in a dose dependent manner and induced apoptosis. High dose of ATRA induced morphologic change in GIST-T1 cells, rounded-up cells, and activated the caspase-3 protein. In further examination, we found that the ATRA-induced apoptosis in GIST-T1 cells was accompanied by the down-regulated expression of survivin and up-regulated expression of Bax protein. Moreover, ATRA suppressed the activity of KIT protein in GIST-T1 cells and its downstream signal, AKT activity, but not MAPK activity. We also have demonstrated that combination of ATRA with imatinib showed additive effect by isobologram, suggesting that the combination of ATRA and imatinib may be a novel potential therapeutic option for GIST treatment. Furthermore, the scracht assay result suggested that ATRA was a potential reagent to prevent the invasion or metastasis of GIST cells.

  15. Effect of Bcl-2 and Bax on survival of side population cells from hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To understand the role and significance of side population (SP) cells from hepatocellular carcinoma (HCC) in hepatocarcinogenesis, development, relapse and metastasis, we simulated the denutrition conditions that cancer cells experience in clinical therapy, observed the different anti-apoptosis ability of SP cells and non-SP cells under such conditions, and established the possible effects of P53, Bcl-2 and Bax on survival of SP cells.METHODS: We used flow cytometry to analyze and sort the SP and non-SP cells in established HCC lines MHCC97and hHCC. We evaluated cell proliferation by methyl thiazolyl tetrazolium (MTT) assay and investigated the expression of p53, bd-2 and bax genes during denutrition,by RT-PCR and immunofluorescence staining.RESULTS: The percentage of SP cells in the two established HCC lines was 0.25% and 0.5%, respectively.SP cells had greater anti-apoptosis and proliferation ability than non-SP cells. Expression of Bcl-2 and Bax in SP and non-SP cells differed during denutrition. The former was up-regulated in SP cells, and the latter was up-regulated in non-SP cells.CONCLUSION: It may be that different upstream molecules acted and led to different expression levels of Bcl-2 and Bax in these two cell lines. There was a direct relationship between up-regulation of Bcl-2 and down-regulation of Bax and higher anti-apoptosis ability in SP cells. It may be that the existence and activity of SP cells are partly responsible for some of the clinical phenomena which are seen in HCC, such as relapse or metastasis. Further research on SP cells may have potential applications in the field of anticancer therapy.

  16. Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis.

    Science.gov (United States)

    Maes, Margaret E; Schlamp, Cassandra L; Nickells, Robert W

    2017-01-01

    The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.

  17. Differential expression of Bcl-2 and Bax during gastric ischemia-reperfusion of rats

    Institute of Scientific and Technical Information of China (English)

    Wei-Li Qiao; Guang-Ming Wang; Yue Shi; Jin-Xia Wu; You-Jian Qi; Jian-Fu Zhang; Hong Sun; Chang-Dong Yan

    2011-01-01

    AIM: To investigate expression of Bcl-2 and Bax in gastric ischemia-reperfusion (GI-R) and involvement of extracellular signal-regulated kinase (ERK) 1/2 activation.METHODS: The GI-R model was established by ligature of the celiac artery for 30 min and reperfusion in Sprague-Dawley rats. Rats were assigned to groups in accordancewith their evaluation period: control, 0, 0.5, 1, 3, 6, 24,48, and 72 h. Expression and distribution of Bcl-2 and Bax proteins were analyzed by immunohistochemistry and western blotting in gastric tissue samples after sacrifice.RESULTS: Compared with controls, the percentage of positive cells and protein levels of Bcl-2 decreased in the early phases of reperfusion, reached its minimumat 1 h (P < 0.05); it then increased, reaching its peak at 24 h of reperfusion (P < 0.05). The pattern of Bax expression was opposite to that of Bcl-2. Bax expressionincreased after reperfusion, with its peak at 1 h of reperfusion (P < 0.05), and then it decreased gradually to a minimum at 24 h after reperfusion (P < 0.05).On the other hand, inhibition of activation of ERK1/2 induced by PD98059, a specific upstream MEK inhibitor,had significant effects on Bcl-2 and Bax in GI-R.Compared with GI-R treatment only at 3 h of reperfusion,PD98059 reduced the number of Bcl-2 positive cells (0.58% of R3h group, P < 0.05) and Bcl-2 proteinlevel (74% of R3h group, P < 0.05) but increased the number of Bax-positive cells (1.33-fold vs R3h group, P< 0.05) and Bax protein level (1.35-fold of R3h group,P < 0.05).CONCLUSION: These results indicated that the Bcl-2 and Bax played a pivotal role in the gastric mucosal I-R injury and repair by activation of ERK1/2.

  18. Conformational Rearrangements in the Pro-apoptotic Protein, Bax, as It Inserts into Mitochondria

    Science.gov (United States)

    Gahl, Robert F.; He, Yi; Yu, Shiqin; Tjandra, Nico

    2014-01-01

    The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro- and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer mitochondrial membrane. Translocation of the pro-apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Förster resonance energy transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurement indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. In addition fluorescence correlation spectroscopy (FCS) showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. Cross-linking the C-terminal helix (α9) to helix α4 reduced the potential of those interactions to occur. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix α2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins necessary for the regulation of apoptosis. PMID:25315775

  19. Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

    Directory of Open Access Journals (Sweden)

    Hyunmi Lee

    Full Text Available Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP, the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM, which we visualize and isolate, into which Bax integrates.MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

  20. Modulation of Bax and mTOR for Cancer Therapeutics.

    Science.gov (United States)

    Li, Rui; Ding, Chunyong; Zhang, Jun; Xie, Maohua; Park, Dongkyoo; Ding, Ye; Chen, Guo; Zhang, Guojing; Gilbert-Ross, Melissa; Zhou, Wei; Marcus, Adam I; Sun, Shi-Yong; Chen, Zhuo G; Sica, Gabriel L; Ramalingam, Suresh S; Magis, Andrew T; Fu, Haian; Khuri, Fadlo R; Curran, Walter J; Owonikoko, Taofeek K; Shin, Dong M; Zhou, Jia; Deng, Xingming

    2017-06-01

    A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non-small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001-12. ©2017 AACR. ©2017 American Association for Cancer Research.

  1. The MUC1-C Oncoprotein Binds to the BH3 Domain of the Pro-apoptotic BAX Protein and Blocks BAX Function*

    Science.gov (United States)

    Ahmad, Rehan; Alam, Maroof; Rajabi, Hasan; Kufe, Donald

    2012-01-01

    The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-C·BAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway. PMID:22544745

  2. Zerumbone induced apoptosis in liver cancer cells via modulation of Bax/Bcl-2 ratio

    Directory of Open Access Journals (Sweden)

    Azimahtol Hawariah LP

    2007-04-01

    Full Text Available Abstract Background Zerumbone is a cytotoxic component isolated from Zingiber zerumbet Smith, a herbal plant which is also known as lempoyang. This new anticancer bioactive compound from Z. zerumbet was investigated for its activity and mechanism in human liver cancer cell lines. Results Zerumbone significantly showed an antiproliferative activity upon HepG2 cells with an IC50 of 3.45 ± 0.026 μg/ml. Zerumbone was also found to inhibit the proliferation of non-malignant Chang Liver and MDBK cell lines. However the IC50 obtained was higher compared to the IC50 for HepG2 cells (> 10 μg/ml. The extent of DNA fragmentation was evaluated by the Tdt-mediated dUTP nick end labelling assay which showed that, zerumbone significantly increased apoptosis in HepG2 cells in a time-course manner. In detail, the apoptotic process triggered by zerumbone involved the up-regulation of pro-apoptotic Bax protein and the suppression of anti-apoptotic Bcl-2 protein expression. The changes that occurred in the levels of this antagonistic proteins Bax/Bcl-2, was independent of p53 since zerumbone did not affect the levels of p53 although this protein exists in a functional form. Western blotting analysis for Bax protein was further confirmed qualitatively with an immunoassay that showed the distribution of Bax protein in zerumbone-treated cells. Conclusion Therefore, zerumbone was found to induce the apoptotic process in HepG2 cells through the up and down regulation of Bax/Bcl-2 protein independently of functional p53 activity.

  3. Effects of Active Components of Fuzi and Gancao Compatibility on Bax, Bcl-2, and Caspase-3 in Chronic Heart Failure Rats

    Directory of Open Access Journals (Sweden)

    Liqin Wang

    2016-01-01

    Full Text Available Hypaconitine (HA and glycyrrhetinic acid (GA are active components of Fuzi (Aconitum carmichaelii and Gancao (Glycyrrhiza uralensis Fisch; they have been used in compatibility for chronic heart failure (CHF from ancient times. The purpose of the present research was to explore whether apoptosis pathways were related with the protective effects of HA + GA against CHF rats or not. The rats were progressed with transverse-aortic constriction (TAC operation for 4 weeks to build the CHF state, and then the Digoxin (1 mg/kg, HA (2.07 mg/kg, GA (25 mg/kg, and HA (2.07 mg/kg + GA (25 mg/kg were orally administrated to rats for 1 week. The levels of BNP and cTnI in the plasma were decreased in the HA + GA group, and the heart/body weight ratio (H/B and left ventricular (LV parameters of transthoracic echocardiography were also declined; moreover, the expressions of Bax, Bcl-2, and caspase-3 were all improved in the HA + GA group than other groups in the immunohistochemistry and western blot methods. In general, the data suggested that Fuzi and Gancao compatibility could protect the CHF rats from apoptosis, which provided a strong evidence for further searching for mechanisms of them.

  4. Occupational health hazards of trichloroethylene among workers in relation to altered mRNA expression of cell cycle regulating genes (p53, p21, bax and bcl-2 and PPARA

    Directory of Open Access Journals (Sweden)

    Meenu Varshney

    2015-01-01

    Full Text Available Trichloroethylene (TCE is widely used as a metal degreaser in industrial processes. The present study reports on the effects of TCE exposure on workers employed in the lock industries. To ensure exposure of the workers to TCE, its toxic metabolites, trichloroacetic acid (TCA, dichloroacetic acid (DCA and trichloroethanol (TCEOH were detected in the plasma of the subjects through solid phase microextraction-gas chromatography-electron capture detection. TCA, DCA and TCEOH were detected in the range of 0.004–2.494 μg/mL, 0.01–3.612 μg/mL and 0.002–0.617 μg/mL, respectively. Quantitative reverse transcription polymerase chain reaction analysis revealed up-regulated expression of p53 (2.4-fold; p < 0.05, p21 (2-fold; p < 0.01, bax (2.9-fold; p < 0.01 mRNAs and down-regulated expression of bcl-2 (67%; p < 0.05 mRNAs, indicating DNA damaging potential of these metabolites. No effects were observed on the levels of p16 and c-myc mRNAs. Further, as TCA and DCA, the ligand of peroxisome proliferator activated receptor alpha (PPARA, are involved in the process of hepatocarcinogenesis in rodents, we examined expression of PPARA mRNA and let-7c miRNA in the workers. No statistically significant differences in expression of PPARA mRNA and let-7c miRNA in patients were observed as compared to values in controls. Dehydroepiandosterone sulfate (DHEAS is a reported endogenous ligand of PPARA so its competitive role was also studied. We observed decreased levels of DHEAS hormone in the subjects. Hence, its involvement in mediation of the observed changes in the levels of various mRNAs analyzed in this study appears unlikely.

  5. Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

    Science.gov (United States)

    Ma, S B; Nguyen, T N; Tan, I; Ninnis, R; Iyer, S; Stroud, D A; Menard, M; Kluck, R M; Ryan, M T; Dewson, G

    2014-01-01

    In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-xL. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family

  6. WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zeilstra, Jurrit; Joosten, Sander P.J. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Wensveen, Felix M. [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Dessing, Mark C.; Schuetze, Denise M. [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Eldering, Eric [Department of Experimental Immunology, Academic Medical Center, Amsterdam (Netherlands); Spaargaren, Marcel [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands); Pals, Steven T., E-mail: s.t.pals@amc.uva.nl [Department of Pathology, Academic Medical Center, University of Amsterdam (Netherlands)

    2011-03-04

    Research highlights: {yields} Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. {yields} Expression profiling of apoptosis-related genes in Apc{sup Min/+} mice revealed the differential expression of pro-apoptotic Bok and Bax. {yields} APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. {yields} Blocking of {beta}-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or {beta}-catenin causes constitutively active {beta}-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the Apc{sup Min/+} mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of {beta}-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which

  7. NOXA-induced alterations in the Bax/Smac axis enhance sensitivity of ovarian cancer cells to cisplatin.

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    Chao Lin

    Full Text Available Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.

  8. NOXA-induced alterations in the Bax/Smac axis enhance sensitivity of ovarian cancer cells to cisplatin.

    Science.gov (United States)

    Lin, Chao; Zhao, Xin-yu; Li, Lei; Liu, Huan-yi; Cao, Kang; Wan, Yang; Liu, Xin-yu; Nie, Chun-lai; Liu, Lei; Tong, Ai-ping; Deng, Hong-xin; Li, Jiong; Yuan, Zhu; Wei, Yu-quan

    2012-01-01

    Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.

  9. Cytosolic BNIP3 Dimer Interacts with Mitochondrial BAX Forming Heterodimers in the Mitochondrial Outer Membrane under Basal Conditions.

    Science.gov (United States)

    Hendgen-Cotta, Ulrike B; Esfeld, Sonja; Rudi, Katharina; Miinalainen, Ilkka; Klare, Johann P; Rassaf, Tienush

    2017-03-23

    The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.

  10. Reconstitution of the anti-apoptotic Bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic Bax protein.

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    Marcus Wallgren

    Full Text Available The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2 protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax, are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23-lauryl-ether (Brij-35 detergent at a level below its critical micelle concentration (CMC. Additional surface plasmon resonance (SPR measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2 to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC. Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

  11. Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells.

    Science.gov (United States)

    Matsumoto, Masaru; Nakajima, Wataru; Seike, Masahiro; Gemma, Akihiko; Tanaka, Nobuyuki

    2016-04-29

    Cisplatin is a highly effective anticancer drug for treatment of various tumors including non-small-cell lung cancer (NSCLC), and is especially useful in cases nonresponsive to molecular-targeted drugs. Accumulating evidence has shown that cisplatin activates the p53-dependent apoptotic pathway, but it also induces apoptosis in p53-mutated cancer cells. Here we demonstrated that DNA-damage inducible proapoptotic BH3 (Bcl-2 homology region 3)-only Bcl-2 family members, Noxa, Puma, Bim and Bid, are not involved in cisplatin-induced apoptosis in human NSCLC cell lines. In contrast, the expression of proapoptotic multidomain Bcl-2-family members, Bak and Bax, was induced by cisplatin in p53-dependent and -independent manners, respectively. Moreover, in wild-type p53-expressing cells, cisplatin mainly used the Bak-dependent apoptotic pathway, but this apoptotic pathway shifted to the Bax-dependent pathway by loss-of-function of p53. Furthermore, both Bak- and Bax-induced apoptosis was enhanced by the antiapoptotic Bcl-2 family member, Bcl-XL knockdown, but not by Mcl-1 knockdown. From this result, we tested the effect of ABT-263 (Navitoclax), the specific inhibitor of Bcl-2 and Bcl-XL, but not Mcl-1, and found that ABT-263 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells in the presence or absence of p53. These results indicate a novel regulatory system in cisplatin-induced NSCLC cell apoptosis, and a candidate efficient combination chemotherapy method against lung cancers.

  12. MicroRNA-650 was a prognostic factor in human lung adenocarcinoma and confers the docetaxel chemoresistance of lung adenocarcinoma cells via regulating Bcl-2/Bax expression.

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    Jia-Yuan Huang

    Full Text Available Increasing evidence shows that dysregulation of microRNAs (miRNAs is involved in malignant transformation. We investigated the clinical significance of miR-650 and its involvement in chemoresistance to docetaxel. Our results showed that the relative expression level of miR-650 was significantly higher in LAD tissues than in corresponding nontumor tissues and high level of miR-650 expression was found to be significantly associated with high incidence of lymph node metastasis, advanced clinical stage and poor prognosis of LAD patients. Univariate and multivariate analyses indicated that high miR-650 expression was an independent prognostic factor for survival. Also, we found that the level of miR-650 in LAD tissues was correlated with the response of patients to docetaxel-based chemotherapy. Silencing of miR-650 could increase the in vitro sensitivity of docetaxel-resistant LAD cells to docetaxel, while upregulation of miR-650 decreased the sensitivity of parental LAD cells to docetaxel both in vitro and in vivo. Additionally, silencing of miR-650 could enhance the caspase-3-dependent apoptosis, which might be correlated with the decreased ratio of Bcl-2/Bax. Further researches suggested that inhibitor of growth 4 (ING4 was a direct target of miR-650. Downregulated or upregulated ING4 expression could partially rescue the effects of miR-650 inhibitor or mimics in docetaxel-resistant or parental LAD cells. Furthermore, we found that ING4 was upregulated in docetaxel-responding LAD tissues, and its expression was inversely correlated with miR-650. Thus, miR-650 is a novel prognostic marker in LAD and its expression is a potential indicator of chemosensitivity to docetaxel-based chemotherapy regimen.

  13. Involvement of nitric oxide signaling in mammalian Bax-induced terpenoid indole alkaloid production of Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bax, a mammalian pro-apoptotic member of the Bcl-2 family, has been demonstrated to be a potential regulatory factor for plant secondary metabolite biosynthesis recently. To investigate the molecular mechanism of Bax-induced secondary metabolite biosynthesis, we determined the contents of nitric oxide (NO) of the transgenic Catharanthus roseus cells overexpressing a mouse Bax protein and checked the effects of NO specific scavenger 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (cPITO) on Bax-induced terpenoid indole alkaloid (TIA) production of the cells. The data showed that overexpression of the mouse Bax in C. roseus cells triggered NO generation of the cells. Treatment of cPITO not only inhibited the Bax-triggered NO burst but also suppressed the Bax-induced TIA production. The results indicated that the mouse Bax might activate the NO signaling in C. roseus cells and induce TIA production through the NO-dependent signal pathway in the cells. Furthermore, the activities of nitric oxide synthase (NOS) were significantly increased in the transgenic Bax cells as compared to those in the control cells, showing that the mouse Bax may induce NOS of C. roseus cells. Treatment of the transgenic Bax cells with NOS inhibitor PBITU blocked both Bax-induced NO generation and TIA production, which suggested that the mouse Bax might trigger NO generation and TIA production through NOS. However, the NOS-like activities and NO generation in the transgenic Bax cells did not match kinetically and the Bax-induced NOS-like activity was much later and lower than NO production. Moreover, the Bax-induced NO generation and TIA production were only partially inhibited by PBITU. Thus, our results suggested that the Bax-induced NO production and secondary metabolite biosynthesis in C. roseus cells was not entirely dependent on NOS or NOS-like enzymes.

  14. Regulating prefrontal cortex activation

    DEFF Research Database (Denmark)

    Aznar, Susana; Klein, Anders Bue

    2013-01-01

    of emotion-based actions, such as addiction and other impulse-related behaviors. In this review, we give an overview of the 5-HT2A receptor distribution (neuronal, intracellular, and anatomical) along with its functional and physiological effect on PFC activation, and how that relates to more recent findings......The prefrontal cortex (PFC) is involved in mediating important higher-order cognitive processes such as decision making, prompting thereby our actions. At the same time, PFC activation is strongly influenced by emotional reactions through its functional interaction with the amygdala...... is highly expressed in the prefrontal cortex areas, playing an important role in modulating cortical activity and neural oscillations (brain waves). This makes it an interesting potential pharmacological target for the treatment of neuropsychiatric modes characterized by lack of inhibitory control...

  15. Bax-deficiency prolongs cerebellar neurogenesis, accelerates medulloblastoma formation and paradoxically increases both malignancy and differentiation

    Science.gov (United States)

    Garcia, Idoia; Crowther, Andrew J.; Gama, Vivian; Miller, C. Ryan; Deshmukh, Mohanish; Gershon, Timothy R.

    2012-01-01

    Neurogenesis requires negative regulation through differentiation of progenitors or their programmed cell death (PCD). Growth regulation is particularly important in the postnatal cerebellum, where excessive progenitor proliferation promotes medulloblastoma, the most common malignant brain tumor in children. We present evidence that PCD operates alongside differentiation to regulate cerebellar granule neuron progenitors (CGNPs) and to prevent medulloblastoma. Here we show that genetic deletion of pro-apoptotic Bax disrupts regulation of cerebellar neurogenesis and promotes medulloblastoma formation. In Bax−/− mice, the period of neurogenesis was extended into the third week of postnatal life, and ectopic neurons and progenitors collected in the molecular layer of the cerebellum and adjacent tectum. Importantly, genetic deletion of Bax in medulloblastoma-prone ND2:SmoA1 transgenic mice greatly accelerated tumorigenesis. Bax-deficient medulloblastomas exhibited strikingly distinct pathology, with reduced apoptosis, increased neural differentiation and tectal migration. Comparing Bax+/+ and Bax−/− medulloblastomas, we were able to identify up-regulation of Bcl-2 and nuclear exclusion of p27 as tumorigenic changes that are required to mitigate the tumor suppressive effect of Bax. Studies on human tumors confirmed the importance of modulating Bax in medulloblastoma pathogenesis. Our results demonstrate that Bax-dependent apoptosis regulates postnatal cerebellar neurogenesis, suppresses medulloblastoma formation, and imposes selective pressure on tumors that form. Functional resistance to Bax-mediated apoptosis, required for medulloblastoma tumorigenesis, may be a tumor-specific vulnerability to be exploited for therapeutic benefit. PMID:22710714

  16. Bax function in the absence of mitochondria in the primitive protozoan Giardia lamblia.

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    Adrian B Hehl

    Full Text Available Bax-induced permeabilization of the mitochondrial outer membrane and release of cytochrome c are key events in apoptosis. Although Bax can compromise mitochondria in primitive unicellular organisms that lack a classical apoptotic machinery, it is still unclear if Bax alone is sufficient for this, or whether additional mitochondrial components are required. The protozoan parasite Giardia lamblia is one of the earliest branching eukaryotes and harbors highly degenerated mitochondrial remnant organelles (mitosomes that lack a genome. Here we tested whether human Bax expressed in Giardia can be used to ablate mitosomes. We demonstrate that these organelles are neither targeted, nor compromised, by Bax. However, specialized compartments of the regulated secretory pathway are completely ablated by Bax. As a consequence, maturing cyst wall proteins that are sorted into these organelles are released into the cytoplasm, causing a developmental arrest and cell death. Interestingly, this ectopic cargo release is dependent on the carboxy-terminal 22 amino acids of Bax, and can be prevented by the Bax-inhibiting peptide Ku70. A C-terminally truncated Bax variant still localizes to secretory organelles, but is unable to permeabilize these membranes, uncoupling membrane targeting and cargo release. Even though mitosomes are too diverged to be recognized by Bax, off-target membrane permeabilization appears to be conserved and leads to cell death completely independently of mitochondria.

  17. Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas.

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    Ramarao Malla

    Full Text Available BACKGROUND: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

  18. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

    Science.gov (United States)

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

    2016-07-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

  19. Expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia

    Institute of Scientific and Technical Information of China (English)

    Sheng-Mian Li; Shu-Kun Yao; Nobuyoshi Yamamura; Toshitsugu Nakamura

    2003-01-01

    AIM: To compare the difference of expression of Bcl-2 and Bax in extrahepatic biliary tract carcinoma and dysplasia, and to analyze the role of Bcl-2 and Bax proteins in the progression from dysplasia to carcinoma and to evaluate the correlation of Bcl-2/Bax protein expression with the biological behaviors.METHODS: Expressions of Bcl-2 and Bax were examined immunohistochemically in 27 cases of extrahepatic biliary tract carcinomas (bile duct carcinoma: n=21, carcinoma of ampulla of Vater: n=6), and 10 cases of atypical dysplasia.Five cases of normal biliary epithelial tissues were used as controls. A semiquantitative scoring system was used to assess the Bcl-2 and Bax reactivity.RESULTS: The expression of Bd-2 was observed in 10 out of 27 (37.0 %) invasive carcinomas, 1 out of 10 clysplasias, none out of 5 normal epithelial tissues. Bax expression rate was 74.1% (20/27) in invasive carcinoma, 30 % (3/10) in dysplasia,and 40 % (2/5) in normal biliary epithelium. Bcl-2 and Bax activities were more intense in carcinoma than in dysplasia,with no significant difference in Bcl-2 expression (P=0.1:10),and significant difference in Bax expression (P=0.038). Level of Bax expression was higher in invasive carcinoma than in dysplasia and normal tissue (P=0.012). Bcl-2 expression was correlated to Bax expression (P=0.0059). However, Bcl-2/Bax expression had no correlation with histological subtype,grade of differentiation, or level of invasion.CONCLUSION: Increased Bcl-2/Bax expression from dysplasia to invasive tumors supports the view that this is the usual route for the development of extrahepatic biliary tract carcinoma. Bcl-2/Bax may be involved, at least in part,in the apoptotic activity in extrahepatic biliary carcinoma.

  20. Dexamethasone protected human glioblastoma U87MG cells from temozolomide induced apoptosis by maintaining Bax:Bcl-2 ratio and preventing proteolytic activities

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    Patel Sunil J

    2004-12-01

    Full Text Available Abstract Background Glioblastoma is the deadliest and most prevalent brain tumor. Dexamethasone (DXM is a commonly used steroid for treating glioblastoma patients for alleviation of vasogenic edema and pain prior to treatment with chemotherapeutic drugs. Temozolomide (TMZ, an alkylating agent, has recently been introduced in clinical trials for treating glioblastoma. Here, we evaluated the modulatory effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells. Results Freshly grown cells were treated with different doses of DXM or TMZ for 6 h followed by incubation in a drug-free medium for 48 h. Wright staining and ApopTag assay showed no apoptosis in cells treated with 40 μM DXM but considerable amounts of apoptosis in cells treated with 100 μM TMZ. Apoptosis in TMZ treated cells was associated with an increase in intracellular free [Ca2+], as determined by fura-2 assay. Western blot analyses showed alternations in the levels of Bax (pro-apoptotic and Bcl-2 (anti-apoptotic proteins resulting in increased Bax:Bcl-2 ratio in TMZ treated cells. Western blot analyses also detected overexpression of calpain and caspase-3, which cleaved 270 kD α-spectrin at specific sites for generation of 145 and 120 kD spectrin break down products (SBDPs, respectively. However, 1-h pretreatment of cells with 40 μM DXM dramatically decreased TMZ induced apoptosis, decreasing Bax:Bcl-2 ratio and SBDPs. Conclusion Our results revealed an antagonistic effect of DXM on TMZ induced apoptosis in human glioblastoma U87MG cells, implying that treatment of glioblastoma patients with DXM prior to chemotherapy with TMZ might result in an undesirable clinical outcome.

  1. Bax and Bif-1 proteins interact on Bilayer Lipid Membrane and form pore.

    Science.gov (United States)

    Gupta, Rajeev; Ghosh, Subhendu

    2015-08-07

    Bax and Bax interacting factor-1(Bif-1) are cytosolic proteins, which translocate towards mitochondria during mitochondria-mediated apoptosis. Bif-1 has been identified to co-immunoprecipitate with Bax in apoptotic cells. We have studied the interaction of Bax and Bif-1 on Bilayer Lipid Membrane (BLM) through electrophysiological experiments. It has been observed that Bax-Bif-1 equimolar mixture can form a pore. The pore conductance is in the range of 4.96-5.41 nS. It also displays a sub-state with a conductance of 2.6 nS. No pore activity is observed on BLM when monomeric Bax and Bif-1 proteins are tested independently. The above-mentioned pore forming activity could be relevant in mitochondria-mediated apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Molecular regulation of osteoclast activity.

    Science.gov (United States)

    Bruzzaniti, Angela; Baron, Roland

    2006-06-01

    Osteoclasts are multinucleated cells derived from hematopoietic precursors that are primarily responsible for the degradation of mineralized bone during bone development, homeostasis and repair. In various skeletal disorders such as osteoporosis, hypercalcemia of malignancy, tumor metastases and Paget's disease, bone resorption by osteoclasts exceeds bone formation by osteoblasts leading to decreased bone mass, skeletal fragility and bone fracture. The overall rate of osteoclastic bone resorption is regulated either at the level of differentiation of osteoclasts from their monocytic/macrophage precursor pool or through the regulation of key functional proteins whose specific activities in the mature osteoclast control its attachment, migration and resorption. Thus, reducing osteoclast numbers and/or decreasing the bone resorbing activity of osteoclasts are two common therapeutic approaches for the treatment of hyper-resorptive skeletal diseases. In this review, several of the key functional players involved in the regulation of osteoclast activity will be discussed.

  3. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.

    Science.gov (United States)

    Lidman, Martin; Pokorná, Šárka; Dingeldein, Artur P G; Sparrman, Tobias; Wallgren, Marcus; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2016-06-01

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Cell death induced by 2-phenylethynesulfonamide uncovers a pro-survival function of BAX.

    Science.gov (United States)

    Mattiolo, Paolo; Barbero-Farran, Ares; Amigó, Josep; Ripamonti, Marta; Ribas, Judit; Boix, Jacint

    2014-11-01

    PES (2-phenylethynesulfonamide) was initially identified as an inhibitor of p53 translocation to mitochondria and named Pifithrin-µ. Further studies showed that PES selectively killed tumour cells and was thus a promising anticancer agent. PES-induced cell death was characterised by a non-apoptotic, autophagosome-rich phenotype. We observed this phenotype via electron microscopy in wild type (wt) and double Bax-/- Bak-/- (DKO) mouse embryonic fibroblasts (MEFs) treated with PES. We excluded the involvement of effector caspases, BAX and BAK, in causing PES-triggered cell death. Therefore, apoptosis was ruled out as the lethal mode of action of PES. Surprisingly, MEFs containing BAX were significantly protected from PES treatments. BAX overexpression in Bax-/- MEFs confirmed this pro-survival effect. Moreover, this protective effect required the ability of BAX to localise to mitochondrial membranes. Conversely, mitochondrial fusion induced by treatment with Mdivi-1 conferred increased resistance to MEFs subjected to PES treatment. The involvement of BAX in the regulation of mitochondrial dynamics has been reported. We propose the promotion of mitochondrial fusion by BAX to be the pro-survival function attributed to BAX. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Integration and oligomerization of Bax protein in lipid bilayers characterized by single molecule fluorescence study.

    Science.gov (United States)

    Luo, Lu; Yang, Jun; Liu, Dongxiang

    2014-11-14

    Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-xL. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-xL can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. PUMA promotes Bax translocation in FOXO3a-dependent pathway during STS-induced apoptosis

    Science.gov (United States)

    Zhang, Yingjie; Chen, Qun

    2009-08-01

    PUMA (p53 up-regulated modulator of apoptosis, also called Bbc3) was first identified as a BH3-only Bcl-2 family protein that is transcriptionally up-regulated by p53 and activated upon p53-dependent apoptotic stimuli, such as treatment with DNA-damaging drugs or UV irradiation. Recently studies have been shown that Puma is also up-regulated in response to certain p53-independent apoptotic stimuli, such as growth factor deprivation or treatment with glucocorticoids or STS (staurosporine). However, the molecular mechanisms of PUMA up-regulation and how PUMA functions in response to p53-independent apoptotic stimuli remain poorly understood. In this study, based on real-time single cell analysis, flow cytometry and western blotting technique, we investigated the function of PUMA in living human lung adenocarcinoma cells (ASTC-a-1) after STS treatment. Our results show that FOXO3a was activated by STS stimulation and then translocated from cytosol to nucleus. The expression of PUMA was up-regulated via a FOXO3a-dependent manner after STS treatment, while p53 had little function in this process. Moreover, cell apoptosis and Bax translocation induced by STS were not blocked by Pifithrin-α (p53 inhibitor), which suggested that p53 was not involved in this signaling pathway. Taken together, these results indicate that PUMA promoted Bax translocation in a FOXO3a-dependment pathway during STS-induced apoptosis, while p53 was dispensable in this process.

  7. Glycogen synthase kinase-3β facilitates cell apoptosis induced by high fluence low-power laser irradiation through acceleration of Bax translocation

    Science.gov (United States)

    Huang, Lei; Wu, Shengnan; Xing, Da

    2011-03-01

    Glycogen synthase kinase-3β (GSK-3β) is a critical activator of cell apoptosis induced by a diverse array of insults. However, the effects of GSK-3β on the human lung adenocarcinoma cell (ASTC-a-1) apoptosis induced by high fluence low-power laser irradiation (HF-LPLI) are not clear. Here, we showed that GSK-3β was constantly translocated from cytoplasm to nucleus and activated during HF-LPLI-induced cell apoptosis. In addition, we found that co-overexpression of YFP-GSK-3β and CFP-Bax in ASTC-a-1 cells accelerated both Bax translocations to mitochondria and cell apoptosis, compared to the cells expressed CFP-Bax only under HF-LPLI treatment, indicating that GSK-3β facilitated ASTC-a-1 cells apoptosis through acceleration mitochondrial translocation of Bax. Our results demonstrate that GSK-3β exerts some of its pro-apoptotic effects in ASTC-a-1 cells by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade.

  8. Role of Bax in death of uninfected retinal cells during murine cytomegalovirus retinitis.

    Science.gov (United States)

    Mo, Juan; Marshall, Brendan; Covar, Jason; Zhang, Nancy Y; Smith, Sylvia B; Atherton, Sally S; Zhang, Ming

    2014-10-08

    Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes. During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  9. EGR-1 forms a complex with YAP-1 and upregulates Bax expression in irradiated prostate carcinoma cells.

    Science.gov (United States)

    Zagurovskaya, M; Shareef, M M; Das, A; Reeves, A; Gupta, S; Sudol, M; Bedford, M T; Prichard, J; Mohiuddin, M; Ahmed, M M

    2009-02-26

    In this study, we investigated the functional role of early growth response-1 (Egr1 gene) in the regulation of radiation-induced clonogenic inhibition and apoptosis in p53 wild-type and mutant prostate cancer cells 22Rv1 and DU145, respectively. 22Rv1 cells were more sensitive to irradiation compared with DU145 cells, and the sensitivity was enhanced by overexpression of EGR-1 in both cells. Dominant-negative EGR-1 mutant (dnEGR-1) or repressor of EGR-1, NGFIA binding protein 1 (NAB1), increased radioresistance of these cells. Significant activation of caspases 3 and 9 and Bcl2-associated X (Bax) with increased poly(ADP-ribose) polymerase (PARP) cleavage and cytochrome c release was observed in radiation-exposed EGR-1 overexpressing cells. Gel shift analysis and chloramphenicol acetyl transferase (CAT) reporter assays indicate that EGR-1 transactivates the promoter of the Bax gene. Interaction of EGR-1 and Yes kinase-associated protein 1 (YAP-1) through the WW domain of YAP-1 enhances the transcriptional activity of EGR-1 on the Bax promoter as shown by chromatin immunoprecipitation and reporter assays. Irradiation of PC3 cell xenografts that were treated with adenoviral EGR-1 showed significant regression in tumor volume. These findings establish the radiation-induced pro-apoptotic action of EGR-1, in a p53-independent manner, by directly transactivating Bax, and prove that alters the B-cell CLL/lymphoma 2 (Bcl-2)/Bax ratio as one of the mechanisms resulting in significant activation of caspases, leading to cell death through the novel interaction of EGR-1 with YAP-1.

  10. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

    Science.gov (United States)

    Große, Lena; Wurm, Christian A; Brüser, Christian; Neumann, Daniel; Jans, Daniel C; Jakobs, Stefan

    2016-02-15

    The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  11. Bak and Bax function to limit adenovirus replication through apoptosis induction.

    Science.gov (United States)

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-05-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.

  12. Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor.

    Directory of Open Access Journals (Sweden)

    Anna V Miller

    Full Text Available Paclitaxel (Taxol-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim(-/- MEFs (mouse embryonic fibroblasts, the bim(-/- mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak (-/- MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax(-/- MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.

  13. 苦参碱对 HaCaT 细胞 Bcl -2/Bax 和Fas/FasL 的调控%Regulation of Bcl-2/Bax and Fas/FasL by matrine in HaCaT cells

    Institute of Scientific and Technical Information of China (English)

    牟宽厚; 周艳; 韩丹; 穆欣

    2014-01-01

    目的:明确苦参碱对 HaCaT 细胞 Bcl-2/ Bax 和 Fas/ FasL 表达的影响。方法:体外培养HaCaT 细胞,选择第二代细胞对数生长期 HaCaT 细胞作为研究对象,将细胞随机分为4组:苦参碱2 mg/ mL、10 mg/ mL 和50 mg/ mL 3组及对照组(加入相同体积的0.9%盐水),孵育48 h 后,MTT 法测定各浓度下细胞增殖,RT-PCR 检测 Bcl-2/ Bax 和 Fas/ FasL 的表达。结果:与对照组相比,当苦参碱浓度为2 mg/ mL 时,HaCaT 细胞增殖活性无明显变化;Bcl -2、Bax、Fas、FasL 表达也无明显变化( P>0.05)。当苦参碱浓度为10 mg/ mL 时 HaCaT 细胞增殖活性较对照组明显下降(P0.05)。结论:苦参碱能够调控上皮细胞致炎因子的表达,抑制细胞的增殖。%To determine the effect of matrine on Bcl-2/ Bax and Fas/ FasL in keratinocytes in vitro. Methods: Second generation cultured HaCaT cells (logarithmic phase cells) were selected and divided into 4 groups:3 matrine groups (2 mg/ mL, 10 mg/ mL and 50 mg/ mL were used in each group) and the control group (0.9% Natrii Chloride). After 48-hour culture, the proliferation of HaCaT were detected by MTT and the levels of Bcl-2/ Bax and Fas/ FasL were measured by RT-PCR. Results: The viability of HaCaT cells was similar in 2 mg/ mL matrine group and control group (P>0.05). In 10 mg/ mL matrine group the proliferation of the cells was significantly decreased (P<0.001) and the Bcl-2 expression was remarkably reduced (P<0.001), while the expression of Bax, Fas and FasL was significantly increased (P<0.01 and P<0.05, respectively). When the concentration of matrine was increased to 50 mL, the viability and the expression of Bcl-2, Bax, Fas and FasL was similar to the results when 10 mL matrine was used. Conclusion: Matrine can inhibit HaCaT cells proliferation (at 10 mg/ mL or more) and may adjust expression of Bcl-2/ Bax and Fas/FasL in HaCaT cells.

  14. Oncogenic Mutations Differentially Affect Bax Monomer, Dimer, and Oligomeric Pore Formation in the Membrane

    Science.gov (United States)

    Zhang, Mingzhen; Zheng, Jie; Nussinov, Ruth; Ma, Buyong

    2016-09-01

    Dysfunction of Bax, a pro-apoptotic regulator of cellular metabolism is implicated in neurodegenerative diseases and cancer. We have constructed the first atomistic models of the Bax oligomeric pore consisting with experimental residue-residue distances. The models are stable, capturing well double electron-electron resonance (DEER) spectroscopy measurements and provide structural details in line with the DEER data. Comparison with the latest experimental results revealed that our models agree well with both Bax and Bak pores, pointed to a converged structural arrangement for Bax and Bak pore formation. Using multi-scale molecular dynamics simulations, we probed mutational effects on Bax transformation from monomer → dimer → membrane pore formation at atomic resolution. We observe that two cancer-related mutations, G40E and S118I, allosterically destabilize the monomer and stabilize an off-pathway swapped dimer, preventing productive pore formation. This observation suggests a mechanism whereby the mutations may work mainly by over-stabilizing the monomer → dimer transformation toward an unproductive off-pathway swapped-dimer state. Our observations point to misfolded Bax states, shedding light on the molecular mechanism of Bax mutation-elicited cancer. Most importantly, the structure of the Bax pore facilitates future study of releases cytochrome C in atomic detail.

  15. [The Mechanisms by which Bax Induces the Apoptosis of Human Ovarian Cancer Cells].

    Science.gov (United States)

    Zeng, Jun; Yang, Jing; Chen, Deng-bang; Cao, Kang

    2015-09-01

    The purpose of this study was to observe the apoptosis of A2780 cells transfected with the recombinant plasmid of pcDNA-Bax and to observe the release of cytochrome C from the mitochondria. The recombinant plasmid of pcDNA-Bax was constructed and transfected into A2784 cells. The Hoechst 33258 stain method was applied to evaluate the apoptosis of the transfected cells and MTT mothod was used to test the cell viability. Western blot analysis was performed to determine the overexpression of Bax and the release of cytochrome C from the mitochondria. The recombinant plasmid of pcDNA-Bax was successfully constructed by using endonuclease digestion and the sequence analysis. The apoptosis of A2780 cells was induced after transfected with pcDNA3. 1-Bax as demonstrated with Hoechst staining. The cell viability were decreased in the pcDNA3. 1-Bax transfected group by MTT assay. The release of cytochrome C from the mitochondria was observed when using Western blotting analysis. And the caspase-9 and the caspase-3 were activated. Our data suggestted that Bax exhibited potent pro-apoptotic activity against the ovarian cancer cells. This study is a foundation for the further research in the pro-apoptotic activity of Bax.

  16. The human septin7 and the yeast CDC10 septin prevent Bax and copper mediated cell death in yeast.

    Science.gov (United States)

    Horowitz, Avital; Lapointe, Jason F; Eid, Rawan; Sheibani, Sara; Gharib, Nada; Jones, Natalie K; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2013-12-01

    The mechanisms of programmed cell death activate genetically encoded intracellular programs in a controlled manner, the most common form being apoptosis. Apoptosis is carried out through a cascade of caspase mediated proteolytic cleavages initiated by the oligomerization of Bax, a cardinal regulator of mitochondrial-mediated apoptosis. Heterologous expression of Bax in yeast causes cell death that shares a number of similarities to processes that occur in mammalian apoptosis. A screen of a cardiac cDNA library for suppressors of Bax-mediated apoptosis identified human septin7, a protein that belongs to the septin superfamily of conserved GTP-binding proteins that share a conserved cdc/septin domain. Analysis of the amino acid sequence deduced from the septin7 clone as well as the corresponding human septin7 gene revealed that a novel alternatively spliced transcript called septin7 variant4 (v4) was uncovered. Yeast cells overexpressing the human septin7 v4 cDNA were also capable of resisting copper-mediated cell death suggesting that it is not only a Bax suppressor but also an anti-apoptotic sequence. Analysis of septin7 function in a MCA1Δ yeast strain suggests that septin7 inhibits apoptosis in a caspase independent pathway. Overexpression of the yeast septin7 ortholog CDC10 also conferred resistance to the negative effects of copper as well as protecting cells from the overexpression of Bax. In contrast, septin7 was unable to prevent the increase in cell size associated with mutants lacking the endogenous yeast CDC10 gene. Taken together, our analysis suggests that anti-apoptosis is a novel yet evolutionarily conserved property of the septin7 sub-family of septins.

  17. HCV upregulates Bim through the ROS/JNK signalling pathway, leading to Bax-mediated apoptosis.

    Science.gov (United States)

    Deng, Lin; Chen, Ming; Tanaka, Motofumi; Ku, Yonson; Itoh, Tomoo; Shoji, Ikuo; Hotta, Hak

    2015-09-01

    We previously reported that hepatitis C virus (HCV) infection induces Bax-triggered, mitochondrion-mediated apoptosis by using the HCV J6/JFH1 strain and Huh-7.5 cells. However, it was still unclear how HCV-induced Bax activation. In this study, we showed that the HCV-induced activation and mitochondrial accumulation of Bax were significantly attenuated by treatment with a general antioxidant, N-acetyl cysteine (NAC), or a specific c-Jun N-terminal kinase (JNK) inhibitor, SP600125, with the result suggesting that the reactive oxygen species (ROS)/JNK signalling pathway is upstream of Bax activation in HCV-induced apoptosis. We also demonstrated that HCV infection transcriptionally activated the gene for the pro-apoptotic protein Bim and the protein expression of three major splice variants of Bim (BimEL, BimL and BimS). The HCV-induced increase in the Bim mRNA and protein levels was significantly counteracted by treatment with NAC or SP600125, suggesting that the ROS/JNK signalling pathway is involved in Bim upregulation. Moreover, HCV infection led to a marked accumulation of Bim on the mitochondria to facilitate its interaction with Bax. On the other hand, downregulation of Bim by siRNA (small interfering RNA) significantly prevented HCV-mediated activation of Bax and caspase 3. Taken together, these observations suggest that HCV-induced ROS/JNK signalling transcriptionally activates Bim expression, which leads to Bax activation and apoptosis induction.

  18. Overexpression of Bax sensitizes prostate cancer cells to TGF-β induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Pei Hui LIN; Zui PAN; Lin ZHENG; Na LI; David DANIELPOUR; Jian Jie MA

    2005-01-01

    NRP-154 is a tumorigenic epithelial cell line derived from the preneoplastic dorsal-lateral prostate of rats. These cells are exquisitely sensitive to TGF-β induced apoptosis. In contrast, we find that NRP-154 cells can sustain overexpression of exogenous Bax protein, which is different from non-tumor cells where Bax functions as a ubiquitous stimulator of apoptosis. NRP-154 cells stably overexpressing Bax show increased sensitivity to TGF-β induced apoptosis. The degree of TGF-β induced apoptosis displays high correlation with cleavage of Bax at the amino-terminus. Our data indicate that prostate cancer cells can host high levels of latent Bax which can be activated through post-translational modification.

  19. Andrographolide reversed 5-FU resistance in human colorectal cancer by elevating BAX expression.

    Science.gov (United States)

    Wang, Weicheng; Guo, Wenjie; Li, Lele; Fu, Zan; Liu, Wen; Gao, Jian; Shu, Yongqian; Xu, Qiang; Sun, Yang; Gu, Yanhong

    2016-12-01

    5-FU is the first line therapy for colorectal cancer, however, treatment effect is often hampered by the development of drug resistance or toxicity at high doses. Andrographolide is a natural diterpenoid from Andrographis paniculata which has anti-bacterial, anti-antiviral and anti-inflammation activities. In the current study, we test the hypothesis that Andrographolide reverses 5-FU resistance in colorectal cancer and examine the underlying mechanism. In vitro and vivo studies indicated that Andrographolide treatment significantly re-sensitizes HCT116/5-FUR cells (HCT116 cells which are 5-FU resistant) to cytotoxicity of 5-FU. Mechanism analysis showed that Andrographolide/5-FU co-treatment elevated apoptosis level of HCT116/5-FUR cells with highly increased level of BAX. By using biotin-Andrographolide pull down and cellular thermal shift assay, we found out that Andrographolide can directly target to BAX. Andrographolide-BAX interaction prevented BAX degradation, enhancing mitochondria-mediated apoptosis thus reversed 5-FU resistance while BAX silence diminished this effect. Further, by analyzing patient samples who received 5-FU involved chemotherapy, we found that expression level of BAX is correlated with PFS. Our results here provide a novel combination treatment strategy, especially for patients with 5-FU-resistant tumors expressing low level of BAX. Meanwhile, we also proposed that BAX expression may be a predicted and prognosis marker of 5-FU involved chemotherapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. The substitution of Proline 168 favors Bax oligomerization and stimulates its interaction with LUVs and mitochondria.

    Science.gov (United States)

    Simonyan, Lilit; Légiot, Alexandre; Lascu, Ioan; Durand, Grégory; Giraud, Marie-France; Gonzalez, Cécile; Manon, Stéphen

    2017-06-01

    Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Isatin decreases Bax protein expression in the substantia nigra of a mouse model of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Jiguo Zhang; Fang Zhang; Yanlong Qiu; Wang Yue

    2011-01-01

    The present study observed the action of 1H-indole-2, 3-dione (isatin) on Bax protein expression in the substantia nigra of a Parkinson's disease animal model. Parkinson's disease-like behaviors were induced in C57BL/6J mice treated with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Bax protein expression was significantly reduced in isatin (100, 200 mg/kg)-pretreated mice. Results demonstrate that isatin plays a neuroprotective role in mice treated with MPTP by down-regulating Bax protein expression.

  2. Dynamin-related protein Drp1 is required for Bax translocation to mitochondria in response to irradiation-induced apoptosis.

    Science.gov (United States)

    Wang, Ping; Wang, Peiguo; Liu, Becky; Zhao, Jing; Pang, Qingsong; Agrawal, Samir G; Jia, Li; Liu, Feng-Ting

    2015-09-08

    Translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria is a crucial step in DNA damage-mediated apoptosis, and is also found to be involved in mitochondrial fragmentation. Irradiation-induced cytochrome c release and apoptosis was associated with Bax activation, but not mitochondrial fragmentation. Both Bax and Drp1 translocated from the cytosol to the mitochondria in response to irradiation. However, Drp1 mitochondrial translocation and oligomerization did not require Bax, and failed to induce apoptosis in Bax deficient diffuse large B-cell lymphoma (DLBCL) cells. Using fluorescent microscopy and the intensity correlation analysis, we demonstrated that Bax and Drp1 were colocalized and the levels of colocalization were increased by UV irradiation. Using co-immuno-precipitation, we confirmed that Bax and Drp1 were binding partners. Irradiation induced a time-associated increase in the interaction between active Bax and Drp1. Knocking down Drp1 using siRNA blocked UV irradiation-mediated Bax mitochondrial translocation. In conclusion, our findings demonstrate for the first time, that Drp1 is required for Bax mitochondrial translocation, but Drp1-induced mitochondrial fragmentation alone is not sufficient to induce apoptosis in DLBCL cells.

  3. Proteinase activity regulation by glycosaminoglycans

    Directory of Open Access Journals (Sweden)

    Tersariol I.L.S.

    2002-01-01

    Full Text Available There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

  4. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1.

    Science.gov (United States)

    Yang, Jiong; Yao, Shukun

    2015-10-27

    Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC). The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1) matrine not only activated caspase and PARP (poly ADP-ribose polymerase) cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2) matrine treatment promoted the JNK-Bcl-2/ Bcl-xL-Bax/Bak pathway; (3) Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4) Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5) finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1.

  5. JNK-Bcl-2/Bcl-xL-Bax/Bak Pathway Mediates the Crosstalk between Matrine-Induced Autophagy and Apoptosis via Interplay with Beclin 1

    Directory of Open Access Journals (Sweden)

    Jiong Yang

    2015-10-01

    Full Text Available Autophagy is associated with drug resistance which has been a threat in chemotherapy of hepatocellular carcinoma (HCC. The interconnected molecular regulators between autophagy and apoptosis serve as switching points critical to the ultimate outcome of the cell. Our study was performed to investigate the crosstalk between autophagy and apoptosis in HCC after the treatment of matrine. Flow cytometry and TUNEL (terminal dexynucleotidyl transferase (TdT-mediated dUTP nick end labeling assay were used to detect apoptosis in vitro and in vivo, respectively. Bax oligomerization and Cytochrome c release assay were performed. Immunoprecipitation and siRNA transfection were used to detect the interplay between Bcl-2/Bcl-xL,Bax, and Beclin 1. Our results showed that: (1 matrine not only activated caspase and PARP (poly ADP-ribose polymerase cleavage, but also triggered autophagy as shown by the increased levels of LC3II, Beclin 1, and PI3KC3, and the decreased level of p62; (2 matrine treatment promoted the JNK-Bcl-2/ Bcl-xL-Bax/Bak pathway; (3 Bax was oligomerized, the mitochondrial membrane potential altered, and Cytochrome c was released subsequently; (4 Bax interacts with Beclin 1 and inhibits autophagy, which may be a new crosstalk point; and (5 finally, we showed that matrine suppressed the growth of a MHCC97L xenograft in vivo for the first time. In conclusion, the JNK-Bcl-2/Bcl-xL-Bax/Bak pathway mediates the crosstalk between matrine-induced autophagy and apoptosis via interplay with Beclin 1.

  6. Bax/bcl-2: cellular modulator of apoptosis in feline skin and basal cell tumours.

    Science.gov (United States)

    Madewell, B R; Gandour-Edwards, R; Edwards, B F; Matthews, K R; Griffey, S M

    2001-01-01

    Bcl-2 and bax are two members of the BCL-2 gene family that play a prominent role in the regulation of apoptosis. Bax and bcl-2 expression were examined immunohistochemically in normal (healthy) feline skin and in 24 benign feline cutaneous basal cell tumours. The tumours were also examined for cellular proliferation by measurement of reactivity for the proliferation marker Ki-67, and for apoptosis by in-situ labelling for fragmented DNA. Bcl-2 was detected in normal basal epithelium and in 23 of 24 basal cell tumours. Bax was detected in both basal and suprabasal epithelium, but in only seven of 24 tumours. For tumours that expressed both bax and bcl-2, the bax:bcl-2 ratio was low. Neither bax nor bcl-2 expression was detected in 14 feline cutaneous squamous cell carcinomas. Basal cell tumours showed modest cellular proliferation (median, 17.5% Ki-67- reactive cells), but few (less than 1%) apoptotic cells. The slow, indolent growth of feline cutaneous basal cells in these benign skin tumours may be a response, at least in part, to opposing regulatory expressions of bcl-2 and bax.

  7. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    XU MaoJun; DONG JuFang

    2007-01-01

    Bax, a mammalian pro-apoptotic member of the Bcl-2 family, triggers hypersensitive reactions when expressed in plants. To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells, we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter. The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied. Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0- and 5.5-fold of the control cells. Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str, two key genes in terpenoid indole alkaloid biosynthetic pathway of Catharanthus roseus cells, and stimulates the accumulation of defense-related protein PR1 in the cells, showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway. Thus, our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  8. Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Bax,a mammalian pro-apoptotic member of the Bcl-2 family,triggers hypersensitive reactions when expressed in plants.To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells,we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter.The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied.Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0-and 5.5-fold of the control cells.Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str,two key genes in terpenoid indole alkaloid bio-synthetic pathway of Catharanthus roseus cells,and stimulates the accumulation of defense-related protein PR1 in the cells,showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway.Thus,our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

  9. 桥本甲状腺炎中细胞凋亡调节蛋白Bcl-2和Bax的免疫组织化学研究%n immunohistochemical study of apoptosis-regulated proteins Bcl-2 and Bax in Hashimoto's thyroiditis

    Institute of Scientific and Technical Information of China (English)

    姬秋和 姬秋和; 张雅萍; 张万会; 张南雁; 宋民喜; 陈健康

    2001-01-01

    目的 了解细胞凋亡调节蛋白Bcl-2和Bax在桥本甲状腺炎(HT)中的分布变化及其意义。方法 以非毒性甲状腺肿(NTG)为对照(17例),采用免疫组织化学方法,检测17例桥本甲状腺炎患者甲状腺标本中Bcl-2和Bax的表达及分布。结果 免疫染色半定量分析及图像分析结果显示,HT中Bcl-2和Bax的免疫染色强度均显著高于对照组(P<0.01),其中Bax免疫染色强阳性甲状腺滤泡细胞多分布于浸润淋巴滤泡附近,Bcl-2免疫染色强阳性细胞则多分布于远离浸润淋巴滤泡的区域,但在Bcl-2免疫反应阴性的淋巴滤泡周围亦有少量分布。结论 HT中细胞凋亡调节蛋白Bcl-2和Bax在甲状腺滤泡细胞中呈有特征性分布的高表达;其表达部位及比例的改变可能对甲状腺滤泡萎缩、破坏具有调控作用%Objective To investigate the significance of expression of the apoptosis-regulated proteins Bcl-2 and Bax in the pathogenesis and pathological changes of Hashimoto's thyroiditis (HT). Methods Expression and distribution of Bcl-2 and Bax proteins in thyroid tissues from 17 patients with HT and 17 patients with nontoxic goiter (NTG) (as controls) were investigated by immunohistochemical methods. Results The intensities of positive immunostaining for Bcl-2 and Bax in thyrocytes from HT patients were significantly higher than those from the NGT patients (P<0.01). The thyrocytes strongly positively immunostained to Bax in HT were mainly distributed in follicles adjacent to lymphocytic infiltrates while the thyrocytes strongly positively stained to Bcl-2 were mainly distributed far away from lymphocytic infiltrates. But in the vicinity of lymphoid follicles negatively immunostained to Bcl-2, some thyrocytes strongly positive stained to Bcl-2 were also observed. Conclusion High characteristic expressions of apoptosis-regulated proteins Bcl-2 and Bax on thyrocytes from HT are observed. These changes seem to lead

  10. Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

    Science.gov (United States)

    D'Orsi, Beatrice; Bonner, Helena; Tuffy, Liam P; Düssmann, Heiko; Woods, Ina; Courtney, Michael J; Ward, Manus W; Prehn, Jochen H M

    2012-02-01

    Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

  11. p53's mitochondrial translocation and MOMP action is independent of Puma and Bax and severely disrupts mitochondrial membrane integrity

    Institute of Scientific and Technical Information of China (English)

    Sonja Wolff; Susan Erster; Gustavo Palacios; Ute M Moll

    2008-01-01

    p53's apoptotic program consists of transcription-dependent and transcription-independent pathways. In the latter, physical interactions between mitochondrial p53 and anti-and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators are central. Using isogenic cell systems with defined deficiencies, we characterize in detail how mitochondrial p53 contributes to mitochondrial permeabilization, to what extent its action depends on other key Bcl2 family members and define its release activity. We show that mitochondrial p53 is highly efficient in inducing the release of soluble and insoluble apoptogenic factors by severely disrupting outer and inner mitochondrial membrane integrity. This action is associated with wild-type p53-induced oligomerization of Bax, Bak and VDAC and the formation of a stress-induced endogenous complex between p53 and cyclophilin D, normally located at the inner membrane. Tumor-derived p53 mutants are deficient in activating the Bax/Bak lipid pore. These actions are independent of Puma and Bax. Importantly, the latter distinguishes the mitochondrial from the cytosolic p53 death pathway.

  12. Bax phosphorylation association with nucleus and oligomerization after neonatal hypoxia-ischemia.

    Science.gov (United States)

    Infante, Smitha Krishna; Oberhauser, Andres F; Perez-Polo, J Regino

    2013-09-01

    Neonatal hypoxia-ischemia (HI) is a common occurrence in preterm and low-birth-weight infants, and the incidence of low-birth-weight and preterm births is increasing. Characterization of brain injury after HI is of critical importance in developing new treatments that more accurately target the injury. After severe HI, neuronal cells undergo necrosis and secondary apoptosis of the surrounding cells as a result of neuroinflammation. We sought to characterize the biochemical pathways associated with cell death after HI. Bax, a cell death signaling protein, is activated after HI and translocates to the nucleus, endoplasmic reticulum, and mitochondria. The translocation patterns of Bax affect the resultant cell death phenotype (necrotic or apoptotic) observed. Although Bax is known to oligomerize once it is activated, less is known about the factors that control its translocation and oligomerization. We hypothesize that Bax kinase-specific phosphorylation determines its oligomerization and intracellular localization. Using well-established in vivo and in vitro models of neonatal HI, we characterized Bax oligomerization and multiorganelle translocation. We found that HI-dependent phosphorylation of Bax determines its oligomerization status and multiorganelle localization, and, ultimately, the cell death phenotype observed. Understanding the mechanisms of Bax translocation will aid in the rational design of therapeutic strategies that decrease the trauma resulting from HI-associated inflammation.

  13. Insights into the structural stability of Bax from molecular dynamics simulations at high temperatures

    Science.gov (United States)

    Rosas-Trigueros, Jorge Luis; Correa-Basurto, José; Guadalupe Benítez-Cardoza, Claudia; Zamorano-Carrillo, Absalom

    2011-01-01

    Bax is a member of the Bcl-2 protein family that participates in mitochondrion-mediated apoptosis. In the early stages of the apoptotic pathway, this protein migrates from the cytosol to the outer mitochondrial membrane, where it is inserted and usually oligomerizes, making cytochrome c-compatible pores. Although several cellular and structural studies have been reported, a description of the stability of Bax at the molecular level remains elusive. This article reports molecular dynamics simulations of monomeric Bax at 300, 400, and 500 K, focusing on the most relevant structural changes and relating them to biological experimental results. Bax gradually loses its α-helices when it is submitted to high temperatures, yet it maintains its globular conformation. The resistance of Bax to adopt an extended conformation could be due to several interactions that were found to be responsible for maintaining the structural stability of this protein. Among these interactions, we found salt bridges, hydrophobic interactions, and hydrogen bonds. Remarkably, salt bridges were the most relevant to prevent the elongation of the structure. In addition, the analysis of our results suggests which conformational movements are implicated in the activation/oligomerization of Bax. This atomistic description might have important implications for understanding the functionality and stability of Bax in vitro as well as within the cellular environment. PMID:21936009

  14. Bax Exists in a Dynamic Equilibrium between the Cytosol and Mitochondria to Control Apoptotic Priming

    Science.gov (United States)

    Schellenberg, Barbara; Wang, Pengbo; Keeble, James A.; Rodriguez-Enriquez, Ricardo; Walker, Scott; Owens, Thomas W.; Foster, Fiona; Tanianis-Hughes, Jolanta; Brennan, Keith; Streuli, Charles H.; Gilmore, Andrew P.

    2013-01-01

    Summary The proapoptotic Bcl-2 protein Bax is predominantly found in the cytosol of nonapoptotic cells and is commonly thought to translocate to mitochondria following an apoptotic stimulus. The current model for Bax activation is that BH3 proteins bind to cytosolic Bax, initiating mitochondrial targeting and outer-membrane permeabilization. Here, we challenge this and show that Bax is constitutively targeted to mitochondria but in nonapoptotic cells is constantly translocated back to the cytosol. Using live-cell spinning-disk confocal imaging with a combination of FLIP, FRAP, and photoactivatable GFP-Bax, we demonstrate that disrupting adhesion-dependent survival signals slows the rate of Bax’s dissociation from mitochondria, leading to its accumulation on the outer mitochondrial membrane. The overall accumulation of mitochondrial Bax following loss of survival signaling sensitizes cells to proapoptotic BH3 proteins. Our findings show that Bax is normally in a dynamic equilibrium between cytosol and mitochondria, enabling fluctuations in survival signals to finely adjust apoptotic sensitivity. PMID:23375500

  15. Bax-PGAM5L-Drp1 complex is required for intrinsic apoptosis execution.

    Science.gov (United States)

    Xu, Wenjuan; Jing, Linlin; Wang, Quanshi; Lin, Chung-Chih; Chen, Xiaoting; Diao, Jianxin; Liu, Yuanliang; Sun, Xuegang

    2015-10-06

    Intrinsic apoptosis eliminates cells with damaged DNA and cells with dysregulated expression of oncogene. PGAM5, a member of the phosphoglycerate mutase family, has two splicing variants: PGAM5L (the long form) and PGAM5S (the short form). It has been well established that PGAM5 is at the convergent point of multiple necrosis pathways. However, the role of PGAM5 in intrinsic apoptosis is still controversial. Here we report that the PGAM5L, but not PGAM5S is a prerequisite for the activation of Bax and dephosphorylation of Drp1 in arenobufagin and staurosporine induced intrinsic apoptosis. Knockdown of PGAM5L inhibits the translocation of Bax to the mitochondria and reduces mitochondrial fission. The interaction between PGAM5L and Drp1 was observed in both arenobufagin and staurosporine treated HCT116 cells, but not in HCT116 Bax(-/-) cells. Bax transfection rescues the formation of the triplex in both arenobufagin and staurosporine stimulated HCT116 Bax(-/-) cells. Arenobufagin shows remarkable anti-cancer effects both in orthotropic and heterotropic CRC models and demonstrates less toxic effects as compared with that of cisplatin. Bax-PGAM5L-Drp1 complex is detected in arenobufagin and staurosporine treated CRC cells in vitro and in arenobufagin and cisplatin treated tumor in vivo as well. In summary, our results demonstrate that Bax-PGAM5L-Drp1 complex is required for intrinsic apoptosis execution.

  16. Bcl-2/Bax protein and mRNA expression in yak (Bos grunniens) placentomes.

    Science.gov (United States)

    Fan, JiangFeng; Yu, SiJiu; Cui, Yan; Xu, Gengquan; Wang, Libin; Pan, Yangyang; He, Honghong

    2017-07-29

    Placental function is complex and influenced by various factors; furthermore, it depends on a delicate balance between cell proliferation, cell differentiation, and cell death. Bcl-2 and Bax proteins are key apoptosis regulators and are considered to play an important role in the maintenance of both dynamic balance and integrity of many tissues. Changes in Bcl-2 and Bax expressions have been described during different developmental stages in normal human placentas. Studies furthermore indicated several pathological placental changes to be related to abnormal Bcl-2 and Bax expressions. In the present study, we investigated both expression and distribution of Bcl-2 and Bax in yak placentas. For this, we collected placentas of 35 yaks at different stages of pregnancy as well as cotyledonary villi of four postpartum yaks. Protein and mRNA expressions of both Bcl-2 and Bax were investigated via immunohistochemistry, Western blot, and real-time PCR. Immunoreactive Bcl-2 protein was mainly localized near the fetal villous trophoblast at various gestational stages and post-partum. The Positive Index (PI) of Bcl-2 protein expression significantly decreased with increasing gestational age. Early during pregnancy (≤2 months), the Bax protein was widely distributed in the fetal villous trophoblast layer, the maternal caruncular crypt epithelium, and the stroma. Subsequently, the Bax protein distribution gradually concentrated in the fetal villous trophoblast layer. The staining intensity of Bax increased from the 3rd month to the prepartum of gestation. The PI reached a minimum of 9.4 ± 2.2 in fetal chorionic villi (FCV) and 1.3 ± 0.8 in maternal caruncular crypts (MCC) of the three months group. Both Bcl-2 and Bax had maximum immunoreactivity in the fetal villous trophoblast layer of placentas collected form postpartum yaks (with PIs of 36.6 ± 5.7 and 38.2 ± 4.8, respectively). Protein and mRNA expression of Bcl-2 and Bax investigated via Western blot and real

  17. Killing of Brain Tumor Cells by Hypoxia-Responsive Element Mediated Expression of BAX

    Directory of Open Access Journals (Sweden)

    Hangjun Ruan

    1999-11-01

    Full Text Available The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE, which can be activated through hypoxia-inducible factor-1 (HIF-1. We transfected plasmids containing multiple copies of HIRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HIRE copy number, and the degree of hypoxia.

  18. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells.

    Science.gov (United States)

    Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  19. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Guomin Niu; Songmei Yin; Shuangfeng Xie; Yiqing Li; Danian Nie; Liping Ma; Xiuju Wang; Yudan Wu

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavo-nol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  20. A non-apoptotic role for BAX and BAK in eicosanoid metabolism

    Science.gov (United States)

    Zhang, Tejia; Walensky, Loren D.; Saghatelian, Alan

    2015-01-01

    BCL-2 proteins are key regulators of programmed cell death. The interplay between pro- and anti-apoptotic BCL-2 members has important roles in many cancers. In addition to their apoptotic function, recent evidence supports key non-apoptotic roles for several BCL-2 proteins. We used an unbiased lipidomics strategy to reveal that the pro-apoptotic proteins BAX, and to a lesser extent BAK, regulate the cellular inflammatory response by mediating COX-2 expression and prostaglandin biosynthesis. COX-2 upregulation in response to the bacterial endotoxin lipopolysaccharide is blunted in the absence of BAX, and Bax−/− mouse embryonic fibroblasts display altered kinetics of NFκB and MAPK signaling following endotoxin treatment. Our approach uncovers a novel, non-apoptotic function for BAX in regulation of the cellular inflammatory response and suggests that inflammation and apoptosis are more tightly connected than previously anticipated. PMID:25815636

  1. Expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins in human retinoblastoma.

    Science.gov (United States)

    Singh, Lata; Pushker, Neelam; Saini, Neeru; Sen, Seema; Sharma, Anjana; Bakhshi, Sameer; Chawla, Bhavna; Kashyap, Seema

    2015-04-01

    Regulation of apoptosis is a complex process that involves a number of genes, including Bcl-2, Bcl-x, Bax and other Bcl-2 family members. The aim of the present study is to assess the expression of Bcl- 2 and Bax in retinoblastoma, and correlate them with clinical and histopathological parameters. The expression of Bcl-2 and Bax proteins were examined using immunohistochemistry, Western blotting and reverse transcriptase-polymerase chain reaction in a series of 60 prospective cases of primary retinoblastoma tissues. Immunohistochemistry showed expression of Bcl-2 in 40/60 (66.6%), whereas Bax expression was found only in 18/60 (30%) cases, and these correlated with mRNA expression. The Western blotting results also correlated well with the immunohistochemical expression of Bcl-2 (25 kDa) and Bax (21 kDa) proteins. Bcl-2 was expressed in 96% (24/25) of invasive tumours and in 45.7% (16/35) of non-invasive tumours. Expression of Bcl-2 significantly correlated with tumour invasiveness (P = 0.0274) and poor differentiation (P = 0.0163), whereas loss of Bax correlated with massive choroidal invasion and Pathological Tumor-Node-Metastasis (pTNM) (P = 0.0341). However, no correlation was found between Bax and Bcl-2 expression. Our findings suggest that these apoptotic regulatory proteins may serve as poor prognostic markers and can be used as a therapeutic target for the treatment of invasive retinoblastoma. Further functional studies are required to explore the role of Bax and Bcl-2 in retinoblastoma. © 2014 Royal Australian and New Zealand College of Ophthalmologists.

  2. Glycosylation regulates prestin cellular activity.

    Science.gov (United States)

    Rajagopalan, Lavanya; Organ-Darling, Louise E; Liu, Haiying; Davidson, Amy L; Raphael, Robert M; Brownell, William E; Pereira, Fred A

    2010-03-01

    Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by

  3. NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

    Directory of Open Access Journals (Sweden)

    Molouki Aidin

    2012-08-01

    Full Text Available Abstract Background Recently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV, the matrix (M protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV. Finding In order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10% at 48 h post-infection. In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells. Conclusion Although the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.

  4. Doxorubicin Changes Bax /Bcl-xL Ratio, Caspase-8 and 9 in Breast Cancer Cells.

    Science.gov (United States)

    Sharifi, Simin; Barar, Jaleh; Hejazi, Mohammad Saeid; Samadi, Nasser

    2015-09-01

    Doxorubicin is administrated as a single agent in first-line therapy of breast cancer to induce apoptosis in tumor cells. Bax, Bcl-xL, Caspase-8 and 9 proteins are involved in induction of apoptosis. The present study describes Bax, Bcl-xL gene expression and Caspase-8 and 9 protein levels in MCF-7 cells incubated with doxorubicin at different doses an incubation times. The cytotoxic effects of doxorubicin were studied using MTT assay. MCF-7 cells were treated with three concentrations of doxorubicin (0.1, 0.5, 1 μM) and incubated for 24, 48 and 72 hours then expression levels of Bax and Bcl-xL genes were elucidated by Real-time RT-PCR technique and protein levels of caspase-8 and caspase-9 proteins were measured using ELISA method. Morphological modifications of the cells were also monitored via light microscopic images. Doxorubicin decreased the anti-apoptotic Bcl-xL and increased pro-apoptotic Bax mRNA levels. Doxorubicin induced a significant increase in Bax /Bcl-xL ratio in all doses and incubation times (pBax /Bcl-xL ratio was revealed after 48 h incubation of the cells with in all doses of doxorubicin. Doxorubicin also increased caspase-9 level in a time and dose-dependent manner, while caspase-8 level didn't follow time and dose dependency pattern. Our results confirm that doxorubicin induces mitochondrial-dependent apoptosis by down-regulation of Bcl-xL and up- regulation of Bax and caspase-9 expressions.

  5. A Single Nucleotide Polymorphism in the Bax Gene Promoter Affects Transcription and Influences Retinal Ganglion Cell Death

    Directory of Open Access Journals (Sweden)

    Sheila J Semaan

    2010-03-01

    Full Text Available Pro-apoptotic Bax is essential for RGC (retinal ganglion cell death. Gene dosage experiments in mice, yielding a single wild-type Bax allele, indicated that genetic background was able to influence the cell death phenotype. DBA/2J Bax+/− mice exhibited complete resistance to nerve damage after 2 weeks (similar to Bax −/− mice, but 129B6 Bax+/− mice exhibited significant cell loss (similar to wild-type mice. The different cell death phenotype was associated with the level of Bax expression, where 129B6 neurons had twice the level of endogenous Bax mRNA and protein as DBA/2J neurons. Sequence analysis of the Bax promoters between these strains revealed a single nucleotide polymorphism (T129B6 to CDBA/2J at position −515. A 1.5- to 2.5-fold increase in transcriptional activity was observed from the 129B6 promoter in transient transfection assays in a variety of cell types, including RGC5 cells derived from rat RGCs. Since this polymorphism occurred in a p53 half-site, we investigated the requirement of p53 for the differential transcriptional activity. Differential transcriptional activity from either 129B6 or DBA/2J Bax promoters were unaffected in p53−/− cells, and addition of exogenous p53 had no further effect on this difference, thus a role for p53 was excluded. Competitive electrophoretic mobility-shift assays identified two DNA-protein complexes that interacted with the polymorphic region. Those forming Complex 1 bound with higher affinity to the 129B6 polymorphic site, suggesting that these proteins probably comprised a transcriptional activator complex. These studies implicated quantitative expression of the Bax gene as playing a possible role in neuronal susceptibility to damaging stimuli.

  6. Loss of BAX by miR-365 Promotes Cutaneous Squamous Cell Carcinoma Progression by Suppressing Apoptosis.

    Science.gov (United States)

    Zhou, Liang; Gao, Ruirui; Wang, Yinghui; Zhou, Meijuan; Ding, Zhenhua

    2017-05-30

    Pro-apoptotic BCL2 associated X (BAX) is traditionally thought to be regulated by anti-apoptotic BCL-2 family members, like BCL2-like 1 (BCL-XL), at the protein level. However, the posttranscriptional regulation of BAX is under explored. In this study, we identified BAX as the novel downstream target of miR-365, which is supported by gain- and loss-of-function studies of onco-miR-365. Loss of BAX by either RNA interference or highly-expressed miR-365 in cells of cutaneous squamous cell carcinoma (CSCC) enhanced the tumor resistance against apoptosis, while repressing cell proliferation, migration, and invasiveness. In vivo experiment confirmed that BAX knockdown promotes the growth of CSCC xenografts. Collectively, our results find a miR-365-BAX axis for alleviating the pro-apoptotic effects of BAX, which promotes CSCC development and may facilitate the generation of novel therapeutic regimens to the clinical treatment of CSCC.

  7. Regulation of p21ras activity

    DEFF Research Database (Denmark)

    Lowy, D R; Zhang, K; DeClue, J E

    1992-01-01

    The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control...... mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange....

  8. A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells.

    Science.gov (United States)

    Huang, Li; Han, Junjie; Ben-Hail, Danya; He, Luwei; Li, Baowei; Chen, Ziheng; Wang, Yueying; Yang, Yanlei; Liu, Lei; Zhu, Yushan; Shoshan-Barmatz, Varda; Liu, Hongwei; Chen, Quan

    2015-09-25

    The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak.

  9. A New Fungal Diterpene Induces VDAC1-dependent Apoptosis in Bax/Bak-deficient Cells*

    Science.gov (United States)

    Huang, Li; Han, Junjie; Ben-Hail, Danya; He, Luwei; Li, Baowei; Chen, Ziheng; Wang, Yueying; Yang, Yanlei; Liu, Lei; Zhu, Yushan; Shoshan-Barmatz, Varda; Liu, Hongwei; Chen, Quan

    2015-01-01

    The pro-apoptotic Bax and Bak proteins are considered central to apoptosis, yet apoptosis occurs in their absence. Here, we asked whether the mitochondrial protein VDAC1 mediates apoptosis independently of Bax/Bak. Upon screening a fungal secondary metabolite library for compounds inducing apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts, we identified cyathin-R, a new cyathane diterpenoid compound able to activate apoptosis in the absence of Bax/Bak via promotion of the VDAC1 oligomerization that mediates cytochrome c release. Diphenylamine-2-carboxilic acid, an inhibitor of VDAC1 conductance and oligomerization, inhibited cyathin-R-induced VDAC1 oligomerization and apoptosis. Similarly, Bcl-2 overexpression conferred resistance to cyathin-R-induced apoptosis and VDAC1 oligomerization. Silencing of VDAC1 expression prevented cyathin-R-induced apoptosis. Finally, cyathin-R effectively attenuated tumor growth and induced apoptosis in Bax/Bak-deficient cells implanted into a xenograft mouse model. Hence, this study identified a new compound promoting VDAC1-dependent apoptosis as a potential therapeutic option for cancerous cells lacking or presenting inactivated Bax/Bak. PMID:26253170

  10. [Molecular mechanisms regulating the activity of macrophages].

    Science.gov (United States)

    Onoprienko, L V

    2011-01-01

    This article reviews modern concepts of the most common types of macrophage activation: classical, alternative, and type II. Molecular mechanisms of induction and regulation of these three types of activation are discussed. Any population of macrophages was shown to change its properties depending on its microenvironment and concrete biological situation (the "functional plasticity of macrophages"). Many intermediate states of macrophages were described along with the most pronounced and well-known activation types (classical activation, alternative activation, and type II activation). These intermediate states are characterized by a variety of combinations of their biological properties, including elements of the three afore mentioned types of activation. Macrophage activity is regulated by a complex network of interrelated cascade mechanisms.

  11. Regulation of ROCK Activity in Cancer

    DEFF Research Database (Denmark)

    Morgan-Fisher, Marie; Wewer, Ulla M; Yoneda, Atsuko

    2013-01-01

    , these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer.......Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key...... regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active...

  12. Bcl-xS and Bax induce different apoptotic pathways in PC12 cells.

    Science.gov (United States)

    Lindenboim, L; Yuan, J; Stein, R

    2000-03-30

    Apoptosis is regulated by the action of the Bcl-2 family of proteins, which includes anti- and pro-apoptotic members such as Bcl-xS and Bax. These proteins may differ from each other in structure, mechanism of action and interactions with anti-apoptotic signaling. The mechanism whereby Bax induces cell death has been studied in some cellular systems, but the mechanism of Bcl-xS-induced apoptosis is largely unknown. In this study we investigated and compared the apoptotic effects of Bcl-xS and Bax in the pheochromocytoma cell line, PC12 (a useful model system for studying neuronal apoptosis), and the extent to which they are protected by the survival factor, nerve growth factor (NGF). PC12 cells express endogenous Bcl-xS, Bax and Bcl-xL proteins. Subcellular fractionation revealed that Bax is presented mainly in the cytosolic and the heavy membrane fractions, Bcl-xS is present only in the cytosol, and the anti-apoptotic protein Bcl-xL is located mainly in the heavy membrane fraction. In contrast to the cytosolic localization of endogenous Bcl-xS, the exogenously overexpressed Bcl-xS is localized to the mitochondria. Overexpression of Bcl-xS or Bax induces cell death in the transfected cells. The cell death induced by overexpression of Bcl-xS was inhibited by coexpression of Bcl-xS with Bcl-2 or Bcl-xL, or by treatment with the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (Z-VAD-FMK) or with NGF. The Bcl-2 mutants deltaC22, which lacks the transmembrane domain, and G145A (mI-3) were able to inhibit the death-inducing effect of Bcl-xS. These results therefore suggest that the apoptotic pathway induced by overexpression of Bcl-xS in PC12 cells can be controlled by Bcl-2 and Bcl-xL, is mediated by caspases, and can be inhibited by the NGF signaling pathway. The Bax-induced cell death was inhibited by co-expression of Bax with Bcl-2 or Bcl-xL, but was not inhibited by Z-VAD-FMK, NGF, or the Bcl-2 ml-3 or deltaC22 mutants. These

  13. Effect of β Radiation on Bcl-2 and Bax Expressions in Benign Prostate Hyperplasia Tissues

    Institute of Scientific and Technical Information of China (English)

    MA Qing-jie; GAO Shi; ZHAO Jie; GU Xin-quan; CAI Shan-yu; ZHAO Guo-qing

    2008-01-01

    The authors chose specimens from nine normal prostate tissues(NP group),15 benign prostate hyperplasia(BPH) prostates(BPH group),and 35 BPH prostates that had been treated with 90Sr/90Y Prostatic Hyperplasia Applicator(exposure group),The expressions of bcl-2 and bax in stroma and epithelia of prostate tissues were demonstrated by means of immunohistochemical staining,and the staining positive rate was semiquantatively determined,so as to observe the expression of bcl-2 and bax genes in the prostate tissues of normal individuals and BPH patients,before and after β radiation,and to evaluate the influence of β radiation on bcl-2 and bax expressions,The expressions of gene bcl-2 in the prostate epithelia of NP and BPH are significantly higher than those in the prostate stroma(P<0.01),However,the expressions of bcl-2 in the prostate epithelia and stroma of the BPH group are obviously higher than those in the NP group(P<0.01),The expression of gene bax in the prostate epithelia of the NP group is higher than that in the BPH group(P<0.05),However,bcl-2 expressions in the prostate epithelia and stroma of the BPH group are significantly higher than the bax expressions(P<0.01),Compared with those of the NP group,the expressions of bcl-2 in the prostate epithelia and stroma of the exposure group decrease remarkably,even as the expressions of the bax notably increase(P<0.01),Thus,the administration of β radiation can remarkably affect bcl-2 and bax gene expressions,to regulate cell apoptosis,in the prostate tissues of BPH.

  14. Traveling Bax and Forth from Mitochondria to Control Apoptosis

    Science.gov (United States)

    Soriano, Maria Eugenia; Scorrano, Luca

    2011-01-01

    Antiapoptotic Bcl-2 proteins on mitochondria inhibit prodeath proteins, such as Bax, which are found primarily in the cytosol. In this issue, Edlich et al., (2011) show that Bax and Bcl-xL interact on the mitochondrial surface and then retrotranslocate to the cytosol, effectively preventing Bax-induced permeabilization of mitochondria. PMID:21458662

  15. The Proapoptotic Bcl-2 Protein Bax Plays an Important Role in the Pathogenesis of Reovirus Encephalitis ▿

    Science.gov (United States)

    Berens, Heather M.; Tyler, Kenneth L.

    2011-01-01

    Encephalitis induced by reovirus serotype 3 (T3) strains results from the apoptotic death of infected neurons. Extrinsic apoptotic signaling is activated in reovirus-infected neurons in vitro and in vivo, but the role of intrinsic apoptosis signaling during encephalitis is largely unknown. Bax plays a key role in intrinsic apoptotic signaling in neurons by allowing the release of mitochondrial cytochrome c. We found Bax activation and cytochrome c release in neurons following infection of neonatal mice with T3 reoviruses. Bax−/− mice infected with T3 Abney (T3A) have reduced central nervous system (CNS) tissue injury and decreased apoptosis, despite viral replication that is similar to that in wild-type (WT) Bax+/+ mice. In contrast, in the heart, T3A-infected Bax−/− mice have viral growth, caspase activation, and injury comparable to those in WT mice, indicating that the role of Bax in pathogenesis is organ specific. Nonmyocarditic T3 Dearing (T3D)-infected Bax−/− mice had delayed disease and enhanced survival compared to WT mice. T3D-infected Bax−/− mice had significantly lower viral titers and levels of activated caspase 3 in the brain despite unaffected transneuronal spread of virus. Cytochrome c and Smac release occurred in some reovirus-infected neurons in the absence of Bax; however, this was clearly reduced compared to levels seen in Bax+/+ wild-type mice, indicating that Bax is necessary for efficient activation of proapoptotic mitochondrial signaling in infected neurons. Our studies suggest that Bax is important for reovirus growth and pathogenesis in neurons and that the intrinsic pathway of apoptosis, mediated by Bax, is important for full expression of disease, CNS tissue injury, apoptosis, and viral growth in the CNS of reovirus-infected mice. PMID:21307199

  16. Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle.

    Science.gov (United States)

    Zhang, Yuan; Iqbal, Sobia; O'Leary, Michael F N; Menzies, Keir J; Saleem, Ayesha; Ding, Shuzhe; Hood, David A

    2013-09-01

    The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.

  17. Ghrelin Administration Increases the Bax/Bcl-2 Gene Expression Ratio in the Heart of Chronic Hypoxic Rats.

    Science.gov (United States)

    Aliparasti, Mohammad Reza; Alipour, Mohammad Reza; Almasi, Shohreh; Feizi, Hadi

    2015-06-01

    Programmed cell death or apoptosis, is a biochemical procedure that initiates due to some conditions, including hypoxia. Bax and Bcl-2 are among the agents that regulate apoptosis. The amplification of the first one triggers the initiation of apoptosis, and the second one prevents it. Ghrelin is an endogenous peptide that antiapoptosis is its new effect. The aim of this study is to examine the effect of ghrelin on the Bax/Bcl-2 ratio. Twenty four wistar rats were divided randomly in three groups; control, hypoxic + saline and hypoxic + ghrelin. Hypoxic animals lived in O2 11% for 2 weeks and received either saline or ghrelin subcutaneously daily. The bax and Bcl-2 gene expression were measured by Real-Time RT-PCR. Chronic hypoxia increased the Bax gene expression significantly compared with normal animals (P = 0.008), but the Bcl-2 was not affected by hypoxia. The Bax/Bcl-2 ratio also amplified significantly (P=0.005). Ghrelin administration significantly increased the Bax/Bcl-2 ratio in the hypoxic animals compared to the hypoxic + saline and normal groups (p=0.042 and P= 0.001, respectively). In the present study, animals' treatment with ghrelin leads to an increment of Bax/Bcl-2 ratio, which indicates a controversy related to cardioprotection of ghrelin.

  18. Ghrelin Administration Increases the Bax/Bcl-2 Gene Expression Ratio in the Heart of Chronic Hypoxic Rats

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Aliparasti

    2015-06-01

    Full Text Available Purpose: Programmed cell death or apoptosis, is a biochemical procedure that initiates due to some conditions, including hypoxia. Bax and Bcl-2 are among the agents that regulate apoptosis. The amplification of the first one triggers the initiation of apoptosis, and the second one prevents it. Ghrelin is an endogenous peptide that antiapoptosis is its new effect. The aim of this study is to examine the effect of ghrelin on the Bax/Bcl-2 ratio. Methods: Twenty four wistar rats were divided randomly in three groups; control, hypoxic + saline and hypoxic + ghrelin. Hypoxic animals lived in O2 11% for 2 weeks and received either saline or ghrelin subcutaneously daily. The bax and Bcl-2 gene expression were measured by Real-Time RT-PCR. Results: Chronic hypoxia increased the Bax gene expression significantly compared with normal animals (P = 0.008, but the Bcl-2 was not affected by hypoxia. The Bax/Bcl-2 ratio also amplified significantly (P=0.005. Ghrelin administration significantly increased the Bax/Bcl-2 ratio in the hypoxic animals compared to the hypoxic + saline and normal groups (p=0.042 and P= 0.001, respectively. Conclusion: In the present study, animals’ treatment with ghrelin leads to an increment of Bax/Bcl-2 ratio, which indicates a controversy related to cardioprotection of ghrelin.

  19. Ghrelin Administration Increases the Bax/Bcl-2 Gene Expression Ratio in the Heart of Chronic Hypoxic Rats

    Science.gov (United States)

    Aliparasti, Mohammad Reza; Alipour, Mohammad Reza; Almasi, Shohreh; Feizi, Hadi

    2015-01-01

    Purpose: Programmed cell death or apoptosis, is a biochemical procedure that initiates due to some conditions, including hypoxia. Bax and Bcl-2 are among the agents that regulate apoptosis. The amplification of the first one triggers the initiation of apoptosis, and the second one prevents it. Ghrelin is an endogenous peptide that antiapoptosis is its new effect. The aim of this study is to examine the effect of ghrelin on the Bax/Bcl-2 ratio. Methods: Twenty four wistar rats were divided randomly in three groups; control, hypoxic + saline and hypoxic + ghrelin. Hypoxic animals lived in O2 11% for 2 weeks and received either saline or ghrelin subcutaneously daily. The bax and Bcl-2 gene expression were measured by Real-Time RT-PCR. Results: Chronic hypoxia increased the Bax gene expression significantly compared with normal animals (P = 0.008), but the Bcl-2 was not affected by hypoxia. The Bax/Bcl-2 ratio also amplified significantly (P=0.005). Ghrelin administration significantly increased the Bax/Bcl-2 ratio in the hypoxic animals compared to the hypoxic + saline and normal groups (p=0.042 and P= 0.001, respectively). Conclusion: In the present study, animals’ treatment with ghrelin leads to an increment of Bax/Bcl-2 ratio, which indicates a controversy related to cardioprotection of ghrelin. PMID:26236657

  20. Effects of cadmium on Bcl-2/ Bax expression ratio in rat cortex brain and hippocampus.

    Science.gov (United States)

    Mahdavi, S; Khodarahmi, P; Roodbari, N H

    2017-01-01

    To investigate the underlying mechanism of neurotoxicity of cadmium, we examined the effects of intraperitoneal injection of cadmium on messenger RNA (mRNA) expression of Bcl-2 (B-cell lymphoma 2) and Bax (Bcl2-associated x) genes and caspase-3/7 activation in rat hippocampus and frontal cortex. Twenty-eight male Wistar rats weighing 200-250 g were randomly divided into four groups. Control group received saline and three other groups received cadmium at doses of 1, 2 and 4 mg/kg (body weight) for 15 successive days. One day after the last injection, the hippocampus and frontal cortex were dissected and removed and then the expression of Bcl-2 and Bax genes was evaluated using real-time polymerase chain reaction and apoptotic studies was done using caspase-3/7 activation assay. Cadmium reduced the mRNA level of Bcl-2 in the control group at doses of 1 ( p Bax increased significantly compared to the control group at the doses of 1 ( p Bax was increased significantly compared to the control group at the doses of 2 and 4 mg/kg ( p Bax mRNA ratio induces cell apoptosis. Apoptotic effect of cadmium may be through the mitochondrial pathway by the activation of caspase-3/7.

  1. Molecular regulation of telomerase activity in aging

    Institute of Scientific and Technical Information of China (English)

    Craig Nicholls; He Li; Jian-Qiu Wang; Jun-Ping Liu

    2011-01-01

    The process of aging is mitigated by the maintenance and repair of chromosome ends (telomeres),resulting in extended lifespan.This review examines the molecular mechanisms underlying the actions and regulation of the enzyme telomerase reverse transcriptase (TERT),which functions as the primary mechanism of telomere maintenance and regulates cellular life expectancy.Underpinning increased cell proliferation,telomerase is also a key factor in facilitating cancer cell immortalization.The review focuses on aspects of hormonal regulations of telomerase,and the intraceilular pathways that converge to regulate telomerase activity with an emphasis on molecular interactions at protein and gene levels.In addition,the basic structure and function of two key telomerase enzyme components-the catalytic subunit TERT and the template RNA (TERC) are discussed briefly.

  2. Expression of the EP300, TP53 and BAX genes in colorectal cancer: Correlations with clinicopathological parameters and survival.

    Science.gov (United States)

    Kowalczyk, Anna E; Krazinski, Bartlomiej E; Godlewski, Janusz; Kiewisz, Jolanta; Kwiatkowski, Przemyslaw; Sliwinska-Jewsiewicka, Agnieszka; Kiezun, Jacek; Sulik, Marian; Kmiec, Zbigniew

    2017-07-01

    E1A binding protein P300 (EP300), tumor protein P53 (TP53) and BCL2 associated X, apoptosis regulator (BAX) genes encode proteins which cooperate to regulate important cellular processes. The present study aimed to determine the expression levels of EP300, TP53 and BAX in colorectal cancer (CRC) and to investigate their prognostic value and association with the progression of CRC. Tumor and matched unchanged colorectal tissues were collected from 121 CRC patients. Quantitative polymerase chain reaction and immunohistochemistry were used to assess the mRNA and protein levels of the studied genes. Altered expression of the studied genes in CRC tissues was observed at both the mRNA and protein levels. The depth of invasion was associated with TP53 mRNA levels and was correlated negatively with BAX mRNA expression. Moreover, a relationship between tumor location and BAX mRNA content was noted. BAX immunoreactivity was correlated positively with the intensity of p300 immunostaining and was associated with lymph node involvement and tumor-node-metastasis (TNM) disease stage. Univariate regression analysis revealed that overexpression of p53 and BAX in CRC tissues was associated with poor patient outcome. In conclusion, dysregulation of the expression of the studied genes was found to contribute to CRC pathogenesis. The association between p300 and BAX levels suggests the existence of an interdependent regulatory mechanism of their expression. Moreover, BAX expression may be regulated alternatively, in a p53-independent manner, since the lack of correlations between expression of these factors was observed.

  3. Expression and Prognostic Significance of EP300, TP53 and BAX in Clear Cell Renal Cell Carcinoma.

    Science.gov (United States)

    Godlewski, Janusz; Krazinski, Bartlomiej E; Kowalczyk, Anna E; Kiewisz, Jolanta; Kiezun, Jacek; Kwiatkowski, Przemyslaw; Sliwińska-Jewsiewicka, Agnieszka; Wierzbicki, Piotr W; Kmieć, Zbigniew

    2017-06-01

    Histone acetyltransferase E1A-binding protein p300 (EP300), tumor protein p53 (TP53) and B-cell lymphoma-2-associated X protein (BAX) contribute to the regulation of the cell cycle and apoptosis, cellular processes that are often impaired in cancer cells. The aim of this study was to determine the expression levels of EP300, TP53 and BAX genes and their respective proteins in clear cell renal cell carcinoma (ccRCC) and evaluate the value of these factors as prognostic factors. EP300, TP53 and BAX expression at the transcript and protein levels were determined by quantitative polymerase-chain reaction (QPCR) and immunohistochemistry (IHC) in paired tumor and kidney specimens from 31 patients with ccRCC. Levels of EP300, TP53 and BAX transcripts were found increased in tumor tissues. Immunoreactivity for TP53 was elevated in cancer cells when compared to unchanged kidney, while EP300 and BAX immunoexpression in ccRCC did not differ from that of normal renal tissue. Immunoreactivity for TP53 was positively associated with larger tumor size. In contrast, stronger BAX immunoexpression correlated with smaller tumor diameters. The average immunoreactivity for BAX was higher in localized, kidney-confined tumor than in advanced/recurrent tumors. None of the analyzed transcripts or proteins correlated with the overall survival of patients. Although TP53 and BAX immunoreactivity levels were associated with some clinicopathological parameters of the patients, the expression of EP300, TP53 and BAX did not reveal any prognostic significance in ccRCC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  4. Regulation of ROCK Activity in Cancer

    Science.gov (United States)

    Morgan-Fisher, Marie; Wewer, Ulla M.

    2013-01-01

    Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)–loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer. PMID:23204112

  5. Low levels of Bax inhibitor-1 gene expression increase tunicamycin-induced apoptosis in human neuroblastoma SY5Y cells

    Institute of Scientific and Technical Information of China (English)

    Dan Wu; Peirong Wang; Shiyao Wang

    2012-01-01

    A human SH-SY5Y neuroblastoma cell line with a low level of Bax inhibitor-1 expression was established by lentivirus-mediated RNA interference and fluorescence-activated cell sorting. In control SH-SY5Y cells, tunicamycin treatment induced endoplasmic reticulum stress-mediated apoptosis; however, after Bax inhibitor-1 gene knockdown, cell survival rates were significantly decreased and the degree of apoptosis was significantly increased following tunicamycin treatment. In addition, chromatin condensation and apparent apoptotic phenomena, such as marginalization and cytoplasmic vesicles, were observed. Our findings indicate that Bax inhibitor-1 can delay apoptosis induced by endoplasmic reticulum stress.

  6. 前列腺癌组织凋亡蛋白酶活化因子1和bax的表达及其临床意义%Expression and clinical significance of apoptotic protease activating factor 1 and bax in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    张丙信; 赵霞; 张金库; 贾文文; 张建树; 孙建梅; 贾喜花

    2016-01-01

    Objective To investigate the expression and clinical significance of apoptotic protease activating factor 1 (Apaf-1) and bax in prostate cancer (PCa) and benign prostatic hyperplasia (BPH). Methods Immunohistochemistry was used to detect the expression of Apaf-1 and bax in the tissues from 45 PCa patients and 60 BPH patients. Results The positive rates of Apaf-1 and bax in PCa tissues were 22.22%(10/45) and 20.00 % (9/45), respectively, while those in BPH tissues were 48.33 % (29/60) and 46.67 % (28/60). There was a statistically significant difference in the expressions of Apaf-1 and bax between two groups (P0.05), but they were correlated with the pathological grade and clinical stage of PCa (P< 0.05). The expressions of Apaf-1 and bax in PCa tissues were lower than those in BPH tissues. There was a positive correlation between the expression of Apaf-1 and bax (r=0.535, P<0.01). Conclusion Apaf-1 and bax might be correlated with the carcinogenesis and development of PCa.%目的:探讨凋亡蛋白酶活化因子1(Apaf-1)和bax在前列腺癌和前列腺增生组织中的表达及其意义。方法采用免疫组织化学法检测45例前列腺癌患者和60例前列腺增生患者Apaf-1和bax的表达。结果前列腺癌中Apaf-1和bax的阳性率分别为22.22%(10/45)、20.00%(9/45),低于前列腺增生组织的48.33%(29/60)、46.67%(28/60),差异均有统计学意义(χ2值分别为7.509、8.013,均P<0.05)。 Apaf-1和bax的表达和前列腺癌患者年龄及是否远处转移无关(P>0.05),与病理分级及临床分期相关(P<0.05),Apaf-1和bax在前列腺癌组织中的表达呈正相关性(r=0.535, P<0.001)。结论 Apaf-1和bax可能与前列腺癌的发生、发展密切相关。

  7. Piracetam ameliorated oxygen and glucose deprivation-induced injury in rat cortical neurons via inhibition of oxidative stress, excitatory amino acids release and P53/Bax.

    Science.gov (United States)

    He, Zhi; Hu, Min; Zha, Yun-hong; Li, Zi-cheng; Zhao, Bo; Yu, Ling-ling; Yu, Min; Qian, Ying

    2014-05-01

    Our previous work has demonstrated that piracetam inhibited the decrease in amino acid content induced by chronic hypoperfusion, ameliorated the dysfunction of learning and memory in a hypoperfusion rat model, down-regulated P53, and BAX protein, facilitated the synaptic plasticity, and may be helpful in the treatment of vascular dementia. To explore the precise mechanism, the present study further evaluated effects of piracetam on Oxygen and glucose deprivation (OGD)-induced neuronal damage in rat primary cortical cells. The addition of piracetam to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release experiments. Piracetam also lowered the levels of malondialdehyde, nitrogen monoxidum, and xanthine oxidase which was increased in the OGD cells, and enhanced the activities of superoxide dismutase and glutathione peroxidase, which were decreased in the OGD cells. We also demonstrated that piracetam could decrease glutamate and aspartate release when cortical cells were subjected to OGD. Furthermore, Western blot study demonstrated that piracetam attenuated the increased expression of P53 and BAX protein in OGD cells. These observations demonstrated that piracetam reduced OGD-induced neuronal damage by inhibiting the oxidative stress and decreasing excitatory amino acids release and lowering P53/Bax protein expression in OGD cells.

  8. Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

    2010-06-15

    CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation.

  9. Bax is not involved in the resveratrol-induced apoptosis in human lung adenocarcinoma cells

    Science.gov (United States)

    Zhang, Wei-wei; Wang, Zhi-ping; Chen, Tong-sheng

    2010-02-01

    Resveratrol (RV) is a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts. Previous studies indicate that RV has an ability to inhibit various stages of carcinogenesis and eliminate preneoplastic cells in vitro and in vivo. However, little is known about the molecular mechanism of RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cell. In this report, we analyzed whether Bax translocation from cytoplasm to mitochondria during RV-induced apoptosis in single living cell using onfocal microscopey. Cells were transfected with GFP-Bax plasmid. Cell counting kit (CCK-8) assay was used to assess the inhibition of RV on the cells viability. Apoptotic activity of RV was detected by Hoechst 33258 and propidium iodide (PI) staining. Our results showed that RV induced a dose-dependent apoptosis in which Bax did not translocate to mitochondrias.

  10. Regulation of ROS in transmissible gastroenteritis virus-activated apoptotic signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Li [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158 (China); Zhao, Xiaomin; Huang, Yong; Du, Qian; Dong, Feng; Zhang, Hongling; Song, Xiangjun; Zhang, Wenlong [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China)

    2013-12-06

    Highlights: •TGEV infection induced ROS accumulation. •ROS accumulation is involved in TGEV-induced mitochondrial integrity impairment. •ROS is associated with p53 activation and apoptosis occurrence in TGEV-infected cells. -- Abstract: Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, causes severe lethal watery diarrhea and dehydration in piglets. Previous studies indicate that TGEV infection induces cell apoptosis in host cells. In this study, we investigated the roles and regulation of reactive oxygen species (ROS) in TGEV-activated apoptotic signaling. The results showed that TGEV infection induced ROS accumulation, whereas UV-irradiated TGEV did not promote ROS accumulation. In addition, TGEV infection lowered mitochondrial transmembrane potential in PK-15 cell line, which could be inhibited by ROS scavengers, pyrrolidinedithiocarbamic (PDTC) and N-acetyl-L-cysteine (NAC). Furthermore, the two scavengers significantly inhibited the activation of p38 MAPK and p53 and further blocked apoptosis occurrence through suppressing the TGEV-induced Bcl-2 reduction, Bax redistribution, cytochrome c release and caspase-3 activation. These results suggest that oxidative stress pathway might be a key element in TGEV-induced apoptosis and TGEV pathogenesis.

  11. Regulators of Slc4 bicarbonate transporter activity

    Directory of Open Access Journals (Sweden)

    Ian M. Thornell

    2015-06-01

    Full Text Available The Slc4 family of transporters is comprised of anion exchangers (AE1-4, Na-coupled bicarbonate transporters (NCBTs including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2, electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2, and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE, as well as a borate transporter (BTR1. These transporters regulate intracellular pH (pHi and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO3– either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO3– transporter contributes to a cell’s ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s (e.g., Na+ or Cl–. In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both classical and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family.

  12. BAX, a novel cell pro-apoptotic protein, involved in hemocytes early antiviral immune response in fresh water crayfish, Procambarus clarkii.

    Science.gov (United States)

    du, Zhi-Qiang

    2016-08-01

    Apoptosis plays an important role in various biological processes and acts as a host defending mechanism by which infected cells are eliminated to restrict the virus propagation scale. Bax is a crucial pro-apoptotic protein, which mediates the release of cytochrome c from mitochondrion to cytosol in mammalian. However, its role in invertebrate is still obscure. Here, a novel pro-apoptotic protein gene was identified from hemocytes of red swamp crayfish. There was a Bcl-2 domain in the C-terminus of Pc-Bax, which possessed 497 amino acids residues. And an important transmembrane region existed in the C-terminus of Pc-Bax, which implied that Pc-Bax located in mitochondrial membrane. Besides, Pc-Bax was expressed at a relative high level in hemocytes, and a relative low expression levels in hepatopancreas, gills, and intestine. In hemocytes, Pc-Bax transcript was rapidly up-regulated from 12 h to 36 h after WSSV infection. And there was the same trend for Pc-Bax protein expression level in hemocytes after WSSV infection. Results of qRT-PCR testing for VP28 gene showed WSSV replication was obviously enhanced after Pc-Bax knockdown. Meantime, hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group. Taken together, these results revealed that crayfish hemocytes apoptosis scale was enhanced to suppress WSSV replication by up-regulating Bax protein expression level after WSSV infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Ack1: activation and regulation by allostery.

    Directory of Open Access Journals (Sweden)

    Ketan S Gajiwala

    Full Text Available The non-receptor tyrosine kinase Ack1 belongs to a unique multi-domain protein kinase family, Ack. Ack is the only family of SH3 domain containing kinases to have an SH3 domain following the kinase domain; others have their SH3 domains preceding the kinase domain. Previous reports have suggested that Ack1 does not require phosphorylation for activation and the enzyme activity of the isolated kinase domain is low relative to other kinases. It has been shown to dimerize in the cellular environment, which augments its enzyme activity. The molecular mechanism of activation, however, remains unknown. Here we present structural and biochemical data on Ack1 kinase domain, and kinase domain+SH3 domain that suggest that Ack1 in its monomeric state is autoinhibited, like EGFR and CDK. The activation of the kinase domain may require N-lobe mediated symmetric dimerization, which may be facilitated by the N-terminal SAM domain. Results presented here show that SH3 domain, unlike in Src family tyrosine kinases, does not directly control the activation state of the enzyme. Instead we speculate that the SH3 domain may play a regulatory role by facilitating binding of the MIG6 homologous region to the kinase domain. We postulate that features of Ack1 activation and regulation parallel those of receptor tyrosine kinase EGFR with some interesting differences.

  14. Bax-induced apoptosis shortens the life span of DNA repair defect Ku70-knockout mice by inducing emphysema.

    Science.gov (United States)

    Matsuyama, Shigemi; Palmer, James; Bates, Adam; Poventud-Fuentes, Izmarie; Wong, Kelvin; Ngo, Justine; Matsuyama, Mieko

    2016-06-01

    Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in aging. However, age-associated diseases, including emphysema and neurodegenerative disorders, are caused by apoptosis of lung alveolar epithelial cells and neurons, respectively. Therefore, enhanced apoptosis also promotes aging and shortens the life span depending on the cell type. Recently, we reported that ku70(-) (/) (-)bax(-) (/) (-) and ku70(-) (/) (-)bax(+/) (-) mice showed significantly extended life span in comparison with ku70(-) (/) (-)bax(+/+) mice. Ku70 is essential for non-homologous end joining pathway for DNA double strand break repair, and Bax plays an important role in apoptosis. Our study suggests that Bax-induced apoptosis has a significant impact on shortening the life span of ku70(-) (/) (-) mice, which are defective in one of DNA repair pathways. The lung alveolar space gradually enlarges during aging, both in mouse and human, and this age-dependent change results in the decrease of respiration capacity during aging that can lead to emphysema in more severe cases. We found that emphysema occurred in ku70(-) (/) (-) mice at the age of three-months old, and that Bax deficiency was able to suppress it. These results suggest that Bax-mediated apoptosis induces emphysema in ku70(-) (/) (-) mice. We also found that the number of cells, including bronchiolar epithelial cells and type 2 alveolar epithelial cells, shows a higher DNA double strand break damage response in ku70 KO mouse lung than in wild type. Recent studies suggest that non-homologous end joining activity decreases with increased age in mouse and rat model. Together, we hypothesize that the decline of Ku70-dependent DNA repair activity in lung alveolar epithelial cells is one of the causes of age-dependent decline of lung function resulting from excess Bax-mediated apoptosis of lung alveolar epithelial cells (and their

  15. Regulation of Aicda expression and AID activity.

    Science.gov (United States)

    Zan, Hong; Casali, Paolo

    2013-03-01

    Activation-induced cytidine deaminase (AID) is expressed in a B cell differentiation stage-specific fashion and is essential for immunoglobulin (Ig) gene class switch DNA recombination (CSR) and somatic hypermutation (SHM). CSR and SHM play a central role in the maturation of antibody and autoantibody responses. AID displays a mutagenic activity by catalyzing targeted deamination of deoxycytidine (dC) residues in DNA resulting in dU:dG mismatches, which are processed into point-mutations in SHM or double-strand breaks (DSBs) in CSR. Although AID specifically targets the Ig gene loci (IgH, Igκ and Igλ), it can also home into a wide array of non-Ig genes in B-and non-B-cell backgrounds. Aberrant expression of AID is associated with multiple diseases such as allergy, inflammation, autoimmunity and cancer. In autoimmune systemic lupus erythematosus, dysregulated AID expression underpins increased CSR, SHM and autoantibody production. As a potent mutator, AID is under stringent transcriptional, post-transcriptional and post-translational regulation. AID is also regulated in its targeting and enzymatic function. In resting naïve or memory B cells, AID transcripts and protein are undetectable. These, however, are readily and significantly up-regulated in B cells induced to undergo CSR and/or SHM. Transcription factors, such as HoxC4 and NF-κB, which are up-regulated in a B cell lineage-and/or differentiation stage-specific manner, regulate the induction of AID. HoxC4 induces AID expression by directly binding to the AID gene promoter through an evolutionarily conserved 5'-ATTT-3' motif. HoxC4 is induced by the same stimuli that induce AID and CSR. It is further up-regulated by estrogen through three estrogen responsive elements in its promoter region. The targeting of AID to switch (S) regions is mediated by 14-3-3 adaptor proteins, which specifically bind to 5'-AGCT-3' repeats that are exist at high frequency in S region cores. Like HoxC4, 14-3-3 adaptors are induced

  16. Modelling Proteasome and Proteasome Regulator Activities

    Directory of Open Access Journals (Sweden)

    Juliane Liepe

    2014-06-01

    Full Text Available Proteasomes are key proteases involved in a variety of processes ranging from the clearance of damaged proteins to the presentation of antigens to CD8+ T-lymphocytes. Which cleavage sites are used within the target proteins and how fast these proteins are degraded have a profound impact on immune system function and many cellular metabolic processes. The regulation of proteasome activity involves different mechanisms, such as the substitution of the catalytic subunits, the binding of regulatory complexes to proteasome gates and the proteasome conformational modifications triggered by the target protein itself. Mathematical models are invaluable in the analysis; and potentially allow us to predict the complex interactions of proteasome regulatory mechanisms and the final outcomes of the protein degradation rate and MHC class I epitope generation. The pioneering attempts that have been made to mathematically model proteasome activity, cleavage preference variation and their modification by one of the regulatory mechanisms are reviewed here.

  17. Phosphorylation regulates coilin activity and RNA association

    Directory of Open Access Journals (Sweden)

    Hanna J. Broome

    2013-02-01

    The Cajal body (CB is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation.

  18. Cytokinins are central regulators of cambial activity.

    Science.gov (United States)

    Matsumoto-Kitano, Miho; Kusumoto, Takami; Tarkowski, Petr; Kinoshita-Tsujimura, Kaori; Václavíková, Katerina; Miyawaki, Kaori; Kakimoto, Tatsuo

    2008-12-16

    The roots and stems of dicotyledonous plants thicken by the cell proliferation in the cambium. Cambial proliferation changes in response to environmental factors; however, the molecular mechanisms that regulate cambial activity are largely unknown. The quadruple Arabidopsis thaliana mutant atipt1;3;5;7, in which 4 genes encoding cytokinin biosynthetic isopentenyltransferases are disrupted by T-DNA insertion, was unable to form cambium and showed reduced thickening of the root and stem. The atipt3 single mutant, which has moderately decreased levels of cytokinins, exhibited decreased root thickening without any other recognizable morphological changes. Addition of exogenously supplied cytokinins to atipt1;3;5;7 reactivated the cambium in a dose-dependent manner. When an atipt1;3;5;7 shoot scion was grafted onto WT root stock, both the root and shoot grew normally and trans-zeatin-type (tZ-type) cytokinins in the shoot were restored to WT levels, but isopentenyladenine-type cytokinins in the shoot remained unchanged. Conversely, when a WT shoot was grafted onto an atipt1;3;5;7 root, both the root and shoot grew normally and isopentenyladenine-type cytokinins in the root were restored to WT levels, but tZ-type cytokinins were only partially restored. Collectively, it can be concluded that cytokinins are important regulators of cambium development and that production of cytokinins in either the root or shoot is sufficient for normal development of both the root and shoot.

  19. CONSTRUCTION OF RECOMBINANT PLASMID PIRES-BAX-HGF%pIRES-Bax-HGF重组质粒的构建

    Institute of Scientific and Technical Information of China (English)

    黄涛; 常青; 徐平

    2011-01-01

    Objective To construct the plasmid vector of pIRES-Bax-HGF. Methods The Bax gene sequence was cloned from ovarian cancer samples, and HGF gene sequence from pBluescript Ⅱ SK+HGF plasmid derived from ATCC. Bax and HGF coding sequence were inserted into pIRES plasmid by step double enzyme digestion. Results Enzyme digestion and sequencing indicated that the construction of recombinant pIRES-Bax-HGF plasmid was successful. Conclusion Obtaining the coding genes of both Bax and HGF, and successfully creating the plasmid vector of plRES-Bax-HGF establish a foundation for further study of the effects of HGF and Bax on vascular endothelial cells, smooth muscle cells, and fibrocytes, which steps out an important step to treat post-CABG restenosis at gene level.%目的 构建pIRES-Bax-HGF质粒载体.方法 采用RT-PCR方法从卵巢癌标本中克隆Bax的基因序列,从ATCC来源的pBlueseriptⅡ SK+HGF质粒中克隆HGF的基因序列.分步双酶切插入到plRES载体质粒中.结果 酶切鉴定及测序表明,重组pIRES-Bax-HGF质粒构建成功.结论 获取HGF及Bax编码基因并成功构建重组pIRES-Bax-HGF质粒载体,为进一步研究HGF及Bax对血管内皮细胞、平滑肌细胞、纤维细胞等的影响奠定了基础,也向基因水平干预冠状动脉旁路移植术术后再狭窄的形成迈出了重要一步.

  20. Resveratrol ameliorates hypoxia/ischemia-induced brain injury in the neonatal rat via the miR-96/Bax axis.

    Science.gov (United States)

    Bian, Hongen; Shan, Haijun; Chen, Tuanying

    2017-07-18

    This study was aimed to investigate the mechanism of resveratrol on amelioration of hypoxia/ischemia (H/I)-induced brain injury. The RT-PCR and western blot were used to detect the mRNA and protein expressions, respectively. The PC12 cell induced by OGD/R was as in vitro H/I brain injury model. The luciferase reporter assay was used to prove the relationship between Bax and miR-96, and the cell apoptosis was detected by MTT assay. The loss of MBP+ area in neonatal rats analyzed by immunohistochemistry was to evaluate the extent of brain injury. The miR-96 expression was decreased in the hippocampus and cerebral cortex of neonatal rats with H/I brain injury and the oxygenglucose deprivation/re-oxygenation (OGD/R)-induced PC12 cell, while Bax expression was opposite. And then the H/I rats and OGD/R-induced PC12 cell were treated with resveratrol (RSV); the results showed that the RSV could reverse the miR-96 and Bax expressions. Next, the luciferase reporter assay proved that Bax was a target of miR-96. We used the miR-96 inhibitor to suppress miR-96 expression in OGD/R-induced PC12 cell, and found that RSV regulated Bax expression and prevented OGD/R-induced PC12 cell apoptosis via miR-96. In addition, the immunohistochemistry was used to analyze the loss of MBP+ area in neonatal rats, and the result showed that the RSV significantly reduced the brain damage, increased miR-96 expression, and decreased Bax expression, while inhibition of miR-96 aggravated the brain damage and reversed the effect of RSV. Resveratrol ameliorates hypoxia/ischemia-induced brain injury in neonatal rat via the miR-96/ Bax axis.

  1. Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions.

    Science.gov (United States)

    Fan, Xiaorui; Xi, Huaming; Zhang, Zhen; Liang, Yajun; Li, Qinghong; He, Junping

    2017-04-01

    The aim of this study was to examine whether an elevated ambient temperature (37-40°C) had an effect on the apoptosis of germ cells and the expression of Bcl-2 and Bax in porcine testis. Six boars were used. Three boars were subjected to an elevated ambient temperature (37-40°C, 7days, 3h per day) as a heat stress (HS) group. The other 3 boars were kept in a room temperature house (20-27°C) as a control group. All boars were castrated and the testes were harvested. TUNEL assay was used for the detection of apoptotic cells. Immunohistochemistry, Western blotting and quantitative real-time PCR were used to analyze protein and mRNA levels of Bcl-2 and Bax in response to heat treatment. The results showed that apoptotic signals increased under heat stress conditions compared with the control (PBax protein and mRNA did not show significant changes between the control and experimental group. Low to moderate Bax immunoreactivity staining was observed in all kinds of germ cells in the control group. Strong staining was observed in spermatogonia, and low to moderate Bax staining was observed in spermatocytes and spermatids. A redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed in the spermatogonia, spermatocytes and spermatids obtained in the heat treated group. These results showed that elevated ambient temperatures induced germ cell apoptosis. In response to heat stress, the expression of Bcl-2 increased and a redistribution of Bax from a cytoplasmic to a perinuclear or nuclear localization. This indicates that Bcl-2 and Bax may be involved in regulation of germ cell apoptosis induced by heat stress in boars. Copyright © 2016. Published by Elsevier GmbH.

  2. The dynamics of Bax channel formation: influence of ionic strength.

    Science.gov (United States)

    Ganesan, Vidyaramanan; Walsh, Timothy; Chang, Kai-Ti; Colombini, Marco

    2012-08-08

    Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, detected by protein release, and more substantial long-lasting permeabilization, measured by the rate of oxidation of added cytochrome c. We found that higher ionic strength causes Bax to form small channels quickly but the expansion of these early channels is impeded. This inhibitory effect of ionic strength is independent of tBid. Channels formed under low ionic strength are not destabilized by raising the ionic strength. Increase in ionic strength also increases the ability of Bcl-xL to inhibit Bax-mediated MOMP. Ionic strength does not affect Bax insertion into mitochondria. Thus, ionic strength influences the assembly of Bax molecules already in membrane into channels. Ionic strength can be used as an effective biophysical tool to study Bax-mediated channel formation.

  3. Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis.

    Science.gov (United States)

    Srivastava, M; Ahmad, N; Gupta, S; Mukhtar, H

    2001-05-04

    Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.

  4. Mitochondrial BAX Determines the Predisposition to Apoptosis in Human AML.

    Science.gov (United States)

    Reichenbach, Frank; Wiedenmann, Cornelius; Schalk, Enrico; Becker, Diana; Funk, Kathrin; Scholz-Kreisel, Peter; Todt, Franziska; Wolleschak, Denise; Döhner, Konstanze; Marquardt, Jens U; Heidel, Florian; Edlich, Frank

    2017-08-15

    Purpose: Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death.Experimental Design: The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort.Results: This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy.Conclusions: In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final "common path." Clin Cancer Res; 23(16); 4805-16. ©2017 AACR. ©2017 American Association for Cancer Research.

  5. p38(MAPK)/p53-Mediated Bax induction contributes to neurons degeneration in rotenone-induced cellular and rat models of Parkinson's disease.

    Science.gov (United States)

    Wu, Feng; Wang, Zhong; Gu, Jin-Hua; Ge, Jian-Bin; Liang, Zhong-Qin; Qin, Zheng-Hong

    2013-09-01

    Rotenone is an environmental neurotoxin that induces degeneration of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc), which ultimately results in parkinsonism, but the molecular mechanisms of selective degeneration of nigral DA neurons are not fully understood. In the present study, we investigated the induction of p38(MAPK)/p53 and Bax in SNpc of Lewis rats after chronic treatment with rotenone and the contribution of Bax to rotenone-induced apoptotic commitment of differentiated PC12 cells. Lewis rats were subcutaneously treated with rotenone (1.5mg/kg) twice a day for 50days and the loss of tyrosine hydroxylase (THase), motor function impairment, and expression of p38(MAPK), P-p38(MAPK), p53, and Bax were assessed. After differentiated PC cells were treated with rotenone (500nM) for 6-36h, protein levels of p38(MAPK) and P-p38(MAPK), p53 nuclear translocation, Bax induction and cell death were measured. The results showed that rotenone administration significantly reduced motor activity and caused a loss of THase immunoreactivity in SNpc of Lewis rats. The degeneration of nigral DA neurons was accompanied by the increases in p38(MAPK), P-p38(MAPK), p53, and Bax protein levels. In cultured PC12 cells, rotenone also induced an upregulation of p38(MAPK), P-p38(MAPK), p53 and Bax. Pharmacological inhibition of p38(MAPK) with SB203580 (25μM) blunted rotenone-induced cell apoptosis. Treatment with SB203580 prevented the p53 nuclear translocation and upregulation of Bax. Inhibition of p53 with pifthrin-alpha or Bax with siRNAs significantly reduced rotenone-induced Bax induction and apoptotic cell death. These results suggest that the p38(MAPK)/p53-dependent induction of Bax contributes to rotenone's neurotoxicity in PD models.

  6. Regulation of signal transducer and activator of transcription 3 and apoptotic pathways by betaine attenuates isoproterenol-induced acute myocardial injury in rats.

    Science.gov (United States)

    Zheng, P; Liu, J; Mai, S; Yuan, Y; Wang, Y; Dai, G

    2015-05-01

    The present study was designed to investigate the cardioprotective effects of betaine on acute myocardial ischemia induced experimentally in rats focusing on regulation of signal transducer and activator of transcription 3 (STAT3) and apoptotic pathways as the potential mechanism underlying the drug effect. Male Sprague Dawley rats were treated with betaine (100, 200, and 400 mg/kg) orally for 40 days. Acute myocardial ischemic injury was induced in rats by subcutaneous injection of isoproterenol (85 mg/kg), for two consecutive days. Serum cardiac marker enzyme, histopathological variables and expression of protein levels were analyzed. Oral administration of betaine (200 and 400 mg/kg) significantly reduced the level of cardiac marker enzyme in the serum and prevented left ventricular remodeling. Western blot analysis showed that isoproterenol-induced phosphorylation of STAT3 was maintained or further enhanced by betaine treatment in myocardium. Furthermore, betaine (200 and 400 mg/kg) treatment increased the ventricular expression of Bcl-2 and reduced the level of Bax, therefore causing a significant increase in the ratio of Bcl-2/Bax. The protective role of betaine on myocardial damage was further confirmed by histopathological examination. In summary, our results showed that betaine pretreatment attenuated isoproterenol-induced acute myocardial ischemia via the regulation of STAT3 and apoptotic pathways.

  7. Immunohistochemical study of epidermal and dermal expression of Bcl-X, Bcl-2 and bax in psoriasis.

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the regulation of cell proliferation and apoptosis in psoriasis. Methods: The expressions of Bcl-X, Bcl-2 and Bax were studied with immunohistochemical technique (SP) in the lesional and non-lesional psoriatic skin. Results: There were significant overexpressions of Bcl-X in all layers of epidermis, inflammatory cells and vascular endothelia in dermis;

  8. Kazrin F is involved in apoptosis and interacts with BAX and ARC

    Institute of Scientific and Technical Information of China (English)

    Qiong Wang; Min Liu; Xin Li; Lu Chen; Hua Tang

    2009-01-01

    Kazrin has recently been identified as a functional protein that is involved in cell-cell junctions and in signal transduction. Here, we identified a new isoform, Kazrin F, which is 518 aa in length and has 97 aa unique at the N-terminus. Knockdown of Kazrin F using siRNA caused cell apoptosis and a marked decrease in cell viability measured by MTT and TUNEL assays. Co-immunoprecipitation analysis revealed that Kazrin F interacts with ARC (apoptosis repressor with caspase recruitment domain) and Bas (Bcl-2-associated X protein). Co-localization of Kazriin F with ARC and Bax in the cytoplasm was determined by immunofluorescence analysis. These results suggested that Kazrin F might play an important role in regulating cellular apoptosis by interacting with ARC and Bax.

  9. Bax-induced apoptosis in Leber's congenital amaurosis: a dual role in rod and cone degeneration.

    Directory of Open Access Journals (Sweden)

    Séverine Hamann

    Full Text Available Pathogenesis in the Rpe65(-/- mouse model of Leber's congenital amaurosis (LCA is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65(-/- mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65, was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65(-/- mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65(-/-/Gnat1(-/- mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65(-/-/Bax(-/- mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65(-/- mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65(-/-/Bax(-/- mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice

  10. 76 FR 12364 - Agency Information Collection Activities: Bonded Warehouse Regulations

    Science.gov (United States)

    2011-03-07

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Bonded Warehouse... Bonded Warehouse Regulations. This request for comment is being made pursuant to the Paperwork Reduction... concerning the following information collection: Title: Bonded Warehouse Regulations. OMB Number:...

  11. Inhibition of nuclear factor-kappaB or Bax prevents endoplasmic reticulum stress- but not nitric oxide-mediated apoptosis in INS-1E cells

    DEFF Research Database (Denmark)

    Tonnesen, Morten F; Grunnet, Lars G; Friberg, Josefine

    2009-01-01

    and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFkappaB activation, as assessed by IkappaB degradation and NFkappaB DNA binding. Inhibition of NFkappaB or the Bcl-2 family member Bax pathways protected beta-cells against TG- but not SNAP....... Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip......-induced beta-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling...

  12. The Dynamics of Bax Channel Formation: Influence of Ionic Strength

    OpenAIRE

    Ganesan, Vidyaramanan; Walsh, Timothy; Chang, Kai-Ti; Colombini, Marco

    2012-01-01

    Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, d...

  13. [Effects of activating silent information regulator 1 on early kidney damage in rats with severe burn].

    Science.gov (United States)

    Bai, X Z; He, T; Liu, Y; Zhang, W; Han, F; Yang, C; Cai, W X; Jia, Y H; Shi, J H; Han, J T; Su, L L; Hu, D H

    2017-06-20

    Objective: To investigate the effects of activating silent information regulator 1 (SIRT1) on the early kidney damage in rats with severe burn. Methods: Thirty healthy male SD rats were divided into sham injury group (SI), pure burn group (PB), and SIRT1 activator group (SA) according to the random number table, with 10 rats in each group. Rats in groups PB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Immediately after injury, rats in group PB were intraperitoneally injected with normal saline in the dosage of 50 mL/kg, and those in group SA with 1 mg/mL (final mass concentration) resveratrol in the dosage of 50 mL/kg. Rats in group SI were sham injured and intraperitoneally injected with normal saline in the dosage of 50 mL/kg immediately after injury. Kidney tissue and abdominal aorta blood of rats in the three groups were collected at 24 hours after injury. The morphology of kidney tissue was observed after HE staining. The serum content of creatinine and urea nitrogen was determined with enzyme-linked immunosorbent assay. Protein expressions of SIRT1, Bax, and Bcl-2 in kidney tissue were determined with Western blotting. mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 in kidney tissue were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with one-way analysis of variance and LSD-t test. Results: (1) In rats of group SI, structures of kidney tubules and glomeruli were intact. In rats of group PB, structures of kidney tubules were not clear with casts in them, and glomeruli showed pyknosis. In rats of group SA, structures of kidney tubules were relatively intact, and the pyknosis of glomeruli were slighter as compared with that of group PB with fewer glomeruli showing pyknosis. (2) The serum content of creatinine and urea nitrogen in rats of group PB was (67±14)

  14. ECONOMIC ACTIVITY REGULATION AND COMPETITION ASSESSMENT

    Directory of Open Access Journals (Sweden)

    Berinde Mihai

    2010-07-01

    Full Text Available In a broad sense, the term „competition” defines the relations between economic operators acting on the same market seeking attainment of certain interests in economic freedom conditions. The need for regulations in the area of competition stems from the nature of free, open market economy which is founded on the existence of fair competition between economic agents, competition which must be observed, maintained and protected by the law. Public authorities who issue various regulations should be cautious about how far this role is played in the economy and they way adopted regulations affect competition in the market. Hence, the need for prior assessment relating to the potential effect of a regulation on competition. It was proven in practice that some regulations may lead to measures that may affect competition directly or indirectly by: limiting the number or range of suppliers; limiting supplier capability to compete and reducing interests of suppliers to compete vigorously.

  15. Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

    Science.gov (United States)

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to G(i), some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

  16. Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release

    National Research Council Canada - National Science Library

    Mikhailov, Valery; Mikhailova, Margarita; Degenhardt, Kurt; Venkatachalam, Manjeri A; White, Eileen; Saikumar, Pothana

    2003-01-01

    ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells...

  17. PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax-caspase9-caspase3 pathway.

    Science.gov (United States)

    Wu, Chun-Yan; Tang, Zhi-Han; Jiang, Lu; Li, Xue-Fei; Jiang, Zhi-Sheng; Liu, Lu-Shan

    2012-01-01

    This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24 h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24 h, cells were treated with ox-LDL for 24 h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.

  18. Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity*

    Science.gov (United States)

    Maity, Biswanath; Yang, Jianqi; Huang, Jie; Askeland, Ryan W.; Bera, Soumen; Fisher, Rory A.

    2011-01-01

    Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨm) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨm was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer. PMID:21041304

  19. Regulator of G protein signaling 6 (RGS6) induces apoptosis via a mitochondrial-dependent pathway not involving its GTPase-activating protein activity.

    Science.gov (United States)

    Maity, Biswanath; Yang, Jianqi; Huang, Jie; Askeland, Ryan W; Bera, Soumen; Fisher, Rory A

    2011-01-14

    Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for Gα subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (ΔΨ(m)) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ΔΨ(m) was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer.

  20. Activation and Regulation of Cellular Eicosanoid Biosynthesis

    Directory of Open Access Journals (Sweden)

    Thomas G. Brock

    2007-01-01

    Full Text Available There is a growing appreciation for the wide variety of physiological responses that are regulated by lipid messengers. One particular group of lipid messengers, the eicosanoids, plays a central role in regulating immune and inflammatory responses in a receptor-mediated fashion. These mediators are related in that they are all derived from one polyunsaturated fatty acid, arachidonic acid. However, the various eicosanoids are synthesized by a wide variety of cell types by distinct enzymatic pathways, and have diverse roles in immunity and inflammation. In this review, the major pathways involved in the synthesis of eicosanoids, as well as key points of regulation, are presented.

  1. The Bax Inhibitor-1 (BI-1) family in apoptosis and tumorigenesis.

    Science.gov (United States)

    Reimers, Kerstin; Choi, Claudia Y U; Bucan, Vesna; Vogt, Peter M

    2008-03-01

    The signaling pathways that determine the fate of a cell regarding death or survival depend on a large number of regulatory proteins. The Bax Inhibitor-1 (BI-1) family is a highly preserved family of small transmembrane proteins located mostly in the endoplasmic reticulum (ER). Although most members of this family are still not characterized an antiapoptotic effect has been described for BI-1, Lifeguard (LFG), and the Golgi anti-apoptotic protein (GAAP). The cytoprotective activity has been associated to the control of ion homeostasis and ER stress but includes other cell death stimuli as well. Recent data describes multiple interactions between the proteins of the BI-1 family and the Bcl-2 family either stimulating the antiapoptotic function of Bcl-2 or inhibiting the proapoptotic effect of Bax. The potent cell death suppression makes this protein family an interesting target for the development of new drugs and gene therapeutic approaches for diseases caused by apoptotic dysregulation, such as cancer.

  2. Erythropoietin can promote survival of cerebral cells by downregulating Bax gene after traumatic brain injury in rats

    Directory of Open Access Journals (Sweden)

    Liao Z

    2009-01-01

    Full Text Available Background : Traumatic brain injury (TBI is an important cause of adult mortality and morbidity. Erythropoietin (Epo has been shown to promote the viability of cerebral cells by upregulating Bcl-2 gene; however, Epo may exert its antiapoptotic effect via the differential regulation of the expression of genes involved in the apoptotic process. Aim : The present study examined the neuroprotective effect of Epo as a survival factor through the regulation of the Bax. Materials and Methods : Wistar rats were randomly divided into three groups: Recombinant human EPO treated (rhEPO TBI, vehicle-treated TBI, and sham-operated. Traumatic brain injury was induced by the Feeney free-falling model. Rats were killed 5, 12, 24, 72, 120, or 168 h after TBI. Regulation of Bcl-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR, western blotting and immunofluorescence. Results : Bax mRNA and protein levels were lower in the rhEPO-treated rat brains than in the vehicle-treated rat brains. Induction of Bax expression peaked at 24 h and remained stable for 72-120 h in vehicle-treated rat brains, whereas induction of Bax expression was only slightly elevated in rhEPO-treated rat brains. The number of TdT-mediated dUTP Nick-End Labeling(TUNEL-positive cells in the rhEPO-treated rat brains was far fewer than in the vehicle-treated rat brains. Conclusions : Epo exerts neuroprotective effect against traumatic brain injury via reducing Bax gene expression involved in inhibiting TBI-induced neuronal cell death.

  3. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation.

    Science.gov (United States)

    Chen, Fang; Zhang, Bing; Sun, Yong; Xiong, Zeng-Xing; Peng, Han; Deng, Ze-Yuan; Hu, Jiang-Ning

    2016-04-25

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  4. 50 CFR 404.7 - Regulated activities.

    Science.gov (United States)

    2010-10-01

    ... vessel engine cooling water, weather deck runoff, and vessel engine exhaust; (f) Discharging or... effluent, cooling water, and engine exhaust; (g) Touching coral, living or dead; (h) Possessing fishing... Wildlife and Fisheries JOINT REGULATIONS (UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF...

  5. Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells.

    Science.gov (United States)

    Coker-Gurkan, Ajda; Arisan, Elif Damla; Obakan, Pinar; Guvenir, Esin; Unsal, Narcin Palavan

    2014-10-15

    The purine-derived analogs, roscovitine and purvalanol are selective synthetic inhibitors of cyclin-dependent kinases (CDKs) induced cell cycle arrest and lead to apoptotic cell death in various cancer cells. Although a number of studies investigated the molecular mechanism of each CDK inhibitor on apoptotic cell death mechanism with their therapeutic potential, their regulatory role on autophagy is not clarified yet. In this paper, our aim was to investigate molecular mechanism of CDK inhibitors on autophagy and apoptosis in wild type (wt) and Bax deficient HCT 116 cells. Exposure of HCT 116 wt and Bax(-/-) cells to roscovitine or purvalanol for 24h decreased cell viability in dose-dependent manner. However, Bax deficient HCT 116 cells were found more resistant against purvalanol treatment compared to wt cells. We also established that both CDK inhibitors induced apoptosis through activating mitochondria-mediated pathway in caspase-dependent manner regardless of Bax expression in HCT 116 colon cancer cells. Concomitantly, we determined that purvalanol was also effective on autophagy in HCT 116 colon cancer cells. Inhibition of autophagy by 3-MA treatment enhanced the purvalanol induced apoptotic cell death in HCT 116 Bax(-/-) cells. Our results revealed that mechanistic action of each CDK inhibitor on cell death mechanism differs. While purvalanol treatment activated apoptosis and autophagy in HCT 116 cells, roscovitine was only effective on caspase-dependent apoptotic pathway. Another important difference between two CDK inhibitors, although roscovitine treatment overcame Bax-mediated drug resistance in HCT 116 cells, purvalanol did not exert same effect. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70-Bax interaction in prostate cancer.

    Science.gov (United States)

    Shukla, Sanjeev; Fu, Pingfu; Gupta, Sanjay

    2014-05-01

    Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70-Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy.

  7. Apigenin induces apoptosis by targeting inhibitor of apoptosis proteins and Ku70–Bax interaction in prostate cancer

    Science.gov (United States)

    Shukla, Sanjeev; Fu, Pingfu; Gupta, Sanjay

    2014-01-01

    Dysfunction of the apoptotic pathway in prostate cancer cells confers apoptosis resistance towards various therapies. A novel strategy to overcome resistance is to directly target the apoptotic pathway in cancer cells. Apigenin, an anticancer agent, selectively toxic to cancer cells induces cell cycle arrest and apoptosis through mechanisms which are not fully explored. In the present study we provide novel insight into the mechanisms of apoptosis induction by apigenin. Treatment of androgen-refractory human prostate cancer PC-3 and DU145 cells with apigenin resulted in dose-dependent suppression of XIAP, c-IAP1, c-IAP2 and survivin protein levels. Apigenin treatment resulted in significant decrease in cell viability and apoptosis induction with the increase of cytochrome C in time-dependent manner. These effects of apigenin were accompanied by decrease in Bcl-xL and Bcl-2 and increase in the active form of Bax protein. The apigenin-mediated increase in Bax was due to dissociation of Bax from Ku70 which is essential for apoptotic activity of Bax. Apigenin treatment resulted in the inhibition of class I histone deacetylases and HDAC1 protein expression, thereby increasing the acetylation of Ku70 and the dissociation of Bax resulting in apoptosis of cancer cells. Furthermore, apigenin significantly reduced HDAC1 occupancy at the XIAP promoter, suggesting that histone deacetylation might be critical for XIAP downregulation. These results suggest that apigenin targets inhibitor of apoptosis proteins and Ku70–Bax interaction in the induction of apoptosis in prostate cancer cells and in athymic nude mouse xenograft model endorsing its in vivo efficacy. PMID:24563225

  8. Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Susmita Mondal

    Full Text Available The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD, a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53-/- cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53-/- over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53-/- cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax-Bak- reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax-Bak+/HCT116Bax+Bak- was only marginally effective after WithaD treatment. In HCT116p53-/- cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.

  9. Sequential Notch activation regulates ventricular chamber development

    OpenAIRE

    D'Amato, Gaetano

    2016-01-01

    Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 15 de enero de 2016 Ventricular chamber morphogenesis is a beautiful example of tissue interactions orchestrating a precise gene regulatory network essential for tissue patterning, cellular proliferation and differentiation that ultimately lead to a fully compacted and functional adult ventricle. The Notch signaling pathway is a crucial regulator ...

  10. Active Power Regulation based on Droop for AC Microgrid

    DEFF Research Database (Denmark)

    Li, Chendan; Coelho, Ernane A. A.; Firoozabadi, Mehdi Savaghebi

    2015-01-01

    In this paper, two different control strategies are proposed to address the active power regulation issue in AC microgrids. The principle of power regulation in the droop controller is firstly introduced. Frequency scheduling and droop gain scheduling on top of droop control is proposed to succes......In this paper, two different control strategies are proposed to address the active power regulation issue in AC microgrids. The principle of power regulation in the droop controller is firstly introduced. Frequency scheduling and droop gain scheduling on top of droop control is proposed...

  11. Modern aspects of tax regulation of investment activity

    Directory of Open Access Journals (Sweden)

    E.S. Podakov

    2016-03-01

    Full Text Available The article investigates the tax regulation of investment activity in modern conditions. Scientists studied different views about the impact of tax regulations on the investment activity in the country. The author determines that the tax regulation of investment activity involves the use of state mechanisms taxation of certain measures to improve investment conditions. The subject is the state tax regulations, and the object is the investment activity of individual and institutional investors of any form of ownership including organizational and legal forms. Such regulation is performed by using complex special tools. The possible methods of tax stimulation of investment processes are described. The article deals with the current results of tax reform in Ukraine and predicts its possible consequences for agricultural producers. The rating positions of Ukraine according to international organizations are showed. The systematic analysis has been carried out and the impact of differential tax rates, tax exemption for a specified period, reducing the tax base, elimination of double taxation on investment activity in certain areas have been researched. The special instruments of investment activity tax regulation are considered. The options for improving investment activity by introducing effective tax regulation are determined.

  12. Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

    Institute of Scientific and Technical Information of China (English)

    Jialong GUO; Kailun ZHANG; Yanmei JI; Xionggang JIANG; Shunqing ZUO

    2008-01-01

    In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups (n=8 in each group): control group was perfused for 120min. In the I/R group, after 30min stabilization the injury was induced by 30min global ischemia followed by 60min reperfusion. Ethyl pyruvate (EP) group was set up with the same protocol as I/R group except that it was supplied with 2mmol/L EP 15min before ischemia and throughout reperfusion. Myocardial malonaldehyde (MDA) content Was measured. Myocardial apoptotic index (AI) was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group (P<0.05). These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

  13. Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status

    OpenAIRE

    Mirjolet, J-F; Barberi-Heyob, M; Didelot, C; Peyrat, J-P; Abecassis, J; Millon, R.; Merlin, J-L

    2000-01-01

    p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53...

  14. Allosteric regulation of deubiquitylase activity through ubiquitination

    Directory of Open Access Journals (Sweden)

    Serena eFaggiano

    2015-02-01

    Full Text Available Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.

  15. Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats

    Institute of Scientific and Technical Information of China (English)

    崔玉芳; 夏国伟; 付小兵; 杨红; 彭瑞云; 张莹; 谷庆阳; 高亚兵; 崔雪梅; 胡文华

    2003-01-01

    Objective: To study the relationship between the expression of Bax, Bcl-2 proteins, and apoptosis in radiation compound wound healing of rats.Methods: Apoptosis, Bax and Bcl-2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods. Results: (1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation-induced delayed wound healing. (2) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bcl-2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level when the apoptosis decreased distinctively. Conclusions: Bax and Bcl-2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.

  16. A novel plant glutathione S-transferase/peroxidase suppresses Bax lethality in yeast

    DEFF Research Database (Denmark)

    Kampranis, S C; Damianova, R; Atallah, M

    2000-01-01

    for the identification of plant genes, which inhibit either directly or indirectly the lethal phenotype of Bax. Using this method a number of cDNA clones were isolated, the more potent of which encodes a protein homologous to the class theta glutathione S-transferases. This Bax-inhibiting (BI) protein was expressed......The mammalian inducer of apoptosis Bax is lethal when expressed in yeast and plant cells. To identify potential inhibitors of Bax in plants we transformed yeast cells expressing Bax with a tomato cDNA library and we selected for cells surviving after the induction of Bax. This genetic screen allows...

  17. Self-regulation as a type of managerial activity.

    Directory of Open Access Journals (Sweden)

    Anna Algazina

    2017-01-01

    Full Text Available УДК 342.9The subject. In the context of the ongoing administrative reform in the Russian Federation the issue of self-regulation is becoming increasingly important.Introduction of Institute of self-regulation is intended to reduce the degree of state intervention in private spheres of professional activity, to eliminate excessive administrative barriers, reduce government expenditures on regulation and control in their respective areas of operation, which is especially important in the current economic conditions.However, in Russian legal science is no recognized definition of "self-regulation", but a unity of views on the question of the relationship between self-regulation and state regulation of business relations.In this regard, the author attempts to examine the concept of "self-regulation" through the prism of knowledge about public administration.The purpose of the article is to identify the essential features and to articulate the concept of self-regulation by comparing it with other varieties of regulation.Methodology. The methodological basis for the study: general scientific methods (analysis, synthesis, comparison, description; private and academic (interpretation, formal-legal.Results, scope. Based on the analysis allocated in the science of administrative law approaches to the system of public administration justifies the conclusion that the notion "regulation" is specific in relation to the generic concept of "management" and is a kind of management, consisting in the drafting of rules of conduct and sanctions for non-compliance or inadequate performance.In addition, the article highlights the problem of the genesis of self-regulation, building a system of principles of self-regulation, comparison of varieties of self-regulatory organizations among themselves.Conclusions. The comparison of self-regulation other types of regulation (such as state regulation and co-regulation highlighted the essential features of this phenomenon

  18. Dietary methanol regulates human gene activity.

    Directory of Open Access Journals (Sweden)

    Anastasia V Shindyapina

    Full Text Available Methanol (MeOH is considered to be a poison in humans because of the alcohol dehydrogenase (ADH-mediated conversion of MeOH to formaldehyde (FA, which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD. There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

  19. Endogenous methanol regulates mammalian gene activity.

    Directory of Open Access Journals (Sweden)

    Tatiana V Komarova

    Full Text Available We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.

  20. Neuronal Activity Regulates Hippocampal miRNA Expression

    NARCIS (Netherlands)

    Eacker, Stephen M.; Keuss, Matthew J.; Berezikov, Eugene; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Neuronal activity regulates a broad range of processes in the hippocampus, including the precise regulation of translation. Disruptions in proper translational control in the nervous system are associated with a variety of disorders that fall in the autistic spectrum. MicroRNA (miRNA) represent a re

  1. Insight into Nek2A activity regulation and its pharmacological ...

    African Journals Online (AJOL)

    Ambuj Kumar

    2012-11-28

    Nov 28, 2012 ... 3. Nek2A activity regulation and associated pathological outcomes . .... vide a detailed insight into how they coordinate the cell cycle .... the adenine subpocket creating steric hindrance in the plane of ... Three dimensional.

  2. Regulation of MDM2 Activity by Nucleolin

    Science.gov (United States)

    2007-06-01

    assistance with FACS analysis, Eric Rubin (UMDNJ) for providing the GST-nucleolin expression vectors, Cris- tina Cardoso for the pENeGFP RPA34 plasmid, and...formation, and functional char- acterization. J. Biol. Chem. 269:11121–11132. 25. Huang, W., and R. L. Erikson . 1994. Constitutive activation of Mek1

  3. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ruizhao, E-mail: liruizhao1979@126.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Li, E-mail: Zhanglichangde@163.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Southern Medical University, Guangzhou, Guangdong (China); Shi, Wei, E-mail: shiwei.gd@139.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Zhang, Bin, E-mail: zhangbinyes@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liang, Xinling, E-mail: xinlingliang@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Liu, Shuangxin, E-mail: mplsxi@yahoo.com.cn [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China); Wang, Wenjian, E-mail: wwjph@yahoo.com [Department of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou, 510080 (China)

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  4. Apoptosis and Bax expression are increased by coal dust in the polycyclic aromatic hydrocarbon-exposed lung

    Energy Technology Data Exchange (ETDEWEB)

    Ghanem, M.M.; Battelli, L.A.; Mercer, R.R.; Scabilloni, J.F.; Kashon, M.L.; Ma, J.Y.C.; Nath, J.; Hubbs, A.F.

    2006-09-15

    Miners inhaling respirable coal dust (CD) frequently develop coal workers' pneumoconiosis. Many coal miners are also exposed to polycyclic aromatic hydrocarbon (PAH) components of diesel engine exhaust and cigarette smoke, which may contribute to lung disease in these workers. Recently, apoptosis was reported to play a critical role in the development of another pneumoconiosis of miners, silicosis. In addition, CID was reported to suppress cytochrome P450 1A1 (CYP1A1) induction by PAHs. We exposed rats intratracheally to 0.0, 2.5, 10.0, 20.0, or 40.0 mg/rat CD and, 11 days later, to intraperitoneal P-naphthoflavone (BNF), a PAH. In another group of rats exposed to CD and BNF, caspase activity was inhibited by injection of the pan-caspase inhibitor Q-VD-OPH (quinoline-Val-Asp (OMe)-CH{sub 2}-OPH). In rats exposed to BNF, CD exposure increased alveolar expression of the proapoptotic mediator Bax but decreased CYP1A1 induction relative to BNF exposure alone. Pan-caspase inhibition decreased CD-associated Bax expression and apoptosis but did not restore CYP1A1 activity. Further, CD-induced lung inflammation and alveolar epithelial cell hypertrophy and hyperplasia were not suppressed by caspase inhibition. It is concluded that combined BNF and CD exposure increased Bax expression and apoptosis in the lung, but Bax and apoptosis were not the major determinants of early lung injury in this model.

  5. Fragile phagocytes: FMRP positively regulates engulfment activity.

    Science.gov (United States)

    Logan, Mary A

    2017-03-06

    Defective immune system function is implicated in autism spectrum disorders, including Fragile X syndrome. In this issue, O'Connor et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201607093) demonstrate that phagocytic activity of systemic immune cells is compromised in a Drosophila melanogaster model of Fragile X, highlighting intriguing new mechanistic connections between FMRP, innate immunity, and abnormal development.

  6. Regulation of APC/C activators in mitosis and meiosis.

    Science.gov (United States)

    Pesin, Jillian A; Orr-Weaver, Terry L

    2008-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ubiquitin ligase that triggers the degradation of multiple substrates during mitosis. Cdc20/Fizzy and Cdh1/Fizzy-related activate the APC/C and confer substrate specificity through complex interactions with both the core APC/C and substrate proteins. The regulation of Cdc20 and Cdh1 is critical for proper APC/C activity and occurs in multiple ways: targeted protein degradation, phosphorylation, and direct binding of inhibitory proteins. During the specialized divisions of meiosis, the activity of the APC/C must be modified to achieve proper chromosome segregation. Recent studies show that one way in which APC/C activity is modified is through the use of meiosis-specific APC/C activators. Furthermore, regulation of the APC/C during meiosis is carried out by both mitotic regulators of the APC/C as well as meiosis-specific regulators. Here, we review the regulation of APC/C activators during mitosis and the role and regulation of the APC/C during female meiosis.

  7. Downregulation of Notch-regulated Ankyrin Repeat Protein Exerts Antitumor Activities against Growth of Thyroid Cancer

    Institute of Scientific and Technical Information of China (English)

    Bing-Feng Chu; Yi-Yu Qin; Sheng-Lai Zhang; Zhi-Wei Quan; Ming-Di Zhang; Jian-Wei Bi

    2016-01-01

    Background:The Notch-regulated ankyrin repeat protein (NRARP) is recently found to promote proliferation of breast cancer cells.The role of NRARP in carcinogenesis deserves extensive investigations.This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation,apoptosis,cell cycle,and invasion in thyroid cancer.Methods:Thirty-four cases with thyroid cancer were collected from the Department of General Surgery,Xinhua Hospital,Shanghai Jiao Tong University School of Medicine between 2011 and 2012.Immunohistochemistry was used to detect the level of NRARP in cancer tissues.Lentivirus carrying NRARP-shRNA (Lenti-NRARP-shRNA) was applied to down-regulate NRARP expression.Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay.Apoptosis and cell cycle distribution were determined by flow cytometry.Cell invasion was tested using Transwell invasion assay.In addition,expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection.Student's t-test,one-way analysis of variance (ANOVA),or Kaplan-Meier were used to analyze the differences between two group or three groups.Results:NRARP was highly expressed in thyroid cancer tissues.Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of 10 or higher (P < 0.05).Lenti-NRARP-shRNA-induced G1 arrest (BHT 101:72.57% ± 5.32%;8305C:75.45% ± 5.26%) by promoting p21 expression,induced apoptosis by promoting bax expression and suppressing bcl-2 expression,and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression.Conclusion:Downregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer,that suggests a potential role of NRARP in thyroid cancer targeted

  8. A Small Group Activity About Bacterial Regulation And Complementation

    Directory of Open Access Journals (Sweden)

    Susan M. Merkel

    2010-11-01

    Full Text Available As teachers, we well understand the need for activities that help develop critical-thinking skills in microbiology. In our experience, one concept that students have difficulty understanding is transcriptional regulation of bacterial genes. To help with this, we developed and evaluated a paper-based activity to help students understand and apply the concepts of bacterial transcriptional regulation. While we don't identify it as such, we use a complementation experiment to assess student understanding of how regulation changes when new DNA is introduced. In Part 1 of this activity, students complete an open book, take-home assignment that asks them to define common terminology related to regulation, and draw the regulatory components of different scenarios involving positive and negative regulation. In Part 2, students work in small groups of 3-4 to depict the regulatory components for a different scenario. They are asked to explain the results of a complementation experiment based on this scenario. They then predict the results of a slightly different experiment. Students who completed the Regulation Activity did significantly better on post-test questions related to regulation, compared to pre-test questions.

  9. Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

    Science.gov (United States)

    Smith, Kelsy F; Bibb, Lori A; Schmitt, Michael P; Oram, Diana M

    2009-03-01

    Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not yet been described for any other zur. An adjacent gene, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.

  10. The Ubiquitin Ligase Siah2 Regulates PPARγ Activity in Adipocytes

    OpenAIRE

    Kilroy, Gail; Kirk-Ballard, Heather; Carter, Lauren E.; Floyd, Z. Elizabeth

    2012-01-01

    Moderate reductions in peroxisome proliferator-activated receptor (PPAR)γ levels control insulin sensitivity as effectively as activation of PPARγ in adipocytes by the thiazolidinediones. That observation suggests that PPARγ activity can be regulated by modulating the amount of PPARγ protein in adipocytes. Activation of PPARγ in adipocytes is linked to changes in PPARγ protein levels via increased degradation of PPARγ proteins by the ubiquitin proteasome system. Identification of the ubiquiti...

  11. Expressão das proteínas BCL-2 e BAX em tumores astrocíticos humanos Expression of BCL-2 and BAX proteins in human astrocytic tumors

    Directory of Open Access Journals (Sweden)

    Mário Henrique Girão Faria

    2006-08-01

    Full Text Available INTRODUÇÃO: Os astrocitomas constituem os mais freqüentes tumores primários do sistema nervoso central (SNC. Admite-se que parte do crescimento tumoral seja resultante da inibição da morte celular programada: a apoptose. Tal fenômeno é basicamente regulado pelo equilíbrio entre moléculas antiapoptóticas (ex.: B-cell lymphoma protein 2 [BCL-2] e pró-apoptóticas (ex.: BCL-2 associated protein X [BAX]. OBJETIVO: O presente estudo objetivou avaliar a expressão de BCL-2 e BAX em tumores astrocíticos humanos. MATERIAL E MÉTODOS: Procedeu-se ao estudo imuno-histoquímico dessas proteínas utilizando-se o método da avidina-biotina-peroxidase em 55 astrocitomas (13 do grau I, 14 do II, sete do III e 21 do grau IV e cinco amostras de tecido cerebral não-tumoral (grupo controle. RESULTADOS: Os índices de positividade para BCL-2 e BAX demonstraram propensão ao acréscimo, de acordo com a gradação tumoral, com positividade geral de 43,26% e 24,67%, respectivamente. Essas proteínas não foram detectadas entre os espécimes não-tumorais. Os escores de marcação para BCL-2 apresentaram tendência ao aumento conforme a progressão histológica, enquanto os para BAX mostraram-se semelhantes nas diversas graduações. A análise conjunta dessas proteínas demonstrou significativa correlação com a gradação tumoral (p BACKGROUND: Astrocytomas represent the most frequent primary tumors of the central nervous system. Admittedly, part of tumor growth is due to inhibition of programmed cell death: the apoptosis. This phenomenon is basically regulated by the balance between anti-apoptotic (e.g.: B-cell lymphoma protein 2 [BCL-2] and pro-apoptotic (e.g.: BCL-2 associated protein X [BAX] molecules. OBJECTIVE: The present study aimed to evaluate the expression of BCL-2 and BAX in human astrocytic tumors of different histopathological grades. MATERIAL AND METHOD: An immunohistochemical study of those proteins using the avidin

  12. Regulation of Activation Induced Deaminase (AID) by Estrogen.

    Science.gov (United States)

    Pauklin, Siim

    2016-01-01

    Regulation of Activation Induced Deaminase (AID) by the hormone estrogen has important implications for understanding adaptive immune responses as well as the involvement of AID in autoimmune diseases and tumorigenesis. This chapter describes the general laboratory techniques for analyzing AID expression and activity induced by estrogen, focusing on the isolation and preparation of cells for hormone treatment and the subsequent analysis of AID responsiveness to estrogen at the RNA level and for determining the regulation of AID activity via estrogen by analyzing Ig switch circle transcripts and mutations in switch region loci.

  13. Absence of canonical active chromatin marks in developmentally regulated genes

    Science.gov (United States)

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  14. Influence of Stress on the Expression of bcl-2/bax Protein in Spontaneously Hypertensive Rats

    Institute of Scientific and Technical Information of China (English)

    刘巍; 李为民; 孙宁玲; 陈源源; 任哲; 虞有智

    2002-01-01

    Objective To explore the influence of stress-induced increased sympathetic nerve activity on cardiomyocyte apoptosis and on the development of congestive heart failure. Methods 45male, 16-week-old spontaneously hypertensive rats (SHRs) were studied, in which 6 as controls. After the 6 controlled SHRs were examined by echocardiography, they were anesthetized and killed by decapitation.The other 39 were divided into the stress group ( n =20) and the control group ( n = 19), and both groups were observed from 16-week-old to 36-week-old. In the stress group, binding- stress model was used. Till 36week, all animals were echocardiographied, weighed and killed as described above. Cardiac bcl-2 and bax protein were quantified by western blot. Circulating catecholamine and angiotensin II (Ang II) were detected by radioimmunoasssy. Results Left ventricular volume ( P < 0.05), left ventricular mass ( P<0.05) and the ratio of ventricular mass to body weight were higher in 36 week than those of the 16 week SHRs, whereas the volumes of eject fraction (EF)manifested the trend of decline, P< 0.05, bindingstress for 20 weeks made this trend significantly, P<0.05. With the increase of age, the serum nore pinephrine (NE), epinephrine (E) and Ang Ⅱ in creased, suggesting that the binding- stress triggered the activity of central sympathetic nerve system. The cardiac bcl-2 protein was higher in 36 week than 16week, P >0.05, whereas the bax protein increased significantly with the increase of age, P < 0.05, and so was the ratio of bax to bcl-2, P < 0.05. Conclusions The model of binding-stress can effectivelyactivate central sympathetic system, thus and mimic the neuroendocrine states. From 16 to 36 week, the process of cardiac apoptosis aggravated and the increased sympathetic activity would exacerbate rather than relieve this trend.

  15. Quercetin regulates bax gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells%五羟黄酮调节肝癌HepG2细胞bax基因表达诱导细胞凋亡的实验研究

    Institute of Scientific and Technical Information of China (English)

    马兴标; 张继红; 梁力建; 黄洁夫

    2007-01-01

    目的 探讨三磷酸肌醇(IP3)和bax基因表达变化在五羟黄酮(Quercetin)诱导肝癌细胞凋亡中的作用.方法 以肝癌HepG2细胞培养72 h为对照,以20,40,60,80μmol/L Quercetin作用于HepG2细胞72 h时和60μ,mol/L Quercetin作用于HepG2细胞6,12,24,48,72 h,应用同位素试剂盒检测细胞IP3含量,RT-PCR分析bax mRNA表达,Western blotting分析细胞bax蛋白表达,流式细胞仪检测细胞凋亡率.结果 各浓度的Quercetin作用于肝癌HepG2细胞72 h,IP3含量显著低于对照组(P<0.01),bax mRNA和bax蛋白表达显著高于对照组,细胞凋亡率显著高于对照组(P<0.01);60 μmol/L Quercetin作用于肝癌HepG2细胞6,12,24,48,72 h,各时相IP3含量显著低于对照组(P<0.01);12 h后bax mRNA和bax蛋白表达显著高于对照组,24 h后各时相细胞凋亡率为显著高于对照组(P<0.01).结论 Quercetin能减少IP3生成,上调bax基因表达,诱导肝癌细胞凋亡.

  16. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

    Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

  17. Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    Sergiy; Kostenko; Gianina; Dumitriu; Kari; Jenssen; Lgreid; Ugo; Moens

    2011-01-01

    Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

  18. Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51.

    Directory of Open Access Journals (Sweden)

    Loro L Kujjo

    Full Text Available BACKGROUND: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell's fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes. METHODS: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays. RESULTS: Our data show that: (i DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii oocytes possess the machinery and capability for repairing such DNA damage; (iii Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51. CONCLUSION/SIGNIFICANCE: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in

  19. Virosecurinine induces apoptosis by affecting Bcl-2 and Bax expression in human colon cancer SW480 cells.

    Science.gov (United States)

    Chen, Chuan-Rong; Xia, Yong-Hui; Yao, Shu-Yan; Zhang, Qing; Wang, Ying; Ji, Zhao-Ning

    2012-04-01

    Virosecurinine, the major alkaloid isolated from Securinega suffruticosa Pall Rehd was found to exhibit growth inhibition and cytotoxicity against huaman colon cancer SW480 cells via the microculture tetrazolium (MTT) assay. Due to its greater cytotoxic potency and selectivity towards SW480 cells, flow cytometry was used to analyze the cell cycle distribution of control and treated SW480 cells whereas Annexin V-FITC/PI flow cytometry analysis was carried out to confirm apoptosis induced by virosecurinine in SW480 cells. Apoptotic regulatory genes were determined by RT-PCR analysis. Virosecurinine was found to induce G1/S cell cycle arrest which led to predominantly apoptotic mode of cell death. Mechanistically, virosecurinine was found to up-regulated the Bax gene expression and down-regulated the Bcl-2 expression in SW480, The ratio of Bcl-2 to Bax was significantly decreased. Hence, we suggest that virosecurinine induced apoptosis in SW480 cells by affecting the expression of bcl-2 and bax.

  20. Effect of hyperbaric oxygen on cytochrome C, Bcl-2 and bax expression after experimental traumatic brain injury in rats

    Institute of Scientific and Technical Information of China (English)

    LIU Zhan; JIAO Qing-fang; YOU Chao; CHE Yan-jun; SU Fang-zhong

    2006-01-01

    Objective: To explore the effects of hyperbaric oxygen (HBO) treatment on the neuronal apoptosis at an earlier stage and the expressions of Cytochrome C (Cyt C), Bcl-2 (B-cell lymphoma-2 family) and Bax (Bcl-2associated X protein) in rat brain tissues after traumatic brain injury (TBI).Methods: Forty adult rats were divided into two groups, i.e., Group A ( the rats with untreated TBI) and Group B ( rats with HBO treatment after TBI). Sections of brain tissues of these two groups were then detected at 3,6,12,24,72 hours after TBI by immunohistochemistry and electronmicroscope, respectively.Results: HBO treatment could up-regulate the expression of Bcl-2 within 72 hours, reduce the release of Cyt C from mitochondria, attenuate the formation of dimeric Bax and alleviate the mitochondrial edema within 24 hours after TBI.Conclusions: HBO treatment can alleviate neuronal apoptosis after TBI by reducing the release of Cyt C and the dimers of Bax and up-regulating the expression of Bcl-2.

  1. Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity

    Directory of Open Access Journals (Sweden)

    Lama Tarayrah

    2015-11-01

    Full Text Available Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. Here we report that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid maintains germline stem cell (GSC mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the Janus kinase signal transducer and activator of transcription (JAK-STAT signaling pathway. Our findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. Our study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities.

  2. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Directory of Open Access Journals (Sweden)

    Jun-Ha Hwang

    Full Text Available Mesenchymal stem cell (MSC differentiation is regulated by the extracellular matrix (ECM through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  3. Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation.

    Science.gov (United States)

    Hwang, Jun-Ha; Byun, Mi Ran; Kim, A Rum; Kim, Kyung Min; Cho, Hang Jun; Lee, Yo Han; Kim, Juwon; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

    2015-01-01

    Mesenchymal stem cell (MSC) differentiation is regulated by the extracellular matrix (ECM) through activation of intracellular signaling mediators. The stiffness of the ECM was shown to be an important regulatory factor for MSC differentiation, and transcriptional coactivator with PDZ-binding motif (TAZ) was identified as an effector protein for MSC differentiation. However, the detailed underlying mechanism regarding the role of ECM stiffness and TAZ in MSC differentiation is not yet fully understood. In this report, we showed that ECM stiffness regulates MSC fate through ERK or JNK activation. Specifically, a stiff hydrogel matrix stimulates osteogenic differentiation concomitant with increased nuclear localization of TAZ, but inhibits adipogenic differentiation. ERK and JNK activity was significantly increased in cells cultured on a stiff hydrogel. TAZ activation was induced by ERK or JNK activation on a stiff hydrogel because exposure to an ERK or JNK inhibitor significantly decreased the nuclear localization of TAZ, indicating that ECM stiffness-induced ERK or JNK activation is important for TAZ-driven osteogenic differentiation. Taken together, these results suggest that ECM stiffness regulates MSC differentiation through ERK or JNK activation.

  4. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells.

    Science.gov (United States)

    Papaianni, Emanuela; El Maadidi, Souhayla; Schejtman, Andrea; Neumann, Simon; Maurer, Ulrich; Marino-Merlo, Francesca; Mastino, Antonio; Borner, Christoph

    2015-01-01

    Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.

  5. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells.

    Directory of Open Access Journals (Sweden)

    Emanuela Papaianni

    Full Text Available Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP. The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1 and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-. Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.

  6. Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells

    Science.gov (United States)

    Papaianni, Emanuela; El Maadidi, Souhayla; Schejtman, Andrea; Neumann, Simon; Maurer, Ulrich; Marino-Merlo, Francesca; Mastino, Antonio; Borner, Christoph

    2015-01-01

    Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells. PMID:26030884

  7. Clinicopathological significance of Bcl-2 and Bax protein expression in human pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Ming Dong; Jian-Ping Zhou; Hao Zhang; Ke-Jian Guo; Yu-Lin Tian; Yu-Ting Dong

    2005-01-01

    AIM: To assess the clinicopathological significance of the expression of the apoptosis-inhibitory Bcl-2 protein (pBcl-2) and the apoptosis-promoting Bax protein (pBax) in human invasive ductal carcinomas (IDCs) of the pancreas. METHODS: Fifty-nine surgical specimens of IDCs of the pancreas were stained immunohistochemically to detectpBcl-2 and pBax expressions whose correlation to tumor classification, staging, and prognosis was analyzed by univariate and multivariate analyses. RESULTS: The expression of pBcl-2 and pBax was detected in 21 of 59 (35.6%) and in 29 of 59 (49.2%) patients with IDCs of the pancreas, respectively. Neither pBcl-2 nor pBax alone was correlated to TNM staging and differentiation degree of IDCs of the pancreas according to univariate analysis. By Mantel-Cox test, the median survival time after surgery for pBcl-2(+) and pBcl-2(-) groups were 14.3 and 7.3 mo, respectively (χ2= 9.357, P = 0.002) and that for pBax(+) and pBax(-) groups were 12.9 and 10.2 mo, respectively (χ2= 0.285, P>0.05).Contingency coefficient between pBd-2 and pBax expression was 0.298, indicating that there was correlation between them (χ2= 5.74, P<0.05). The median survival time after surgery for pBd-2(+)pBax(+) and pBcl-2(+)pBax(-) groups were 14.3 and 14.1 mo, respectively, and that for pBcl-2 (-)pBax(+) and pBcl-2(-)pBax(-) groups were 5.9 and 9.9 mo, respectively. There was a significant difference between pBcl-2(+)pBax(+) and pBcl-2(-)pBax(+) (χ2 = 5.06,P<0.05), such was the case for pBcl-2(+)pBax(+) andpBcl-2(-)pBax(-) (χ2= 7.18, P<0.01). Cox proportional hazards model for multivariate analysis was applied, indicating that pBcl-2, TNM staging, age and pBax were high risk factors of post-surgical survival time. CONCLUSION: Both pBcl-2 and pBax have high expression in IDCs of the pancreas, indicating that co-expression of pBcl-2 and pBax is a good indicator of favorable prognosis in IDCs of the pancreas.

  8. THE EUROPEAN MODEL OF STATE REGULATION OF TOURISM ACTIVITIES

    Directory of Open Access Journals (Sweden)

    О. Davydova

    2013-11-01

    Full Text Available In the article the existing model of state regulation of the development of tourism. Expediency of the European model of state regulation of tourism development in Ukraine. It is noted that the European model of state regulation of tourism activities based on the coordination of marketing activities and the development of cooperation between the public and private sectors. The basic forms of public-private partnerships and the advantages of using cluster model of development of tourism, namely, contracts, production sharing agreement, lease, joint venture. Promising areas of application of the PPP identified the transport sector, housing and utilities, energy and tourism sector. The features of cluster formations in the country and the prospects for tourism clusters.

  9. Pentraxins in the activation and regulation of innate immunity.

    Science.gov (United States)

    Daigo, Kenji; Inforzato, Antonio; Barajon, Isabella; Garlanda, Cecilia; Bottazzi, Barbara; Meri, Seppo; Mantovani, Alberto

    2016-11-01

    Humoral fluid phase pattern recognition molecules (PRMs) are a key component of the activation and regulation of innate immunity. Humoral PRMs are diverse. We focused on the long pentraxin PTX3 as a paradigmatic example of fluid phase PRMs. PTX3 acts as a functional ancestor of antibodies and plays a non-redundant role in resistance against selected microbes in mouse and man and in the regulation of inflammation. This molecule interacts with complement components, thus modulating complement activation. In particular, PTX3 regulates complement-driven macrophage-mediated tumor progression, acting as an extrinsic oncosuppressor in preclinical models and selected human tumors. Evidence collected over the years suggests that PTX3 is a biomarker and potential therapeutic agent in humans, and pave the way to translation of this molecule into the clinic. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Neural progenitor cells regulate microglia functions and activity.

    Science.gov (United States)

    Mosher, Kira I; Andres, Robert H; Fukuhara, Takeshi; Bieri, Gregor; Hasegawa-Moriyama, Maiko; He, Yingbo; Guzman, Raphael; Wyss-Coray, Tony

    2012-11-01

    We found mouse neural progenitor cells (NPCs) to have a secretory protein profile distinct from other brain cells and to modulate microglial activation, proliferation and phagocytosis. NPC-derived vascular endothelial growth factor was necessary and sufficient to exert at least some of these effects in mice. Thus, neural precursor cells may not only be shaped by microglia, but also regulate microglia functions and activity.

  11. E2F is involved in radioresistance of carbon ion induced apoptosis via Bax/caspase 3 signal pathway in human hepatoma cell.

    Science.gov (United States)

    Xie, Yi; Si, Jing; Wang, Yu-Pei; Li, Hong-Yan; Di, Cui-Xia; Yan, Jun-Fang; Ye, Yan-Cheng; Zhang, Yan-Shan; Zhang, Hong

    2017-05-13

    Deletion of p53, most common genetic alteration, is observed in human tumors and reported to lead to improve in cell radioresistance. Heavy-ion irradiation (IR) could induce p53(-/-) cancer cells apoptosis. However, little is known regarding the molecular mechanism in this type of cell apoptosis. The present studies have focused on mechanisms state of signaling pathways as an activator of the cell fate decisions induced by heavy ion IR without p53. Carbon ion IR could induce up-regulation of E2F1 expression in cancer cells. This phenomenon was not observed in X-ray IR group. Up-regulation of E2F1 could cause a higher reduction in clonogenic survival, low level of cellular activity, G2 /M phase arrest, promotion of apoptosis rate, up-regulation of phosphor-Rb, Bax, and cleaved-caspase 3 proteins expressions without p53. Changes of E2F1 expressions could partly alter radioresistance in cancer cells. The results were suggested that heavy ion IR could induce p53(-/-) cancer cells apoptosis via E2F1 signal pathway. Our study provides a scientific rationale for the clinical use of heavy ion as radiotherapy in patients with p53-deficient tumors, which are often resistant to radiotherapy. © 2017 Wiley Periodicals, Inc.

  12. Active galactic nuclei activity: self-regulation from backflow

    Science.gov (United States)

    Antonuccio-Delogu, V.; Silk, Joseph

    2010-06-01

    We study the internal circulation within the cocoon carved out by the relativistic jet emanating from an active galactic nucleus (AGN) within the interstellar medium (ISM) of its host galaxy. First, we develop a model for the origin of the internal flow, noticing that a significant increase of large-scale velocity circulation within the cocoon arises as significant gradients in the density and entropy are created near the hotspot (a consequence of Crocco's vorticity generation theorem). We find simple and accurate approximate solutions for the large-scale flow, showing that a backflow towards the few inner parsec region develops. We solve the appropriate fluid dynamic equations, and we use these solutions to predict the mass inflow rates towards the central regions. We then perform a series of 2D simulations of the propagation of jets using FLASH 2.5, in order to validate the predictions of our model. In these simulations, we vary the mechanical input power between 1043 and 1045 ergs-1, and assume a Navarro-Frenk-White (NFW) density profile for the dark matter halo, within which an isothermal diffuse ISM is embedded. The backflows which arise supply the central AGN region with very low angular-momentum gas, at average rates of the order of , the exact value seen to be strongly dependent on the central ISM density (for fixed input jet power). The time-scales of these inflows are apparently weakly dependent on the jet/ISM parameters, and are of the order of . We then argue that these backflows could (at least partially) feed the AGN, and provide a self-regulatory mechanism of AGN activity, that is not directly controlled by, but instead controls, the star formation rate within the central circumnuclear disc.

  13. Signal integration by Ca2+ regulates intestinal stem cell activity

    Science.gov (United States)

    Deng, Hansong; Gerencser, Akos A.; Jasper, Heinrich

    2015-01-01

    Summary Somatic stem cells (SCs) maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here, we identify Ca2+ signaling as a central regulator of intestinal SC (ISC) activity in Drosophila. We find that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response and for an associated modulation of cytosolic Ca2+ oscillations that results in sustained high cytosolic Ca2+ concentrations. High cytosolic Ca2+ induces ISC proliferation by regulating Calcineurin and CREB - regulated transcriptional co-activator (CRTC). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca2+ oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca2+ levels allows effective integration of diverse mitogenic signals in ISCs to tailor their proliferative activity to the needs of the tissue. PMID:26633624

  14. How is AMPK activity regulated in skeletal muscles during exercise?

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian Beck; Rose, Adam John

    2008-01-01

    discuss the influence of reactive oxygen species produced within the muscle as well as muscle glycogen and TAK1 in regulating AMPK during exercise. Currently, during intensive contraction, activation of alpha2-AMPK seems mainly to rely on AMP accumulating from ATP-hydrolysis whereas calcium signaling may...

  15. Role of PDI in regulating tissue factor: FVIIa activity.

    Science.gov (United States)

    Popescu, Narcis I; Lupu, Cristina; Lupu, Florea

    2010-04-01

    Cell exposed tissue factor (TF) is generally in a low procoagulant ("cryptic") state, and requires an activation step (decryption) to exhibit its full procoagulant potential. Recent data suggest that TF decryption may be regulated by the redox environment through the oxidoreductase activity of protein disulfide isomerase (PDI). In this article we review PDI contribution to different models of TF decryption, namely the disulfide switch model and the phosphatidylserine dynamics, and hypothesize on PDI contribution to TF self-association and association with lipid domains. Experimental evidence debate the disulfide switch model of TF decryption and its regulation by PDI. More recently we showed that PDI oxidoreductase activity regulates the phosphatidylserine equilibrium at the plasma membrane. Interestingly, PDI reductase activity could maintain TF in the reduced monomeric form, while also maintaining low exposure of PS, both states correlated with low procoagulant function. In contrast, PDI inhibition or oxidants may promote the adverse effects with a net increase in coagulation. The relative contribution of disulfide isomerization and PS exposure needs to be further analyzed to understand the redox control of TF procoagulant function. For the moment however TF regulation remains cryptic.

  16. Neuronal Activity Regulates Hippocampal miRNA Expression

    Science.gov (United States)

    Eacker, Stephen M.; Keuss, Matthew J.; Berezikov, Eugene; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Neuronal activity regulates a broad range of processes in the hippocampus, including the precise regulation of translation. Disruptions in proper translational control in the nervous system are associated with a variety of disorders that fall in the autistic spectrum. MicroRNA (miRNA) represent a relatively recently discovered player in the regulation of translation in the nervous system. We have conducted an in depth analysis of how neuronal activity regulates miRNA expression in the hippocampus. Using deep sequencing we exhaustively identify all miRNAs, including 15 novel miRNAs, expressed in hippocampus of the adult mouse. We identified 119 miRNAs documented in miRBase but less than half of these miRNA were expressed at a level greater than 0.1% of total miRNA. Expression profiling following induction of neuronal activity by electroconvulsive shock demonstrates that most miRNA show a biphasic pattern of expression: rapid induction of specific mature miRNA expression followed by a decline in expression. These results have important implications into how miRNAs influence activity-dependent translational control. PMID:21984899

  17. Neuronal activity regulates hippocampal miRNA expression.

    Directory of Open Access Journals (Sweden)

    Stephen M Eacker

    Full Text Available Neuronal activity regulates a broad range of processes in the hippocampus, including the precise regulation of translation. Disruptions in proper translational control in the nervous system are associated with a variety of disorders that fall in the autistic spectrum. MicroRNA (miRNA represent a relatively recently discovered player in the regulation of translation in the nervous system. We have conducted an in depth analysis of how neuronal activity regulates miRNA expression in the hippocampus. Using deep sequencing we exhaustively identify all miRNAs, including 15 novel miRNAs, expressed in hippocampus of the adult mouse. We identified 119 miRNAs documented in miRBase but less than half of these miRNA were expressed at a level greater than 0.1% of total miRNA. Expression profiling following induction of neuronal activity by electroconvulsive shock demonstrates that most miRNA show a biphasic pattern of expression: rapid induction of specific mature miRNA expression followed by a decline in expression. These results have important implications into how miRNAs influence activity-dependent translational control.

  18. 76 FR 28801 - Agency Information Collection Activities: Bonded Warehouse Regulations

    Science.gov (United States)

    2011-05-18

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Bonded Warehouse... approval in accordance with the Paperwork Reduction Act: Bonded Warehouse Regulations. This is a proposed..., mechanical, or other technological techniques or other forms of information. Title: Bonded...

  19. Commission for Energy regulation (CRE) - Activity report June 2004

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2004-07-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2004 activity report of CRE. Content: A - Opening of the gas and electricity markets for professional customers on 1 July 2004; B - Regulation of the gas market: Gas markets and players (The European environment, The French gas market); Regulation of the gas market (Implementing regulation, Works planned for the coming year; C - Regulation of the electricity market: The electricity markets and players (The European electricity markets, The French electricity market, Monitoring the electricity market); Regulation of the French electricity market (Access to public grid, Cross-border exchanges, Un-bundled accounting principles); The public electricity service in the regulated market (Content of the public service, Public service charges, Electricity production public service financing, Electricity sales tariffs) D - The working of CRE: How CRE exercises its jurisdiction, Tools; E - Appendices: Glossary, Units and conversions, Council of European Energy Regulators, Index of tables and figures.

  20. The IRE1/bZIP60 pathway and bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana Plants

    DEFF Research Database (Denmark)

    Gaguancela, Omar Arias; Zúñiga, Lizbeth Peña; Arias, Alexis Vela

    2016-01-01

    The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that reg......The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor...

  1. Apoptosis Induction by Targeting Interferon Gamma Receptor 2 (IFNgammaR2) in Prostate Cancer: Ligand (IFNgamma)-Independent Novel Function of IFNgammaR2 as a Bax Inhibitor

    Science.gov (United States)

    2015-08-01

    inhibitor of Bax. Bax is a key mediator of apoptosis. We found that IFNγR2 is overexpressed in prostate cancer, and we hypothesize that abnormally high...We found that IFNγR2 levels are abnormally elevated in prostate cancer cell lines. Short hairpin (sh) RNA- mediated knockdown of IFNγR2 was able to... enhances Bax activation. (Months 1-24) Task 2: To identify the subtype of prostate cancer that can be effectively treated by IFNγR2-targeting

  2. Bz-423 superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax.

    Science.gov (United States)

    Blatt, Neal B; Boitano, Anthony E; Lyssiotis, Costas A; Opipari, Anthony W; Glick, Gary D

    2009-10-15

    Bz-423 is a pro-apoptotic 1,4-benzodiazepine with therapeutic properties in murine models of lupus demonstrating selectivity for autoreactive lymphocytes. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. In order to understand some of the features that contribute to the increased sensitivity of lymphocytes, we report the signaling pathway engaged by Bz-423 in a Burkitt lymphoma cell line (Ramos). Following the generation of superoxide, Bz-423-induced apoptosis requires the activation of Bax and Bak to induce mitochondrial outer membrane permeabilization and cytochrome c release. Knockdown of the BH3-only proteins Bad, Bim, Bik, and Puma inhibits Bz-423 apoptosis, suggesting that these proteins serve as upstream sensors of the oxidant stress induced by Bz-423. Treatment with Bz-423 results in superoxide-dependent Mcl-1 degradation, implicating this protein as the link between Bz-423-induced superoxide and Bax and Bak activation. In contrast to fibroblasts, B cell death induced by Bz-423 is independent of c-Jun N-terminal kinase. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a specific apoptotic response that differs across cell types.

  3. Impact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK.

    Science.gov (United States)

    Ke, F; Grabow, S; Kelly, G L; Lin, A; O'Reilly, L A; Strasser, A

    2015-10-22

    It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok(-/-)Bax(-/-)Bak(-/-) triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax(-/-)Bak(-/-) double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.

  4. Complement system part I - molecular mechanisms of activation and regulation

    Directory of Open Access Journals (Sweden)

    Nicolas eMerle

    2015-06-01

    Full Text Available Complement is a complex innate immune surveillance system, playing a key role in defense against pathogens and in host homeostasis. The complement system is initiated by conformational changes in recognition molecular complexes upon sensing danger signals. The subsequent cascade of enzymatic reactions is tightly regulated to assure that complement is activated only at specific locations requiring defense against pathogens, thus avoiding host tissue damage. Here we discuss the recent advances describing the molecular and structural basis of activation and regulation of the complement pathways and their implication on physiology and pathology. This article will review the mechanisms of activation of alternative, classical and lectin pathways, the formation of C3 and C5 convertases, the action of anaphylatoxins and the membrane attack complex. We will also discuss the importance of structure-function relationships using the example of atypical hemolytic uremic syndrome. Lastly we will discuss the development and benefits of therapies using complement inhibitors.

  5. Histological structure and expression of Bax protein in Pavo cristatus kidney%白孔雀肾脏的组织结构及Bax蛋白在肾脏中的表达

    Institute of Scientific and Technical Information of China (English)

    俞诗源; 王小勇; 吴勍

    2012-01-01

    To study Pavo cristatus kidney histological structure and the expression of relevant active substances, histological structure is obvserved by light microscopy and expression of Bax protein is examined by immunohistochemical methods. The results indicate that Pavo cristatus kidney consists of many nephrons, collecting ducts and small amounts of areolar tissue. The glomerular capillary is simpler, renal tubules arrange closely and the quantity is more. The proximal tubule consists of monolayer cuboidal epithelium cells, with brush border and deeper color. Bax protein immune response positive material is mainly distributed in proximal tubule epithelium. Bax protein may be involved in the cell apoptosis of Pavo cristatus kidney and plays an important regulation role in the development of bird kidney.%为了搞清白孔雀(Pavo cristatus)肾脏的结构特征和相关活性物质的表达问题,利用生物显微技术观察了白孔雀肾脏的组织结构,用免疫组织化学方法检测了Bax蛋白在肾组织中的表达.结果显示白孔雀肾脏主要由许多肾单位、集合管和少量结缔组织组成.白孔雀肾小球毛细血管网较简单,肾小管之间排列紧密,数量较多,近端小管由单层立方上皮细胞组成,细胞顶端有刷状缘,细胞着色较深;Bax蛋白免疫反应阳性物质主要分布在近端小管上皮细胞;Bax蛋白可能与白孔雀肾脏细胞的凋亡有关.

  6. Molecular and immunohistochemical expression of apoptotic proteins Bax, Bcl-2 and Caspase 3 in infantile hemangioma tissues as an effect of propranolol treatment.

    Science.gov (United States)

    Wnęk, Aneta; Andrzejewska, Ewa; Kobos, Józef; Taran, Katarzyna; Przewratil, Przemysław

    2017-05-01

    Infantile hemangiomas (IHs) are the most common benign tumors of childhood. They are characterized by a unique clinical course with two phases, proliferation and involution, which are followed by regression. The therapy of infantile hemangiomas was revolutionized in 2008 by the introduction of propranolol, however, the mechanism of its influence on hemangiomas remains unclear. The study included 71 patients with IHs, 27 of whom were treated with propranolol while the remaining 44 were used as a comparative group. The expression of Bcl-2, Bax and Caspase3 was determined with immunohistochemistry and mRNA of Bax, Bcl-2 and Caspase3 were assessed with the use of RT-PCR. Both methods revealed a statistically significant decrease in Bcl-2 expression and an increase in Bax in IHs tissues after propranolol treatment. The results obtained for Bax and Bcl-2 proteins may indicate a link between the effect of propranolol and apoptosis. Higher Bax and lower Bcl-2 expression in the propranolol treated group indicates a strong pro- apoptotic action countering any anti-apoptotic activity; apoptosis was indicted in IH tissue as a potential result of propranolol treatment, with potential clinical impact in other tumors. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  7. Mode of cell death induced by the HSP90 inhibitor 17-AAG (tanespimycin) is dependent on the expression of pro-apoptotic BAX

    Science.gov (United States)

    Powers, Marissa V; Valenti, Melanie; Miranda, Susana; Maloney, Alison; Eccles, Suzanne A.; Thomas, George; Clarke, Paul A; Workman, Paul

    2013-01-01

    Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both in vitro and in tumor xenografts in vivo. Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents. PMID:24185264

  8. Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis.

    Directory of Open Access Journals (Sweden)

    Keqing Wang

    Full Text Available Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

  9. Associations of NF-kappaB and Bax with Apoptosis in Varicose Veins of Women of Different Age Groups

    Directory of Open Access Journals (Sweden)

    Helle Evi Simovart

    2011-01-01

    Full Text Available The study aimed at detecting apoptotic endothelial cells (ECs and smooth muscle cells (SMCs together with determining expression of NF-kappaB (p105/p50 and Bax in varicose vein walls. Women (n=35 undergoing the excision of varicose veins were divided into 3 groups: younger than 35 years (I, 36–50 years (II, and older than 50 years (III. Apoptosis was determined by the TUNEL method, NF-kappaB and Bax expression by immunohistochemistry. The percentage of apoptotic ECs and SMCs in the layers of varicose vein wall increased in groups II and III. NF-kappaB expression had the lowest level in Group II with particularly low level in the media. Contrariwise, Bax expression levels in Group II were increased. The study revealed that in varicose veins ECs and SMCs apoptosis increased with advancing age. If increase in apoptosis during earlier stages of varicosities is probably regulated by intrinsic pathway, then in older patients other signaling pathways may be involved.

  10. Bromelain inhibits COX-2 expression by blocking the activation of MAPK regulated NF-kappa B against skin tumor-initiation triggering mitochondrial death pathway.

    Science.gov (United States)

    Bhui, Kulpreet; Prasad, Sahdeo; George, Jasmine; Shukla, Yogeshwer

    2009-09-18

    Chemoprevention impels the pursuit for either single targeted or cocktail of multi-targeted agents. Bromelain, potential agent in this regard, is a pharmacologically active compound, present in stems and fruits of pineapple (Ananas cosmosus), endowed with anti-inflammatory, anti-invasive and anti-metastatic properties. Herein, we report the anti tumor-initiating effects of bromelain in 2-stage mouse skin tumorigenesis model. Pre-treatment of bromelain resulted in reduction in cumulative number of tumors (CNT) and average number of tumors per mouse. Preventive effect was also comprehended in terms of reduction in tumor volume up to a tune of approximately 65%. Components of the cell signaling pathways, connecting proteins involved in cell death were targeted. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in Bcl-2. A marked inhibition in cyclooxygenase-2 (Cox-2) expression and inactivation of nuclear factor-kappa B (NF-kappaB) was recorded, as phosphorylation and consequent degradation of I kappa B alpha was blocked by bromelain. Also, bromelain treatment curtailed extracellular signal regulated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and Akt activity. The basis of anti tumor-initiating activity of bromelain was revealed by its time dependent reduction in DNA nick formation and increase in percentage prevention. Thus, modulation of inappropriate cell signaling cascades driven by bromelain is a coherent approach in achieving chemoprevention.

  11. Cbl negatively regulates JNK activation and cell death

    Institute of Scientific and Technical Information of China (English)

    Andrew A Sproul; Zhiheng Xu; Michael Wilhelm; Stephen Gire; Lloyd A Greene

    2009-01-01

    Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apopto-sis--nerve growth factor (NGF) deprivation and DNA damage--cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation (and most likely, activa-tion) of c-Cbl. Targeting e-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase (JNK) signaling. We demonstrate that Cbl pro-teins interact with the JNK pathway components mixed lineage kinase (MLK) 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK acti-vation and on cell death.

  12. The effect of octreotide and bromocriptine on expression of a pro-apoptotic Bax protein in rat prolactinoma.

    Directory of Open Access Journals (Sweden)

    Jolanta Kunert-Radek

    2004-03-01

    Full Text Available It is well established that disruption of apoptosis may lead to tumor initiation, progression or metastasis. It is also well documented that many anticancer drugs induce apoptosis. In the earlier studies, the dopamine D2 receptor agonist bromocriptine (BC and somatostatin analog octreotide (OCT were found to inhibit the growth of the estrogen-induced rat prolactinoma. Our previous investigations, applying the TUNEL method showed the involvement of the pro-apoptotic effect in the action of BC, and to a lesser degree, in the action of OCT. The aim of the present study was to investigate whether the pro-apoptotic action of these drugs involves the increased expression of Bax--a member of Bcl-2 protein family which is known to play an important role in the regulation of apoptosis. Male four-week Fisher 344 rats were used in the experiment. Capsules containing diethylstilboestrol (DES were implanted subcutaneously. Six weeks after the implantation the rats were given OCT (2 x 25 microg/animal/24, BC (3 mg/kg b.w./24 h or OCT and BC at the above doses for 10 days. Bax expression was detected by immunohistochemistry. Prolactin (PRL in blood serum was measured by radioimmunoassay (RIA. It has been found that both OCT and BC, alone or in combination, significantly reduce the tumor weight. Both OCT and BC suppressed PRL levels, but the inhibitory effect of BC was stronger than that of OCT. It has been found that the treatment with OCT and BC, alone or in combination, causes a significant increase in Bax expression in the rat prolactinoma cells. Our findings indicate that anti-tumoral action of bromocriptine and to some extent the action of octreotide in the experimental rat prolactinoma is connected with the induction of apoptosis and is associated with increased Bax expression.

  13. CYLD regulates RhoA activity by modulating LARG ubiquitination.

    Directory of Open Access Journals (Sweden)

    Yunfan Yang

    Full Text Available Rho family guanosine triphosphatases (GTPases, such as RhoA, Cdc42, and Rac1, play a fundamental role in various cellular processes. The activation of Rho proteins is catalyzed by guanine nucleotide-exchange factors (GEFs, which promote the exchange of GDP for GTP. The precise mechanisms regulating the activation of Rho proteins are not fully understood. Herein, we demonstrate that RhoA activity is regulated by cylindromatosis (CYLD, a deubiquitinase harboring multiple functions. In addition, we find that RhoA-mediated cytoskeletal rearrangement, chromosome separation, and cell polarization are altered in CYLD-depleted cells. Mechanistically, CYLD does not interact with RhoA; instead, it interacts with and deubiquitinates leukemia-associated RhoGEF (LARG. Our data further show that CYLD-mediated deubiquitination of LARG enhances its ability to stimulate the GDP/GTP exchange on RhoA. These data thus identify LARG as a new substrate of CYLD and provide novel insights into the regulation of RhoA activation. Our results also suggest that the LARG-RhoA signaling pathway may play a role in diverse CYLD-mediated cellular events.

  14. CYLD regulates RhoA activity by modulating LARG ubiquitination.

    Science.gov (United States)

    Yang, Yunfan; Sun, Lei; Tala; Gao, Jinmin; Li, Dengwen; Zhou, Jun; Liu, Min

    2013-01-01

    Rho family guanosine triphosphatases (GTPases), such as RhoA, Cdc42, and Rac1, play a fundamental role in various cellular processes. The activation of Rho proteins is catalyzed by guanine nucleotide-exchange factors (GEFs), which promote the exchange of GDP for GTP. The precise mechanisms regulating the activation of Rho proteins are not fully understood. Herein, we demonstrate that RhoA activity is regulated by cylindromatosis (CYLD), a deubiquitinase harboring multiple functions. In addition, we find that RhoA-mediated cytoskeletal rearrangement, chromosome separation, and cell polarization are altered in CYLD-depleted cells. Mechanistically, CYLD does not interact with RhoA; instead, it interacts with and deubiquitinates leukemia-associated RhoGEF (LARG). Our data further show that CYLD-mediated deubiquitination of LARG enhances its ability to stimulate the GDP/GTP exchange on RhoA. These data thus identify LARG as a new substrate of CYLD and provide novel insights into the regulation of RhoA activation. Our results also suggest that the LARG-RhoA signaling pathway may play a role in diverse CYLD-mediated cellular events.

  15. Lhx8 regulates primordial follicle activation and postnatal folliculogenesis.

    Science.gov (United States)

    Ren, Yu; Suzuki, Hitomi; Jagarlamudi, Krishna; Golnoski, Kayla; McGuire, Megan; Lopes, Rita; Pachnis, Vassilis; Rajkovic, Aleksandar

    2015-06-16

    The early stages of ovarian follicle formation-beginning with the breakdown of germ cell cysts and continuing with the formation of primordial follicles and transition to primary and secondary follicles-are critical in determining reproductive life span and fertility. Previously, we discovered that global knockouts of germ cell-specific transcriptional co-regulators Sohlh1, Sohlh2, Lhx8, and Nobox, cause rapid oocyte loss and ovarian failure. Also factors such as Nobox and Sohlh1 are associated with human premature ovarian failure. In this study, we developed a conditional knockout of Lhx8 to study oocyte-specific pathways in postnatal folliculogenesis. The conditional deficiency of Lhx8 in the oocytes of primordial follicles leads to massive primordial oocyte activation, in part, by indirectly interacting with the PI3K-AKT pathway, as shown by synergistic effects on FOXO3 nucleocytoplasmic translocation and rpS6 activation. However, LHX8 does not directly regulate members of the PI3K-AKT pathway; instead, we show that LHX8 represses Lin28a expression, a known regulator of mammalian metabolism and of the AKT/mTOR pathway. LHX8 can bind to the Lin28a promoter, and the depletion of Lin28a in Lhx8-deficient oocytes partially suppresses primordial oocyte activation. Moreover, unlike the PI3K-AKT pathway, LHX8 is critical beyond primordial follicle activation, and blocks the primary to secondary follicle transition. Our results indicate that the LHX8-LIN28A pathway is essential in the earliest stages of primordial follicle activation, and LHX8 is an important oocyte-specific transcription factor in the ovary for regulating postnatal folliculogenesis.

  16. Alexithymia influences brain activation during emotion perception but not regulation.

    Science.gov (United States)

    van der Velde, Jorien; Gromann, Paula M; Swart, Marte; Wiersma, Durk; de Haan, Lieuwe; Bruggeman, Richard; Krabbendam, Lydia; Aleman, André

    2015-02-01

    Alexithymia is a psychological construct that can be divided into a cognitive and affective dimension. The cognitive dimension is characterized by difficulties in identifying, verbalizing and analysing feelings. The affective dimension comprises reduced levels of emotional experience and imagination. Alexithymia is widely regarded to arise from an impairment of emotion regulation. This is the first functional magnetic resonance imaging (fMRI) study to critically evaluate this by investigating the neural correlates of emotion regulation as a function of alexithymia levels. The aim of the current study was to investigate the neural correlates underlying the two alexithymia dimensions during emotion perception and emotion regulation. Using fMRI, we scanned 51 healthy subjects while viewing, reappraising or suppressing negative emotional pictures. The results support the idea that cognitive alexithymia, but not affective alexithymia, is associated with lower activation in emotional attention and recognition networks during emotion perception. However, in contrast with several theories, no alexithymia-related differences were found during emotion regulation (neither reappraisal nor suppression). These findings suggest that alexithymia may result from an early emotion processing deficit rather than compromised frontal circuits subserving higher-order emotion regulation processes.

  17. Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling

    Directory of Open Access Journals (Sweden)

    Dyugovskaya Larissa

    2012-10-01

    Full Text Available Abstract Background Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA characterized by nightly intermittent hypoxia (IH. This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated. Methods Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis. Results Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated

  18. Neuronal activity-induced regulation of Lingo-1.

    Science.gov (United States)

    Trifunovski, Alexandra; Josephson, Anna; Ringman, Andreas; Brené, Stefan; Spenger, Christian; Olson, Lars

    2004-10-25

    Axonal regeneration after injury can be limited in the adult CNS by the presence of inhibitory proteins such as Nogo. Nogo binds to a receptor complex that consists of Nogo receptor (NgR), p75NTR, and Lingo-1. Nogo binding activates RhoA, which inhibits axonal outgrowth. Here we assessed Lingo-1 and NgR mRNA levels after delivery of BDNF into the rat hippocampal formation, Lingo-1 mRNA levels in rats subjected to kainic acid (KA) and running in running wheels. Lingo-1 mRNA was not changed by running. However, we found that Lingo-1 mRNA was strongly up-regulated while NgR mRNA was down-regulated in the dentate gyrus in both the BDNF and the KA experiments. Our data demonstrate inverse regulation of NgR and Lingo-1 in these situations, suggesting that Lingo-1 up-regulation is one characteristic of activity-induced neural plasticity responses.

  19. Commission for Energy regulation (CRE) - Activity report June 2007

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2007-07-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2007 activity report of CRE. Content: A - Towards a single European energy market: Birth of a single European energy market (Origins of Europe of Energy, Emergence of a European energy policy); Main European Community guiding lines (European governance as regards energy, Guiding principles for the internal energy market); European Community activities (European Commission reports, Electricity and gas Regional Initiatives); Organisation and coordination of European regulators (Joint organisation of European regulators, CRE's relations with European Community institutions); CRE's European activities (Regional integration of gas markets, Regional integration of electricity markets, Operation of the European interconnected electricity grid and security of supply, CRE's other European activities); B - CRE action at national level: Grids/networks and infrastructures (General information, Electricity grids, Regulation of gas networks and infrastructures); Markets (Changes in the regulatory and legislative contexts of electricity and natural gas markets, Electricity markets

  20. Regulation of burstiness by network-driven activation

    CERN Document Server

    García-Pérez, Guillermo; Serrano, M Ángeles

    2014-01-01

    We prove that complex networks of interactions have the capacity to regulate and buffer unpredictable fluctuations in production events. We show that non-bursty network-driven activation dynamics can effectively regulate the level of burstiness in the production of nodes, which can be enhanced or reduced. Burstiness can be induced even when the endogenous inter-event time distribution of nodes' production is non-bursty. We found that hubs tend to be less controllable than low degree nodes, which are more susceptible to the networked regulatory effects. Our results have important implications for the analysis and engineering of bursty activity in a range of systems, from telecommunication networks to transcription and translation of genes into proteins in cells.

  1. Shape regulation generates elastic interaction between active force dipoles

    CERN Document Server

    Golkov, Roman

    2016-01-01

    The organization of live cells to tissues is associated with the mechanical interaction between cells, which is mediated through their mechanical environment. We model live cells as spherical active force dipoles surrounded by an infinite elastic matrix, and analytically evaluate their elastic interaction energy for different scenarios of their regulatory behavior. For purely dilational eigenstrains the elastic interaction energy between any two bodies vanishes. We identify mechanical interactions between active cells applying non isotropic displacements with a regulation mechanism designed so that they will preserve their spherical shape. We express the resultant non-isotropic deformation field by a multipole expansion in terms of spherical harmonics. Mechanical self-regulation of live cells is not fully understood, and we compare homeostatic (set point) force applied by the cells on their environment versus homeostatic displacements on their surface. By including or excluding the first term of the expansion...

  2. JNK3 phosphorylates Bax protein and induces ability to form pore on bilayer lipid membrane

    Directory of Open Access Journals (Sweden)

    Rajeev Gupta

    2017-06-01

    Full Text Available Bax is a pro-apoptotic cytosolic protein. In this work native (unphosphorylated and JNK3 phosphorylated Bax proteins are studied on artificial bilayer membranes for pore formation. Phosphorylated Bax formed pore on the bilayer lipid membrane whereas native one does not. In cells undergoing apoptosis the pore formed by the phosphorylated Bax could be important in cytochrome c release from the mitochondrial intermembrane space to the cytosol. The low conductance (1.5 nS of the open state of the phosphorylated Bax pore corresponds to pore diameter of 0.9 nm which is small to release cytochrome c (∼3.4 nm. We hypothesized that JNK3 phosphorylated Bax protein can form bigger pores after forming complexes with other mitochondrial proteins like VDAC, t-Bid etc. to release cytochrome c.

  3. A Rewriting Framework and Logic for Activities Subject to Regulations

    Science.gov (United States)

    2015-02-28

    Lincoln, N. Martı́-Oliet, J. Meseguer, and C. Talcott. All About Maude: A High-Performance Logical Framework. Springer , 2007. R. Corin, S. Etalle, P. H...Under consideration for publication in Math. Struct. in Comp. Science A Rewriting Framework and Logic for Activities Subject to Regulations M A X K A...whenever applied. We present a formal semantics of our model based on focused proofs of linear logic with definitions. We also determine the

  4. Preparation and Photocatalytic Properties of Sr2−xBaxTa3O10−yNz Nanosheets

    Directory of Open Access Journals (Sweden)

    Tatsumi Ishihara

    2013-01-01

    Full Text Available Sr2−xBaxTa3O10−yNz (x = 0.0, 0.5, 1.0 nanosheets were prepared by exfoliating layered perovskite compounds (CsSr2−xBaxTa3O10−yNz. The Sr1.5Ba0.5Ta3O9.7N0.2 nanosheet showed the highest photocatalytic activity for H2 production from the water/methanol system among the Sr2−xBaxTa3O9.7N0.2 nanosheets prepared. In addition, Rh-loaded Sr1.5Ba0.5Ta3O9.6N0.3 nanosheet showed the photocatalytic activity for oxygen and hydrogen production from water. The ratio of hydrogen to oxygen evolved was around two. These results indicate that the Rh-loaded Sr1.5Ba0.5Ta3O9.6N0.3 nanosheet is a potential catalyst for photocatalytic water splitting.

  5. Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

    KAUST Repository

    Radogna, Flavia

    2015-03-01

    Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10-9 vs. 10-5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10-5M, but drops to 10-9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

  6. Harvester ants use interactions to regulate forager activation and availability.

    Science.gov (United States)

    Pinter-Wollman, Noa; Bala, Ashwin; Merrell, Andrew; Queirolo, Jovel; Stumpe, Martin C; Holmes, Susan; Gordon, Deborah M

    2013-07-01

    Social groups balance flexibility and robustness in their collective response to environmental changes using feedback between behavioural processes that operate at different timescales. Here we examine how behavioural processes operating at two timescales regulate the foraging activity of colonies of the harvester ant, Pogonomyrmex barbatus, allowing them to balance their response to food availability and predation. Previous work showed that the rate at which foragers return to the nest with food influences the rate at which foragers leave the nest. To investigate how interactions inside the nest link the rates of returning and outgoing foragers, we observed outgoing foragers inside the nest in field colonies using a novel observation method. We found that the interaction rate experienced by outgoing foragers inside the nest corresponded to forager return rate, and that the interactions of outgoing foragers were spatially clustered. Activation of a forager occurred on the timescale of seconds: a forager left the nest 3-8 s after a substantial increase in interactions with returning foragers. The availability of outgoing foragers to become activated was adjusted on the timescale of minutes: when forager return was interrupted for more than 4-5 min, available foragers waiting near the nest entrance went deeper into the nest. Thus, forager activation and forager availability both increased with the rate at which foragers returned to the nest. This process was checked by negative feedback between forager activation and forager availability. Regulation of foraging activation on the timescale of seconds provides flexibility in response to fluctuations in food abundance, whereas regulation of forager availability on the timescale of minutes provides robustness in response to sustained disturbance such as predation.

  7. PLAP-1/Asporin Positively Regulates FGF-2 Activity.

    Science.gov (United States)

    Awata, T; Yamada, S; Tsushima, K; Sakashita, H; Yamaba, S; Kajikawa, T; Yamashita, M; Takedachi, M; Yanagita, M; Kitamura, M; Murakami, S

    2015-10-01

    PLAP-1 is an extracellular matrix protein that is predominantly expressed in the periodontal ligament within periodontal tissue. It was previously revealed that PLAP-1 negatively regulates bone morphogenetic protein 2 and transforming growth factor β activity through direct interactions. However, the interaction between PLAP-1 and other growth factors has not been defined. Here, we revealed that PLAP-1 positively regulates the activity of fibroblast growth factor 2 (FGF-2), a critical growth factor in tissue homeostasis and repair. In this study, we isolated mouse embryonic fibroblasts (MEFs) from Plap-1(-/-) mice generated in our laboratory. Interestingly, Plap-1(-/-) MEFs exhibited enhanced responses to bone morphogenetic protein 2 but defective responses to FGF-2, and Plap-1 transfection into Plap-1(-/-) MEFs rescued these defective responses. In addition, binding assays revealed that PLAP-1 promotes FGF-2-FGF receptor 1 (FGFR1) complex formation by direct binding to FGF-2. Immunocytochemistry analyses revealed colocalization of PLAP-1 and FGF-2 in wild-type MEFs and reduced colocalization of FGF-2 and FGFR1 in Plap-1(-/-) MEFs compared with wild-type MEFs. Taken together, PLAP-1 positively regulates FGF-2 activity through a direct interaction. Extracellular matrix-growth factor interactions have considerable effects; thus, this approach may be useful in several regenerative medicine applications.

  8. Length regulation of active biopolymers by molecular motors.

    Science.gov (United States)

    Johann, Denis; Erlenkämper, Christoph; Kruse, Karsten

    2012-06-22

    For biopolymers like cytoskeletal actin filaments and microtubules, assembly and disassembly are inherently dissipative processes. Molecular motors can affect the rates of subunit removal at filament ends. We introduce a driven lattice-gas model to study the effects of motor-induced depolymerization on the length of active biopolymers and find that increasing motor activity sharpens unimodal steady-state length distributions. Furthermore, for sufficiently fast moving motors, the relative width of the length distribution is determined only by the attachment rate of motors. Our results show how established molecular processes can be used to robustly regulate the size of cytoskeletal structures like mitotic spindles.

  9. Regulation of eNOS enzyme activity by posttranslational modification.

    Science.gov (United States)

    Heiss, Elke H; Dirsch, Verena M

    2014-01-01

    The regulation of endothelial NO synthase (eNOS) employs multiple different cellular control mechanisms impinging on level and activity of the enzyme. This review aims at summarizing the current knowledge on the posttranslational modifications of eNOS, including acylation, nitrosylation, phosphorylation, acetylation, glycosylation and glutathionylation. Sites, mediators and impact on enzyme localization and activity of the single modifications will be discussed. Moreover, interdependence, cooperativity and competition between the different posttranslational modifications will be elaborated with special emphasis on the susceptibility of eNOS to metabolic cues.

  10. Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila.

    Directory of Open Access Journals (Sweden)

    Fan Xia

    2008-05-01

    Full Text Available The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf, which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.

  11. ATPase activity of the cystic fibrosis transmembrane conductance regulator.

    Science.gov (United States)

    Li, C; Ramjeesingh, M; Wang, W; Garami, E; Hewryk, M; Lee, D; Rommens, J M; Galley, K; Bear, C E

    1996-11-08

    The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.

  12. Methamphetamine Regulation of Firing Activity of Dopamine Neurons.

    Science.gov (United States)

    Lin, Min; Sambo, Danielle; Khoshbouei, Habibeh

    2016-10-05

    Methamphetamine (METH) is a substrate for the dopamine transporter that increases extracellular dopamine levels by competing with dopamine uptake and increasing reverse transport of dopamine via the transporter. METH has also been shown to alter the excitability of dopamine neurons. The mechanism of METH regulation of the intrinsic firing behaviors of dopamine neurons is less understood. Here we identified an unexpected and unique property of METH on the regulation of firing activity of mouse dopamine neurons. METH produced a transient augmentation of spontaneous spike activity of midbrain dopamine neurons that was followed by a progressive reduction of spontaneous spike activity. Inspection of action potential morphology revealed that METH increased the half-width and produced larger coefficients of variation of the interspike interval, suggesting that METH exposure affected the activity of voltage-dependent potassium channels in these neurons. Since METH has been shown to affect Ca(2+) homeostasis, the unexpected findings that METH broadened the action potential and decreased the amplitude of afterhyperpolarization led us to ask whether METH alters the activity of Ca(2+)-activated potassium (BK) channels. First, we identified BK channels in dopamine neurons by their voltage dependence and their response to a BK channel blocker or opener. While METH suppressed the amplitude of BK channel-mediated unitary currents, the BK channel opener NS1619 attenuated the effects of METH on action potential broadening, afterhyperpolarization repression, and spontaneous spike activity reduction. Live-cell total internal reflection fluorescence microscopy, electrophysiology, and biochemical analysis suggest METH exposure decreased the activity of BK channels by decreasing BK-α subunit levels at the plasma membrane.

  13. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Wei-Jie; Wang, Sheng [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Hu, Zhuang, E-mail: zhuanghu475000@sina.com [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China); Zhou, Zhen-Yu; Song, Cai-Juan [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China)

    2015-11-20

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

  14. The IRE1/bZIP60 pathway and Bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants

    Science.gov (United States)

    The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor and when activated it splices the bZIP60 mRNA producing a truncated transcription factor that upregulates expression of genes involved in the unfolded protein response (UPR). Bax inhibitor 1 (BI-1) is another ER stre...

  15. Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

    DEFF Research Database (Denmark)

    Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina

    2004-01-01

    Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S-transfer......Bax-induced lethality in yeast is accompanied by morphological changes in mitochondria, giving rise to a reduced number of swollen tubules. Although these changes are completely abolished upon coexpression of the Bax inhibitor, Bcl-2, coexpression of Bax with Bax inhibiting-glutathione S......-transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria. In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic. While Bcl-2 coexpression only partially corrects...

  16. Schiff Base Metal Derivatives Enhance the Expression of HSP70 and Suppress BAX Proteins in Prevention of Acute Gastric Lesion

    Directory of Open Access Journals (Sweden)

    Shahram Golbabapour

    2013-01-01

    Full Text Available Schiff base complexes have appeared to be promising in the treatment of different diseases and disorders and have drawn a lot of attention to their biological activities. This study was conducted to evaluate the regulatory effect of Schiff base metal derivatives on the expression of heat shock proteins (HSP 70 and BAX in protection against acute haemorrhagic gastric ulcer in rats. Rats were assigned to 6 groups of 6 rats: the normal control (Tween 20 5% v/v, 5 mL/kg, the positive control (Tween 20 5% v/v, 5 mL/kg, and four Schiff base derivative groups named Schiff_1, Schiff_2, Schiff_3, and Schiff_4 (25 mg/kg. After 1 h, all of the groups received ethanol 95% (5 mL/kg but the normal control received Tween 20 (Tween 20 5% v/v, 5 mL/kg. The animals were euthanized after 60 min and the stomachs were dissected for histology (H&E, immunohistochemistry, and western blot analysis against HSP70 and BAX proteins. The results showed that the Schiff base metal derivatives enhanced the expression of HSP70 and suppressed the expression of BAX proteins during their gastroprotection against ethanol-induced gastric lesion in rats.

  17. Near infrared radiation protects against oxygen-glucose deprivation-induced neurotoxicity by down-regulating neuronal nitric oxide synthase (nNOS) activity in vitro.

    Science.gov (United States)

    Yu, Zhanyang; Li, Zhaoyu; Liu, Ning; Jizhang, Yunneng; McCarthy, Thomas J; Tedford, Clark E; Lo, Eng H; Wang, Xiaoying

    2015-06-01

    Near infrared radiation (NIR) has been shown to be neuroprotective against neurological diseases including stroke and brain trauma, but the underlying mechanisms remain poorly understood. In the current study we aimed to investigate the hypothesis that NIR may protect neurons by attenuating oxygen-glucose deprivation (OGD)-induced nitric oxide (NO) production and modulating cell survival/death signaling. Primary mouse cortical neurons were subjected to 4 h OGD and NIR was applied at 2 h reoxygenation. OGD significantly increased NO level in primary neurons compared to normal control, which was significantly ameliorated by NIR at 5 and 30 min post-NIR. Neither OGD nor NIR significantly changed neuronal nitric oxide synthase (nNOS) mRNA or total protein levels compared to control groups. However, OGD significantly increased nNOS activity compared to normal control, and this effect was significantly diminished by NIR. Moreover, NIR significantly ameliorated the neuronal death induced by S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor. Finally, NIR significantly rescued OGD-induced suppression of p-Akt and Bcl-2 expression, and attenuated OGD-induced upregulation of Bax, BAD and caspase-3 activation. These results suggest NIR may protect against OGD at least partially through reducing NO production by down-regulating nNOS activity, and modulating cell survival/death signaling.

  18. Hsp90 regulation of fibroblast activation in pulmonary fibrosis

    Science.gov (United States)

    Sontake, Vishwaraj; Wang, Yunguan; Kasam, Rajesh K.; Sinner, Debora; Reddy, Geereddy B.; Naren, Anjaparavanda P.; McCormack, Francis X.; Jegga, Anil G.; Madala, Satish K.

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease associated with fibroblast activation that includes excessive proliferation, tissue invasiveness, myofibroblast transformation, and extracellular matrix (ECM) production. To identify inhibitors that can attenuate fibroblast activation, we queried IPF gene signatures against a library of small-molecule-induced gene-expression profiles and identified Hsp90 inhibitors as potential therapeutic agents that can suppress fibroblast activation in IPF. Although Hsp90 is a molecular chaperone that regulates multiple processes involved in fibroblast activation, it has not been previously proposed as a molecular target in IPF. Here, we found elevated Hsp90 staining in lung biopsies of patients with IPF. Notably, fibroblasts isolated from fibrotic lesions showed heightened Hsp90 ATPase activity compared with normal fibroblasts. 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a small-molecule inhibitor of Hsp90 ATPase activity, attenuated fibroblast activation and also TGF-β–driven effects on fibroblast to myofibroblast transformation. The loss of the Hsp90AB, but not the Hsp90AA isoform, resulted in reduced fibroblast proliferation, myofibroblast transformation, and ECM production. Finally, in vivo therapy with 17-AAG attenuated progression of established and ongoing fibrosis in a mouse model of pulmonary fibrosis, suggesting that targeting Hsp90 represents an effective strategy for the treatment of fibrotic lung disease. PMID:28239659

  19. Commission for Energy regulation (CRE) - Activity report june 2006

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2006-07-01

    CRE is the French commission for energy regulation. CRE's remit is to assist in ensuring the proper operation of the electricity and natural gas markets for the benefit of the end-user. In particular, CRE ensures that the conditions of access to electricity and natural gas transmission and distribution systems do not hinder the development of competition. It monitors, for the electricity and natural gas sectors, all transactions made between suppliers, traders and producers, all transactions made on the organised markets and cross-border trading. It ensures that suppliers, traders and producers propose offers that are consistent with their financial and technical constraints. It monitors the implementation of and compliance with regulations giving consumers the right to choose their supplier in a competitive market, and allowing new suppliers to enter the market. This document is the 2006 activity report of CRE. Content: A - Opening of the electricity and natural gas markets to household consumers on 1 July 2007: CRE at the service of eligible customers (Information for eligible customers, Improved knowledge of non-household customers); Monitoring of the non-discrimination, transparency and independence of system operators (Drafting and distribution of codes of good conduct for system operators, The necessary improvement of system operator independence); Preparing the practical methods of opening: GTE 2007 and GTG 2007 (The necessary simplification of relations between operators and customers, Achieving a greater level of consumer information and protection, The clearly defined stages of the 'customer pathway', Profiling and settlement mechanisms: turning experience feedback from 2004 to good account); Persisting uncertainties and hurdles (The need for a suitable regulatory and legislative platform, Hurdles to the opening of the household market); B - Regulation of the natural gas market: The gas market in the European context (Increasing weight of

  20. Active pre-filters for dc/dc Boost regulators

    Directory of Open Access Journals (Sweden)

    Carlos Andrés Ramos-Paja

    2014-07-01

    Full Text Available This paper proposes an active pre-filter to mitigate the current harmonics generated by classical dc/dc Boost regulators, which generate current ripples proportional to the duty cycle. Therefore, high output voltage conditions, i.e., high voltage conversion ratios, produce high current harmonics that must be filtered to avoid damage or source losses. Traditionally, these current components are filtered using electrolytic capacitors, which introduce reliability problems because of their high failure rate. The solution introduced in this paper instead uses a dc/dc converter based on the parallel connection of the Boost canonical cells to filter the current ripples generated by the Boost regulator, improving the system reliability. This solution provides the additional benefits of improving the overall efficiency and the voltage conversion ratio. Finally, the solution is validated with simulations and experimental results.

  1. Epigenetic regulation of hepatic stellate cell activation and liver fibrosis.

    Science.gov (United States)

    El Taghdouini, Adil; van Grunsven, Leo A

    2016-12-01

    Chronic liver injury to hepatocytes or cholangiocytes, when left unmanaged, leads to the development of liver fibrosis, a condition characterized by the excessive intrahepatic deposition of extracellular matrix proteins. Activated hepatic stellate cells constitute the predominant source of extracellular matrix in fibrotic livers and their transition from a quiescent state during fibrogenesis is associated with important alterations in their transcriptional and epigenetic landscape. Areas covered: We briefly describe the processes involved in hepatic stellate cell activation and discuss our current understanding of alterations in the epigenetic landscape, i.e DNA methylation, histone modifications and the functional role of non-coding RNAs that accompany this key event in the development of chronic liver disease. Expert commentary: Although great progress has been made, our understanding of the epigenetic regulation of hepatic stellate cell activation is limited and, thus far, insufficient to allow the development of epigenetic drugs that can selectively interrupt liver fibrosis.

  2. Mcl-1和Bax基因在2-甲氧基雌二醇诱导骨髓增生异常综合征细胞凋亡中的调控作用%Regulation of 2-Methoxyestradiol-induced Cell Apoptosis by Mcl-1 and Bax Genes in Myelodisplastic Syndrome

    Institute of Scientific and Technical Information of China (English)

    夏国华; 陈宝安; 芦慧霞; 邵泽叶; 丁家华; 高冲; 胡玲莉

    2009-01-01

    本研究探讨bcl-2基因家族成员调控2-甲氧基雌二醇(2-ME)谤导骨髓增生异常综合征(MDS)细胞凋亡的机制.用2-ME预处理MUTZ-1细胞,荧光比色法检测凋亡信号蛋白半胱氨酸蛋白酶-3(caspase-3)活性,用逆转录聚合酶链反应(RT-PCR)检测凋亡相关基因-髓细胞白血病基因-1(mcl-1)和bcl-2相关X蛋白(bax)mRNA的表达.结果表明:与时照组相比,2-ME增强MUTZ-1细胞内caspase-3活性,并呈浓度和时间依赖性(P0.05).结论:2-ME可能通过下调mcl-1表达和增强caspase-3活性的途径来调控MDS细胞凋亡.

  3. DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

    DEFF Research Database (Denmark)

    Nguyen, Thanh Hung; Kugler, Jan-Michael; Cohen, Stephen Michael

    2017-01-01

    /TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family...... deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor...

  4. [Polymethoxylated flavonoids activate cystic fibrosis transmembrane conductance regulator chloride channel].

    Science.gov (United States)

    Cao, Huan-Huan; Fang, Fang; Yu, Bo; Luan, Jian; Jiang, Yu; Yang, Hong

    2015-04-25

    Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 μmol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation.

  5. Expression of apoptosis related genes bax, bcl-2 and bcl-X in human gastric cancer: early results of an investigation

    Directory of Open Access Journals (Sweden)

    Domenico D'Ugo

    2005-03-01

    Full Text Available

    Background. Evidences indicate an involvement of apoptosis related genes in gastric carcinogenesis. We studied the gene and protein expression patterns of bcl-2, bax and bcl-X in samples of gastric adenocarcinoma. The apoptotic index values, histological type, differentiation grade, cancer stage and lymph node status were statistically analysed for possible correlations with expressional data.

    Methods. Thirty specimens of gastric cancer and respective normal control gastric mucosa were collected from patients with the diagnosis of gastric adenocarcinoma who underwent a curative gastrectomy. bcl-2, bax and bcl-XL mRNA and protein levels were respectively determined by reverse transcription PCR (RT-PCR and western blot using monoclonal antibodies for immunodetection.

    Results and conclusions.We observed a significant suppression of bax with an increase of bcl-XL at protein and mRNA levels. The presence of lymph node metastases was statistically related to the loss of bax overexpression. Bcl-XL was mostly up-regulated in intestinal/mixed types of gastric carcinoma. The expression patterns described confirm the role for these apoptosis genes in gastric adenocarcinoma.

  6. 75 FR 5100 - Agency Information Collection Activities: NAFTA Regulations and Certificate of Origin

    Science.gov (United States)

    2010-02-01

    ... SECURITY Customs and Border Protection Agency Information Collection Activities: NAFTA Regulations and... collection requirement concerning the NAFTA Regulations and Certificate of Origin. This request for comment... CBP is soliciting comments concerning the following information collection: Title: NAFTA Regulations...

  7. Epac Activation Regulates Human Mesenchymal Stem Cells Migration and Adhesion.

    Science.gov (United States)

    Yu, Jiao-Le; Deng, Ruixia; Chung, Sookja K; Chan, Godfrey Chi-Fung

    2016-04-01

    How to enhance the homing of human mesenchymal stem cells (hMSCs) to the target tissues remains a clinical challenge nowadays. To overcome this barrier, the mechanism responsible for the hMSCs migration and engraftment has to be defined. Currently, the exact mechanism involved in migration and adhesion of hMSCs remains unknown. Exchange protein directly activated by cAMP (Epac), a novel protein discovered in cAMP signaling pathway, may have a potential role in regulating cells adhesion and migration by triggering the downstream Rap family signaling cascades. However, the exact role of Epac in cells homing is elusive. Our study evaluated the role of Epac in the homing of hMSCs. We confirmed that hMSCs expressed functional Epac and its activation enhanced the migration and adhesion of hMSCs significantly. The Epac activation was further found to be contributed directly to the chemotactic responses induced by stromal cell derived factor-1 (SDF-1) which is a known chemokine in regulating hMSCs homing. These findings suggested Epac is connected to the SDF-1 signaling cascades. In conclusion, our study revealed that Epac plays a role in hMSCs homing by promoting adhesion and migration. Appropriate manipulation of Epac may enhance the homing of hMSCs and facilitate their future clinical applications.

  8. The cytoskeletal protein Ndel1 regulates dynamin 2 GTPase activity.

    Directory of Open Access Journals (Sweden)

    Mathieu Chansard

    Full Text Available Cytoskeleton dynamics, membranes trafficking and positioning are essential for the proper functioning of any mammalian cell. The identification of the molecules and mechanisms that allow these cellular processes to interface is vital for understanding cell behaviors. Ndel1, the mammalian homolog of the Aspergillus nidulans NudE, organizes the cytoskeleton and regulates molecular motors, thereby impacting on the positioning of membranes. Hypothetically, Ndel1 can act in concert with enzymes controlling membrane trafficking (vesicle-mediated transport per se, but this idea has never been investigated. We now report that a pool of Ndel1 associates directly with Dynamin 2 (Dyn2, a large cytosolic GTPase involved in the trafficking of the AMPA receptor subunit GluR1. In vitro, Ndel1 enhances Dyn2 GTPase activity in its unassembled and assembled forms, without promoting oligomerization of the enzyme. In cells, gain and loss of function of Ndel1 recapitulate the effects of overexpression of Dyn2 and Dyn2 dominant negative with reduced GTPase activity on the intracellular localization of GluR1, respectively, without affecting the stability of microtubules. Together, these results indicate that Ndel1 regulates Dyn2 GTPase activity and impacts GluR1-containing membranes distribution in a manner reminiscent of Dyn2.

  9. Fbxw7 controls angiogenesis by regulating endothelial Notch activity.

    Directory of Open Access Journals (Sweden)

    Nanae Izumi

    Full Text Available Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7(iECKO mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs. Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.

  10. Negative regulation of lymphocyte activation by the adaptor protein LAX.

    Science.gov (United States)

    Zhu, Minghua; Granillo, Olivia; Wen, Renren; Yang, Kaiyong; Dai, Xuezhi; Wang, Demin; Zhang, Weiguo

    2005-05-01

    The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

  11. Dynamic regulation of Polycomb group activity during plant development.

    Science.gov (United States)

    Bemer, Marian; Grossniklaus, Ueli

    2012-11-01

    Polycomb group (PcG) complexes play important roles in phase transitions and cell fate determination in plants and animals, by epigenetically repressing sets of genes that promote either proliferation or differentiation. The continuous differentiation of new organs in plants, such as leaves or flowers, requires a highly dynamic PcG function, which can be induced, modulated, or repressed when necessary. In this review, we discuss the recent advance in understanding PcG function in plants and focus on the diverse molecular mechanisms that have been described to regulate and counteract PcG activity in Arabidopsis.

  12. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    Science.gov (United States)

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-08

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.

  13. Regulation of dopamine transporter activity by carboxypeptidase E

    Directory of Open Access Journals (Sweden)

    Zhang Heping

    2009-05-01

    Full Text Available Abstract Background The dopamine transporter (DAT plays a critical role in terminating the action of dopamine by rapid reuptake into the presynaptic neuron. Previous studies have revealed that the DAT carboxyl terminus (DAT-CT can directly interact with other cellular proteins and regulate DAT function and trafficking. Results Here, we have identified that carboxypeptidase E (CPE, a prohormone processing exopeptidase and sorting receptor for the regulated secretory pathway, interacts with the DAT-CT and affects DAT function. Mammalian cell lines coexpressing CPE and DAT exhibited increased DAT-mediated dopamine uptake activity compared to cells expressing DAT alone. Moreover, coexpression of an interfering DAT-CT minigene inhibited the effects of CPE on DAT. Functional changes caused by CPE could be attributed to enhanced DAT expression and subsequent increase in DAT cell surface localization, due to decreased DAT degradation. In addition, CPE association could reduce the phosphorylation state of DAT on serine residues, potentially leading to reduced internalization, thus stabilizing plasmalemmal DAT localization. Conclusion Taken together, our results reveal a novel role for CPE in the regulation of DAT trafficking and DAT-mediated DA uptake, which may provide a novel target in the treatment of dopamine-governed diseases such as drug addiction and obesity.

  14. SUMOylation of Argonaute-2 regulates RNA interference activity

    Science.gov (United States)

    Josa-Prado, Fernando; Henley, Jeremy M.; Wilkinson, Kevin A.

    2015-01-01

    Post-translational modification of substrate proteins by small ubiquitin-like modifier (SUMO) regulates a vast array of cellular processes. SUMOylation occurs through three sequential enzymatic steps termed E1, E2 and E3. Substrate selection can be determined through interactions between the target protein and the SUMO E2 conjugating enzyme Ubc9 and specificity can be enhanced by substrate interactions with E3 ligase enzymes. We used the putative substrate recognition (PINIT) domain from the SUMO E3 PIAS3 as bait to identify potential SUMO substrates. One protein identified was Argonaute-2 (Ago2), which mediates RNA-induced gene silencing through binding small RNAs and promoting degradation of complimentary target mRNAs. We show that Ago2 can be SUMOylated in mammalian cells by both SUMO1 and SUMO2. SUMOylation occurs primarily at K402, and mutation of the SUMO consensus site surrounding this lysine reduces Ago2-mediated siRNA-induced silencing in a luciferase-based reporter assay. These results identify SUMOylation as a potential regulator of Ago2 activity and open new avenues for research into the mechanisms underlying the regulation of RNA-induced gene silencing. PMID:26188511

  15. Erk1 positively regulates osteoclast differentiation and bone resorptive activity.

    Directory of Open Access Journals (Sweden)

    Yongzheng He

    Full Text Available The extracellular signal-regulated kinases (ERK1 and 2 are widely-expressed and they modulate proliferation, survival, differentiation, and protein synthesis in multiple cell lineages. Altered ERK1/2 signaling is found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, Neurofibromatosis type 1, and Cardio-facio-cutaneous syndrome, suggesting that MEK-ERK signals regulate human skeletal development. Here, we examine the consequence of Erk1 and Erk2 disruption in multiple functions of osteoclasts, specialized macrophage/monocyte lineage-derived cells that resorb bone. We demonstrate that Erk1 positively regulates osteoclast development and bone resorptive activity, as genetic disruption of Erk1 reduced osteoclast progenitor cell numbers, compromised pit formation, and diminished M-CSF-mediated adhesion and migration. Moreover, WT mice reconstituted long-term with Erk1(-/- bone marrow mononuclear cells (BMMNCs demonstrated increased bone mineral density as compared to recipients transplanted with WT and Erk2(-/- BMMNCs, implicating marrow autonomous, Erk1-dependent osteoclast function. These data demonstrate Erk1 plays an important role in osteoclast functions while providing rationale for the development of Erk1-specific inhibitors for experimental investigation and/or therapeutic modulation of aberrant osteoclast function.

  16. Estrogen receptor β regulates endometriotic cell survival through serum and glucocorticoid-regulated kinase activation.

    Science.gov (United States)

    Monsivais, Diana; Dyson, Matthew T; Yin, Ping; Navarro, Antonia; Coon, John S; Pavone, Mary Ellen; Bulun, Serdar E

    2016-05-01

    To determine the expression and biological roles of serum and glucocorticoid-regulated kinase (SGK1) in tissues and cells from patients with endometriosis and from healthy control subjects. Case-control. University research setting. Premenopausal women. Endometriotic tissues were obtained from women with ovarian endometriosis, and normal endometrial tissues were obtained from women undergoing hysterectomy for benign conditions. Expression levels of SGK1, the role of SGK1 in endometriosis pathology, and regulation of SGK1 by estrogen receptor (ER) β. Transcript and protein levels of SGK1 were significantly higher in endometriotic tissues and cells compared with normal endometrium. SGK1 mRNA and protein levels were stimulated by E2, by the ERβ-selective agonist diarylpropionitrile, and by prostaglandin E2. SGK1 was transcriptionally regulated by ERβ based on small interfering RNA knockdown and chromatin immunoprecipitation of ERβ followed by quantitative polymerase chain reaction. SGK1 knockdown led to increased cleavage of poly(ADP-ribose) polymerase, and SGK1 activation was correlated with the phosphorylation of FOXO3a, a proapoptotic factor. ERβ leads to SGK1 overexpression in endometriosis, which contributes to the survival of endometriotic lesions through inhibition of apoptosis. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  17. The Expression of Bcl-2 and Bax in Normal,Hyperplastic,and Malignant Endometrium

    Institute of Scientific and Technical Information of China (English)

    ZHONGGang; TANLingfang; 等

    2002-01-01

    Objective:To investigate the expression of Bcl-2 and Bax proteins in normal,hyperplastic,and malignant endometrium.Methods:Endometrial tissues were obtained from 14 proliferative endometrial samples;simple(n=30)and complex hyperplasia without atypia(n=13);complex hyperplasia with atypia(n=20)and endometrial adenocarcinoma(n=17).The expression of Bcl-2and Bax proteins was detected by using immunohistochemical staining with appropriate antibodies.Results:The intensity of Bcl-2 staining was gradually increased from proliferative to simple and complex hyperplasia,but it was gradually decreased from atypia hyperplasia to endometrial adenocarcinoma(P<0.05).The intensity of Bax staining was gradually increased from proliferative endometrium to simple and complex hyperplasia,but in atypia hyperplasia it was obviously lower than simple hyperplasia,the ratio of Bco-2;Bax staining intensity was changed with the endometrium from proliferative,hyperplastic endo-metrium to endometrial adenocarcinoma.The ratio of Bcl-2;Bax staining intensity was obviously decreased in atypia hyperplasia and endometrial adenocarcinoma.Conclusion:The survival time of the cells in hyperplasia expressing Bcl-2 might be prolonged.Neoplastic cells in atypia hyperplasia and adenocarcinoma might show a decreased expression of Bcl-2 and Bax,suggesting that Bcl-2 and Bax might be important indexes and prognosis factors and the expression of Bcl-2 and Bax might be correlated with carcinogenesis in the uterine endometrium of hu-mans.

  18. Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

    Science.gov (United States)

    Sharawat, Surender Kumar; Raina, Vinod; Kumar, Lalit; Sharma, Atul; Bakhshi, Radhika; Vishnubhatla, Sreenivas; Gupta, Ritu; Bakhshi, Sameer

    2014-10-01

    Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.

  19. Effect of Helicobacter pylori infection on Bax protein expression in patients with gastric precancerous lesions

    Institute of Scientific and Technical Information of China (English)

    Hai-Feng Liu; Wei-Wen Liu; Guo-An Wang; Xiao-Chun Teng

    2005-01-01

    AIM: To investigate the effect of Helicobacter pylori (H pylori) infection on Bax protein expression, and explore the role of H pylori in gastric carcinogenesis.METHODS: H pylori was assessed by rapid urease test and Warthin-Starry method, and expression of Bax protein was examined immunohistochemically in 72 patients with pre-malignant lesions.RESULTS: Bax protein was differently expressed in intestinal metaplasia and gastric dysplasia, and showed 63.99% positivity. The positivity of Bax protein expression in H pylori-positive gastric precancerous lesions (72.3%) was significantly higher than that in H pylori-negative gastric precancerous lesions (48.0%, χ2 = 4.191, P<0.05).H pylori infection was well correlated with the expression of Bax protein in gastric precancerous lesions (r = 0.978,P<0.01). After eradication of H pylori, the positivity of Bax protein expression significantly decreased in H pylori-positive gastric precancerous lesions (χ2= 5.506,P<0.05). In the persisting H pylori-infected patients,the positivity of Bax protein expression was not changed.CONCLUSION: H pylori infection may be involved in the upregulation of Bax gene, which might be one of the mechanisms of H pylori infection-induced gastric epithelial cell apoptosis. H pylori might act as a tumor promoter in the genesis of gastric carcinoma and eradication of H pylori could inhibit gastric carcinogenesis.

  20. Higher expression of Bax in regulatory T cells increases vascular inflammation.

    Science.gov (United States)

    Xiong, Zeyu; Song, Jian; Yan, Yan; Huang, Yajue; Cowan, Alan; Wang, Hong; Yang, Xiao-Feng

    2008-05-01

    This study is to examine our hypothesis that CD4+CD25(high)Foxp3+ regulatory T cells (Tregs) have an interleukin-2 (IL-2) withdrawal-triggered apoptosis pathway, and modulation of Treg apoptosis pathway affects development of vascular inflammation. We found that pro-apoptotic protein Bax upregulation in Tregs is induced by IL-2 withdrawal. Treg apoptosis induced by IL-2 withdrawal is inhibited by a Bax inhibitor, suggesting that highly expressed Bax is functional. To define the role of upregulated Bax in Treg apoptosis, we established a Tregs-specific Bax transgenic mouse model. Enforced expression of Bax in Tregs promotes Treg apoptosis triggered by IL-2 withdrawal and other apoptosis stimuli, suggesting pro-apoptotic role of highly expressed Bax in wild-type Tregs. Finally, higher expression of Bax in Tregs decreases the striking threshold of vascular inflammation due to the failure of suppression of inflammatory cells resulting from Treg apoptosis. These results have demonstrated the proof of principle that the modulation of Tregs apoptosis/survival could be used as a new therapeutic approach for inflammatory cardiovascular diseases.

  1. Fas-Induced Apoptosis of Renal Cell Carcinoma is Mediated by Apoptosis Signal-Regulating Kinase 1 via Mitochondrial Damage-Dependent Caspase-8 Activation

    Directory of Open Access Journals (Sweden)

    Mohamed Hassan

    2009-01-01

    Full Text Available Renal cell carcinoma (RCC is a prototype of a chemo refractory tumour. It remains the most lethal of the common urologic cancers and is highly resistant to conventional therapy. Here, we confirmed the efficiency of anti-Fas monoclonal antibody (CH11 as alternative therapeutic approach for the treatment of RCC and investigated the molecular mechanism(s, whereby CH11 induces apoptosis of RCC cells. The present study shows an essential role for apoptosis signal-regulating kinase 1 (ASK1, together with both c-jun-N-terminal kinase (JNK and p38 pathways, and caspase-8 in this process. Furthermore, CH11-dependent induction of the ASK1–JNK/p38 pathways was found to activate the transcription factors AP-1 and ATF-2, and FADD-caspase-8-Bid signalling, resulting in the translocation of both Bax and Bak proteins, and subsequently mitochondrial dysregulation that is characterized by the loss of mitochondrial membrane potential (ΔΨm, cytochrome c release and cleavage of caspase-9, caspase-3 and PARP. Thus, the described molecular mechanisms of CH11-induced apoptosis suggest the reliability of Fas activation as an alternative therapeutic approach for the treatment of patients with advanced renal cell carcinoma.

  2. ROMK1 channel activity is regulated by monoubiquitination.

    Science.gov (United States)

    Lin, Dao-Hong; Sterling, Hyacinth; Wang, Zhijian; Babilonia, Elisa; Yang, Baofeng; Dong, Ke; Hebert, Steven C; Giebisch, Gerhard; Wang, Wen-Hui

    2005-03-22

    The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 microA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.

  3. The regulation of ant colony foraging activity without spatial information.

    Science.gov (United States)

    Prabhakar, Balaji; Dektar, Katherine N; Gordon, Deborah M

    2012-01-01

    Many dynamical networks, such as the ones that produce the collective behavior of social insects, operate without any central control, instead arising from local interactions among individuals. A well-studied example is the formation of recruitment trails in ant colonies, but many ant species do not use pheromone trails. We present a model of the regulation of foraging by harvester ant (Pogonomyrmex barbatus) colonies. This species forages for scattered seeds that one ant can retrieve on its own, so there is no need for spatial information such as pheromone trails that lead ants to specific locations. Previous work shows that colony foraging activity, the rate at which ants go out to search individually for seeds, is regulated in response to current food availability throughout the colony's foraging area. Ants use the rate of brief antennal contacts inside the nest between foragers returning with food and outgoing foragers available to leave the nest on the next foraging trip. Here we present a feedback-based algorithm that captures the main features of data from field experiments in which the rate of returning foragers was manipulated. The algorithm draws on our finding that the distribution of intervals between successive ants returning to the nest is a Poisson process. We fitted the parameter that estimates the effect of each returning forager on the rate at which outgoing foragers leave the nest. We found that correlations between observed rates of returning foragers and simulated rates of outgoing foragers, using our model, were similar to those in the data. Our simple stochastic model shows how the regulation of ant colony foraging can operate without spatial information, describing a process at the level of individual ants that predicts the overall foraging activity of the colony.

  4. Regulated O2 activation in flavin-dependent monooxygenases.

    Science.gov (United States)

    Frederick, Rosanne E; Mayfield, Jeffery A; DuBois, Jennifer L

    2011-08-17

    Flavin-dependent monooxygenases (FMOs) are involved in important biosynthetic pathways in diverse organisms, including production of the siderophores used for the import and storage of essential iron in serious pathogens. We have shown that the FMO from Aspergillus fumigatus, an ornithine monooxygenase (Af-OMO), is mechanistically similar to its well-studied distant homologues from mammalian liver. The latter are highly promiscuous in their choice of substrates, while Af-OMO is unusually specific. This presents a puzzle: how do Af-OMO and other FMOs of the biosynthetic classes achieve such specificity? We have discovered substantial enhancement in the rate of O(2) activation in Af-OMO in the presence of L-arginine, which acts as a small molecule regulator. Such protein-level regulation could help explain how this and related biosynthetic FMOs manage to couple O(2) activation and substrate hydroxylation to each other and to the appropriate cellular conditions. Given the essentiality of Fe to Af and the avirulence of the Af-OMO gene knock out, inhibitors of Af-OMO are likely to be drug targets against this medically intractable pathogen.

  5. Osteoblast differentiation and migration are regulated by dynamin GTPase activity.

    Science.gov (United States)

    Eleniste, Pierre P; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W; Bruzzaniti, Angela

    2014-01-01

    Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0-21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation.

  6. Activating transcription factor 4 regulates osteoclast differentiation in mice

    Science.gov (United States)

    Cao, Huiling; Yu, Shibing; Yao, Zhi; Galson, Deborah L.; Jiang, Yu; Zhang, Xiaoyan; Fan, Jie; Lu, Binfeng; Guan, Youfei; Luo, Min; Lai, Yumei; Zhu, Yibei; Kurihara, Noriyoshi; Patrene, Kenneth; Roodman, G. David; Xiao, Guozhi

    2010-01-01

    Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4–/– bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4–/– BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity. PMID:20628199

  7. GARP regulates the bioavailability and activation of TGFβ.

    Science.gov (United States)

    Wang, Rui; Zhu, Jianghai; Dong, Xianchi; Shi, Minlong; Lu, Chafen; Springer, Timothy A

    2012-03-01

    Glycoprotein-A repetitions predominant protein (GARP) associates with latent transforming growth factor-β (proTGFβ) on the surface of T regulatory cells and platelets; however, whether GARP functions in latent TGFβ activation and the structural basis of coassociation remain unknown. We find that Cys-192 and Cys-331 of GARP disulfide link to the TGFβ1 prodomain and that GARP with C192A and C331A mutations can also noncovalently associate with proTGFβ1. Noncovalent association is sufficiently strong for GARP to outcompete latent TGFβ-binding protein for binding to proTGFβ1. Association between GARP and proTGFβ1 prevents the secretion of TGFβ1. Integrin α(V)β(6) and to a lesser extent α(V)β(8) are able to activate TGFβ from the GARP-proTGFβ1 complex. Activation requires the RGD motif of latent TGFβ, disulfide linkage between GARP and latent TGFβ, and membrane association of GARP. Our results show that GARP is a latent TGFβ-binding protein that functions in regulating the bioavailability and activation of TGFβ.

  8. Physical Activity Plays an Important Role in Body Weight Regulation

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Chaput

    2011-01-01

    Full Text Available Emerging literature highlights the need to incorporate physical activity into every strategy intended to prevent weight gain as well as to maintain weight loss over time. Furthermore, physical activity should be part of any plan to lose weight. The stimulus of exercise provides valuable metabolic adaptations that improve energy and macronutrient balance regulation. A tight coupling between energy intake and energy expenditure has been documented at high levels of physical exercise, suggesting that exercise may improve appetite control. The regular practice of physical activity has also been reported to reduce the risk of stress-induced weight gain. A more personalized approach is recommended when planning exercise programs in a clinical weight loss setting in order to limit the compensatory changes associated to exercise-induced weight loss. With modern environment promoting overeating and sedentary behavior, there is an urgent need for a concerted action including legislative measures to promote healthy active living in order to curb the current epidemic of chronic diseases.

  9. Activating transcription factor 4 regulates osteoclast differentiation in mice.

    Science.gov (United States)

    Cao, Huiling; Yu, Shibing; Yao, Zhi; Galson, Deborah L; Jiang, Yu; Zhang, Xiaoyan; Fan, Jie; Lu, Binfeng; Guan, Youfei; Luo, Min; Lai, Yumei; Zhu, Yibei; Kurihara, Noriyoshi; Patrene, Kenneth; Roodman, G David; Xiao, Guozhi

    2010-08-01

    Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4-/- bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4-/- BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.

  10. Neuroligin-1 links neuronal activity to sleep-wake regulation

    Science.gov (United States)

    El Helou, Janine; Bélanger-Nelson, Erika; Freyburger, Marlène; Dorsaz, Stéphane; Curie, Thomas; La Spada, Francesco; Gaudreault, Pierre-Olivier; Beaumont, Éric; Pouliot, Philippe; Lesage, Frédéric; Frank, Marcos G.; Franken, Paul; Mongrain, Valérie

    2013-01-01

    Maintaining wakefulness is associated with a progressive increase in the need for sleep. This phenomenon has been linked to changes in synaptic function. The synaptic adhesion molecule Neuroligin-1 (NLG1) controls the activity and synaptic localization of N-methyl-d-aspartate receptors, which activity is impaired by prolonged wakefulness. We here highlight that this pathway may underlie both the adverse effects of sleep loss on cognition and the subsequent changes in cortical synchrony. We found that the expression of specific Nlg1 transcript variants is changed by sleep deprivation in three mouse strains. These observations were associated with strain-specific changes in synaptic NLG1 protein content. Importantly, we showed that Nlg1 knockout mice are not able to sustain wakefulness and spend more time in nonrapid eye movement sleep than wild-type mice. These changes occurred with modifications in waking quality as exemplified by low theta/alpha activity during wakefulness and poor preference for social novelty, as well as altered delta synchrony during sleep. Finally, we identified a transcriptional pathway that could underlie the sleep/wake-dependent changes in Nlg1 expression and that involves clock transcription factors. We thus suggest that NLG1 is an element that contributes to the coupling of neuronal activity to sleep/wake regulation. PMID:23716671

  11. Activation of epithelial STAT3 regulates intestinal homeostasis.

    Science.gov (United States)

    Neufert, Clemens; Pickert, Geethanjali; Zheng, Yan; Wittkopf, Nadine; Warntjen, Moritz; Nikolaev, Alexei; Ouyang, Wenjun; Neurath, Markus F; Becker, Christoph

    2010-02-15

    The intestinal epithelium that lines the mucosal surface along the GI-tract is a key player for the intestinal homeostasis of the healthy individual. In case of a mucosal damage or a barrier defect as seen in patients with inflammatory bowel disease, the balance is disturbed, and translocation of intestinal microbes to the submucosa is facilitated. We recently demonstrated a pivotal role of STAT3 activation in intestinal epithelial cells (IEC) for the restoration of the balance at the mucosal surface of the gut in an experimental colitis model. STAT3 was rapidly induced in intestinal epithelial cells upon challenge of mice in both experimental colitis and intestinal wound healing models. STAT3 activation was found to be dispensable in the steady-state conditions but was important for efficient regeneration of the epithelium in response to injury. Here, we extend our previous findings by showing epithelial STAT3 activation in human patients suffering from IBD and provide additional insights how the activation of epithelial STAT3 by IL-22 regulates intestinal homeostasis and mucosal wound healing. We also demonstrate that antibody-mediated neutralization of IL-22 has little impact on the development of experimental colitis in mice, but significantly delays recovery from colitis. Thus, our data suggest that targeting the STAT3 signaling pathway in IEC is a promising therapeutic approach in situations when the intestinal homeostasis is disturbed, e.g., as seen in Crohn's disease or Ulcerative colitis.

  12. Melatonin may play a role in modulation of bax and bcl-2 expression levels to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Mohseni, Mehran [Department of Radiology and Medical Physics, Faculty of Paramedicine, Kashan University of Medical Sciences, Kashan (Iran, Islamic Republic of); Mihandoost, Ehsan, E-mail: mihandoost.e@gmail.com [Department of Medical Radiation Engineering, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of); Shirazi, Alireza [Department of Medical Physics and Biomedical Engineering, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Sepehrizadeh, Zargham [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Bazzaz, Javad Tavakkoly [Department of Medical Genetics, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Ghazi-khansari, Mahmoud [Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2012-10-15

    The close relationship between free radicals effects and apoptosis process has been proved. Melatonin has been reported as a direct free radical scavenger. We investigated the capability of melatonin in the modification of radiation-induced apoptosis and apoptosis-associated upstream regulators expression in rat peripheral blood lymphocytes. Rats were irradiated with a single whole body Cobalt 60-gamma radiation dose of 8 Gy at a dose rate of 101 cGy/min with or without melatonin pretreatments at different concentrations of 10 and 100 mg/kg body weight. The rats were divided into eight groups of control, irradiation-only, vehicle-only, vehicle plus irradiation, 10 mg/kg melatonin alone, 10 mg/kg melatonin plus irradiation, 100 mg/kg melatonin alone and 100 mg/kg melatonin plus irradiation. Rats were given an intraperitoneal (IP) injection of melatonin or the same volume of vehicle alone 1 h prior to irradiation. Blood samples were taken 4, 24, 48 and 72 h after irradiation for evaluation of flow cytometric analysis of apoptotic lymphocytes using Annexin V/PI assay and measurement of bax and bcl-2 expression using quantitative real-time PCR (RT{sup 2}qPCR). Irradiation-only and vehicle plus irradiation showed an increase in the percentage of apoptotic lymphocytes significantly different from control group (P < 0.01), while melatonin pretreatments in a dose-dependent manner reduced it as compared with the irradiation-only and vehicle plus irradiation groups (P < 0.01) in all time points. This reduced apoptosis by melatonin was related to the downregulation of bax, upregulation of bcl-2, and therefore reduction of bax/bcl-2 ratio. Our results suggest that melatonin in these doses may provide modulation of bax and bcl-2 expression as well as bax/bcl-2 ratio to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis.

  13. Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

    Science.gov (United States)

    Kim, Sang-Woo; Schifano, Matthew; Oleksyn, David; Jordan, Craig T; Ryan, Daniel; Insel, Richard; Zhao, Jiyong; Chen, Luojing

    2014-10-01

    Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

  14. Sesamin suppresses STZ induced INS-1 cell apoptosis through inhibition of NF-κB activation and regulation of Bcl-2 family protein expression.

    Science.gov (United States)

    Zheng, Shuguo; Zhao, Mengqiu; Ren, Younan; Wu, Yuanjie; Yang, Jieren

    2015-03-05

    Diverse risk factors for diabetes can induce oxidative stress, leading to pancreatic beta cell damage and insulin secretion dysfunction. In the present study, we evaluated the effect of sesamin on streptozotocin (STZ) induced apoptosis in INS-1 cells and the possible mechanisms implicated. After preincubation with indicated concentrations of sesamin (0.1, 1.0 and 10.0μmol/l) for 24h, INS-1 cells were exposed to STZ (3mmol/l) for 12h. Sesamin effectively improved STZ induced cell damage as determined by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] assay and insulin secretion capacity, and suppressed STZ induced cell apoptosis as evaluated by flow cytometry using annexin V and propidium iodide double staining. Western blot analysis demonstrated that sesamin markedly suppressed STZ induced nuclear factor kappa B (NF-κB) activation, with Bax protein down-regulated and Bcl-2 protein up-regulated significantly. Preincubation with sesamin resulted in an evident enhancement of total antioxidant capacity in INS-1 cells, accompanied by a significant reduction of intracellular reactive oxygen species and malondialdehyde, an end product of lipid peroxidation. Taken together, these findings suggested that sesamin was capable of suppressing STZ induced INS-1 cell apoptosis, which might be ascribed, at least partly, to the inhibition of NF-κB activation and subsequent regulation of Bcl-2 family protein expression. This study would provide a potential target for treatment of diabetes with sesamin as well as other antioxidants.

  15. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  16. Influence of BCL2-938C>A and BAX-248G>A promoter polymorphisms in the development of AML: case-control study from South India.

    Science.gov (United States)

    Cingeetham, Anuradha; Vuree, Sugunakar; Dunna, Nageswara Rao; Gorre, Manjula; Nanchari, Santhoshi Rani; Edathara, Prajitha Mohandas; Meka, Phannibhushann; Annamaneni, Sandhya; Digumarthi, Raghunadharao; Sinha, Sudha; Satti, Vishnupriya

    2015-09-01

    B-cell lymphoma 2 (BCL2) and BCL2-associated X protein (BAX) proteins are anti-apoptotic and pro-apoptotic determinants of mitochondrial-mediated apoptosis, and their relative expression determines the cell fate. The promoter polymorphisms in these genes were shown to alter the protein function or expression and exert an impact on apoptosis regulation. Deregulation in the expression of any of these genes leads to disruption of cellular homeostasis and malignant transformation. The present study was aimed to determine the association of BCL2-938C>A and BAX-248G>A promoter polymorphisms with origin and progression of acute myeloid leukemia (AML). We also have performed combined genotype analysis to evaluate the cumulative effect of risk genotypes in the AML development. These polymorphisms were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) in 221 AML patients and 305 age- and sex-matched healthy controls. Our study revealed that BCL2-938CA (p = 0.018) and BAX-248GG (0.043) genotypes were significantly associated with increased risk for AML occurrence. BAX-248A allele had shown decreased risk for AML. The combined analysis had shown that BCL2-938CA+AA-BAX-248GG group had a 1.63-fold (95 % CI: 1.08-2.45, p = 0.02) increased risk for AML. None of the clinical variables had shown any significant association with both polymorphisms. With respect to complete remission (CR) rate, BAX-248GG genotype (p = 0.002) and G allele (p = 0.009) had conferred significant risk for complete remission failure. Although the log rank test was not significant, survival analysis had shown a trend where BCL2-938CA genotype, and BAX-248GG had reduced median disease-free survival (DFS) of 9 and 10 months, respectively. In conclusion, BCL2-938C>A and BAX-248G>A gene polymorphisms might contribute to the origin of AML. Moreover, influence of BAX-248GG genotype on CR and DFS rate suggests that the BAX-248G>A polymorphism can serve as

  17. Spatial regulation and the rate of signal transduction activation.

    Directory of Open Access Journals (Sweden)

    Nizar N Batada

    2006-05-01

    Full Text Available Of the many important signaling events that take place on the surface of a mammalian cell, activation of signal transduction pathways via interactions of cell surface receptors is one of the most important. Evidence suggests that cell surface proteins are not as freely diffusible as implied by the classic fluid mosaic model and that their confinement to membrane domains is regulated. It is unknown whether these dynamic localization mechanisms function to enhance signal transduction activation rate or to minimize cross talk among pathways that share common intermediates. To determine which of these two possibilities is more likely, we derive an explicit equation for the rate at which cell surface membrane proteins interact based on a Brownian motion model in the presence of endocytosis and exocytosis. We find that in the absence of any diffusion constraints, cell surface protein interaction rate is extremely high relative to cytoplasmic protein interaction rate even in a large mammalian cell with a receptor abundance of a mere two hundred molecules. Since a larger number of downstream signaling events needs to take place, each occurring at a much slower rate than the initial activation via association of cell surface proteins, we conclude that the role of co-localization is most likely that of cross-talk reduction rather than coupling efficiency enhancement.

  18. Ribosomal Protein S14 Negatively Regulates c-Myc Activity*

    Science.gov (United States)

    Zhou, Xiang; Hao, Qian; Liao, Jun-ming; Liao, Peng; Lu, Hua

    2013-01-01

    The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA. PMID:23775087

  19. Regulation of nucleus accumbens activity by the hypothalamic neuropeptide MCH

    Science.gov (United States)

    Sears, Robert M.; Liu, Rong-Jian; Narayanan, Nandakumar S.; Sharf, Ruth; Yeckel, Mark F.; Laubach, Mark; Aghajanian, George K.; DiLeone, Ralph J.

    2010-01-01

    The lateral hypothalamus (LH) and the nucleus accumbens shell (AcbSh) are brain regions important for food intake. The AcbSh contains high levels of receptor for melanin-concentrating hormone (MCH), a lateral hypothalamic peptide critical for feeding and metabolism. MCH receptor (MCHR1) activation in the AcbSh increases food intake while AcbSh MCHR1 blockade reduces feeding. Here biochemical and cellular mechanisms of MCH action in the rodent AcbSh are described. A reduction of phosphorylation of GluR1 at Serine 845 (pSer845) is shown to occur after both pharmacological and genetic manipulations of MCHR1 activity. These changes depend upon signaling through Gi/o, and result in decreased surface expression of GluR1-containing AMPA receptors (AMPARs). Electrophysiological analysis of medium spiny neurons (MSNs) in the AcbSh revealed decreased amplitude of AMPAR-mediated synaptic events (mEPSC) with MCH treatment. In addition, MCH suppressed action potential firing MSNs through K+ channel activation. Finally, in vivo recordings confirmed that MCH reduces neuronal cell firing in the AcbSh in freely moving animals. The ability of MCH to reduce cell firing in the AcbSh is consistent with a general model from other pharmacological and electrophysiological studies whereby reduced AcbSh neuronal firing leads to food intake. The current work integrates the hypothalamus into this model, providing biochemical and cellular mechanisms whereby metabolic and limbic signals converge to regulate food intake. PMID:20554878

  20. Substrate regulation of ascorbate transport activity in astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. (Univ. of Western Ontario, London (Canada))

    1990-10-01

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

  1. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

    Science.gov (United States)

    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival.

  2. BAK1 Directly Regulates Brassinosteroid Perception and BRI1 Activation

    Institute of Scientific and Technical Information of China (English)

    Kai He; Shengbao Xu; Jia Li

    2013-01-01

    Plants utilize plasma membrane-localized receptor-like kinases (RLKs) to sense extracellular signals to coordinate growth, development, and innate immune responses. BAK1 regulates multiple signaling pathways acting as a co-receptor of several distinct ligand-binding RLKs. It has been debated whether BAK1 serves as an essential regulatory component or only a signal amplifier without pathway specificity. This issue has been clarified recently. Genetic and structural analyses indicated that BAK1 and its homologs play indispensible roles in mediating brassinosteroid (BR) signaling pathway by directly perceiving the ligand BR and activating the receptor of BR, BRI1. The mechanism revealed by these studies now serves as a paradigm for how a pair of RLKs can function together in ligand binding and subsequent initiation of signaling.

  3. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

    Science.gov (United States)

    Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

  4. Involvement of P53 and Bax/Bad triggering apoptosis in thioacetamide-induced hepatic epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Li-Hsuen Chen; Chia-Yu Hsu; Ching-Feng Weng

    2006-01-01

    AIM: Thioacetamide (TAA) has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS: The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAAinduced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAAinduced clone 9 cells were measured by Western blot. RESULTS: The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died (MTT assay) within 24 h after 100 mg/L TAA was applied. Apoptotic cell percentage (TUNEL assay) and caspase 3 activities were highest after 100 mg/L TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspasedependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment.CONCLUSION: These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents,perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.

  5. Effects of Online Self-Regulation Activities on Physical Activity Among Pregnant and Early Postpartum Women.

    Science.gov (United States)

    Kim, Hye Kyung; Niederdeppe, Jeff; Graham, Meredith; Olson, Christine; Gay, Geri

    2015-01-01

    Physical and psychological changes that occur during pregnancy present a unique challenge for women's physical activity. Using a theory-based prospective design, this study examines the effects of pregnant women's (a) physical activity cognitions (self-efficacy, outcome expectancy, and safety beliefs) and (b) online self-regulation activities (goal-setting and self-monitoring) on subsequent changes in their physical activity intentions and behavior during pregnancy and immediately postpartum. The authors used data from three panel surveys administered to pregnant women enrolled in a web-based intervention to promote healthy pregnancy and postpartum weight, as well as log data on their use of self-regulatory features on the intervention website. Perceived self-efficacy and perceived safety of physical activity in pregnancy enhanced subsequent intentions to be physically active. Repeated goal-setting and monitoring of those goals helped to maintain positive intentions during pregnancy, but only repeated self-monitoring transferred positive intentions into actual behavior. Theoretically, this study offers a better understanding of the roles of self-regulation activities in the processes of goal-striving. The authors also discuss practical implications for encouraging physical activity among pregnant and early postpartum women.

  6. Deacetylation of Ku70 by SIRT6 attenuates Bax-mediated apoptosis in hepatocellular carcinoma.

    Science.gov (United States)

    Tao, Na-Na; Ren, Ji-Hua; Tang, Hua; Ran, Long-Kuan; Zhou, Hong-Zhong; Liu, Bo; Huang, Ai-Long; Chen, Juan

    2017-04-15

    SIRT6 is a class III histone deacetylase that has been implicated in HCC development. We previously reported that SIRT6 potentiated apoptosis evasion in hepatocellular carcinoma by inhibiting both Bax expression and mitochondrial translocalization. However, the mechanism underlying SIRT6-mediated inhibition of Bax mitochondrial localization remains elusive. In this study, we found that although SIRT6 had no effect on the expression level of Ku70, SIRT6 could interact with Ku70 and deacetylate it. The increased acetylation of Ku70 in SIRT6-depleted cells disrupt its interaction with Bax, which finally resulted in Bax mitochondrial translocalization. Furthermore, lysine K542 on Ku70 was the target for deacetylation by SIRT6. Ku70(K542Q) mutation abolished suppression of association between Ku70 and Bax and caused redistribution of Bax to the cytosol in SIRT6-depleted cells. Finally, Ku70(K542Q) mutation could reversed the inhibition of growth and apoptosis promotion mediated by SIRT6 silencing. Together, our findings revealed SIRT6 could block the mitochondrial translocation of Bax and decrease the apoptotic ratio of HCC cells by deacetylation of Ku70. SIRT6 may serve as a promising target for developing targeted therapies for HCC in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. 复方参芩对犬细小病毒致心肌组织Bcl-2和Bax mRNA的影响%Effect of Shenqin Compound on mRNA Expression of Bcl-2 and Bax Genes in Canine Myocardium Infected by Canine Parvovirus

    Institute of Scientific and Technical Information of China (English)

    刘俊玮; 刘娟; 杜林林; 郭志兴; 凌榕镔

    2013-01-01

    为了探讨复方参芩对犬细小病毒致犬心肌组织Bcl-2和Bax基因表达的影响,将丹参、黄芩、甘草等中药配伍并制备成为复方参芩针剂,人工感染犬细小病毒建立模型;将犬分为空白对照组、模型组、黄芪多糖组、复方参芩组;给药7d后,接种病毒,观察各组犬临床症状,取心肌组织,电镜观察心脏组织超微结构变化,采用荧光实时定量PCR法检测Bcl-2和Bax mRNA表达.结果表明,模型组犬死亡率高,心肌组织结构损伤严重,与空白对照组比较,心肌组织细胞Bcl-2 mRNA表达下调(P<0.05),Bax mRNA表达增加(P<0.01).复方参芩组犬存活率较高,心肌组织损伤轻,与模型组相比,心肌组织细胞Bax mRNA表达下调(P<0.01),Bcl-2 mRNA表达上调(P<0.01).通过本试验证明复方参芩可通过上调犬心肌组织细胞Bcl-2 mRNA表达,下调Bax mRNA的表达,抑制细小病毒引起的心肌细胞凋亡,保护犬心肌组织免受细小病毒损害.%To investigate the effect of Shenqin compound to Bcl-2 and Bax genes in canine myocardium infected by canine parvovirus. Compatibility of salvia, scutellaria, glycyrrhiza and other Chinese herbal to prepare Shenqin compound injection, and to build model by artificial infection of canine parvovirus, the canines were divided into blank control group, model group, astragalus polysaccharide group and Shenqin compound group, after injecting for 7 days, inoculated canine parvovirus, observed clinical symptom in each group, the change of ultrastructure in myocardium was observed through electron microscope. The RT-PCR method were used to test the expression of Bcl-2 and Bax mRNA. The results showed: the model group had high mortality and the myocardium was seriously damaged, compared with blank control group, the expression of Bcl-2 mRNA was down-regulated at P<0. 05 level, and the expression of Bax mRNA was up-regulated at P<0. 01 level. In Shenqin compound group, the protective rates were high

  8. Regulation of ALF promoter activity in Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Dan Li

    Full Text Available BACKGROUND: In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter. PRINCIPAL FINDINGS: The promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems. CONCLUSIONS: Overall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

  9. MK-STYX, a catalytically inactive phosphatase regulating mitochondrially dependent apoptosis.

    Science.gov (United States)

    Niemi, Natalie M; Lanning, Nathan J; Klomp, Jeff A; Tait, Stephen W; Xu, Yong; Dykema, Karl J; Murphy, Leon O; Gaither, L Alex; Xu, H Eric; Furge, Kyle A; Green, Douglas R; MacKeigan, Jeffrey P

    2011-04-01

    Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism.

  10. Carry-over of self-regulation for physical activity to self-regulating eating in women with morbid obesity.

    Science.gov (United States)

    Annesi, James J; Porter, Kandice J; Johnson, Ping H

    2015-01-01

    Poor outcomes from behavioral treatments of severe obesity have led to a dependence on invasive medical interventions, including surgery for morbidly obese individuals. Improved methods to self-regulate eating will be required to reduce obesity. The use of self-regulation methods for completing physical activity may carry over to increased self-regulation for eating through improved feelings of competence (self-efficacy) and mood. The study recruited women (Meanage = 43 years) with morbid obesity (MeanBMI = 44 kg/m(2)) to participate in 26 weeks of cognitive-behavioral support of physical activity paired with either nutrition education (n = 51) or cognitive-behavioral nutrition (n = 51) methods. Data collected were from 2011 and 2012. Significant improvements in self-regulation for physical activity, self-regulation for eating, overall mood, and self-efficacy for eating, with greater improvement in self-regulation for eating, were observed in the cognitive-behavioral nutrition group. Changes in mood and self-efficacy for eating significantly mediated the relationship between changes in self-regulation for physical activity and self-regulation for eating. When subscales of overall mood and self-efficacy were entered into separate regression equations as mediators, the only significant mediators were vigor, and controlling eating when socially pressured and when increased cues to overeat were present.

  11. After the slippery slope: Dutch experiences on regulating active euthanasia.

    Science.gov (United States)

    Boer, Theo A

    2003-01-01

    "When a country legalizes active euthanasia, it puts itself on a slippery slope from where it may well go further downward." If true, this is a forceful argument in the battle of those who try to prevent euthanasia from becoming legal. The force of any slippery slope argument, however, is by definition limited by its reference to future developments which cannot empirically be sustained. Experience in the Netherlands--where a law regulating active euthanasia was accepted in April 2001--may shed light on the strengths as well as the weaknesses of the slippery slope argument in the context of the euthanasia debate. This paper consists of three parts. First, it clarifies the Dutch legislation on euthanasia and explains the cultural context in which it originated. Second, it looks at the argument of the slippery slope. A logical and an empirical version are distinguished, and the latter, though philosophically less interesting, proves to be most relevant in the discussion on euthanasia. Thirdly, it addresses the question whether Dutch experiences in the process of legalizing euthanasia justify the fear of the slippery slope. The conclusion is that Dutch experiences justify some caution.

  12. Activated Type 2 Innate Lymphoid Cells regulate Beige Fat Biogenesis

    Science.gov (United States)

    Lee, Min-Woo; Odegaard, Justin I.; Mukundan, Lata; Qiu, Yifu; Molofsky, Ari B.; Nussbaum, Jesse C.; Yun, Karen; Locksley, Richard M.; Chawla, Ajay

    2014-01-01

    SUMMARY Type 2 innate lymphoid cells (ILC2s), an innate source of the type 2 cytokines interleukin (IL)-5 and -13, participate in the maintenance of tissue homeostasis. Although type 2 immunity is critically important for mediating metabolic adaptations to environmental cold, the functions of ILC2s in beige or brown fat development are poorly defined. We report here that activation of ILC2s by IL-33 is sufficient to promote the growth of functional beige fat in thermoneutral mice. Mechanistically, ILC2 activation results in the proliferation of bipotential adipocyte precursors (APs) and their subsequent commitment to the beige fat lineage. Loss- and gain-of-function studies reveal that ILC2-and eosinophil-derived type 2 cytokines stimulate signaling via the IL-4Rα in PDGFRα+ APs to promote beige fat biogenesis. Together, our results highlight a critical role for ILC2s and type 2 cytokines in the regulation of adipocyte precursor numbers and fate, and as a consequence, adipose tissue homeostasis. PMID:25543153

  13. Sucking pump activity in feeding behaviour regulation in carpenter ants.

    Science.gov (United States)

    Falibene, Agustina; Gontijo, Alberto de Figueiredo; Josens, Roxana

    2009-06-01

    Modulation of liquid feeding-rate would allow insects to ingest more food in the same time when this was required. Ants can vary nectar intake rate by increasing sucking pump frequency according to colony requirements. We analysed electrical signals generated by sucking pump activity of ants during drinking solutions of different sucrose concentrations and under different carbohydrate-deprivation levels. Our aim was to define parameters that characterize the recordings and analyse their relationship with feeding behaviour. Signals showed that the initial and final frequencies of sucking pump activity, as well as the difference between them were higher in sugar-deprived ants. However, these parameters were not influenced by sucrose solution concentration, which affected the number of pump contractions and the volume per contraction. Unexpectedly, we found two different responses in feeding behaviour of starved and non-starved ants depending on concentration. Starved ants drank dilute solutions for the same length of time as non-starved ants but ingested higher volumes. While drinking the concentrated solutions, starved ants drank the same volume, but did so in a shorter time than the non-starved ones. Despite these differences, for each analysed concentration the total number of pump contractions remained constant independently of sugar-deprivation level. These results are discussed in the frame of feeding regulation and decision making in ant foraging behaviour.

  14. Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

    Directory of Open Access Journals (Sweden)

    Jeon Hyejin

    2012-06-01

    Full Text Available Abstract Background Plasminogen activator inhibitor type 1 (PAI-1 is the primary inhibitor of urokinase type plasminogen activators (uPA and tissue type plasminogen activators (tPA, which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system. Methods In this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated by in vitro scratch-wound healing assay or Boyden chamber assay and an in vivo stab wound injury model. Phagocytic activity was measured by uptake of zymosan particles. Results The levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-γ stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP 1/Janus kinase (JAK/signal transducer and activator of transcription (STAT1 axis. PAI-1 also increased microglial migration in vivo when injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner. Conclusion Our results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

  15. Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

    Science.gov (United States)

    Marcel, Nimi; Sarin, Apurva

    2016-01-01

    Cell survival is one of several processes regulated by the Notch pathway in mammalian cells. Here we report functional outcomes of non-nuclear Notch signaling to activate autophagy, a conserved cellular response to nutrient stress, regulating survival in murine natural T-regulatory cells (Tregs), an immune subset controlling tolerance and inflammation. Induction of autophagy required ligand-dependent, Notch intracellular domain (NIC) activity, which controlled mitochondrial organization and survival of activated Tregs. Consistently, NIC immune-precipitated Beclin and Atg14, constituents of the autophagy initiation complex. Further, ectopic expression of an effector of autophagy (Atg3) or recombinant NIC tagged to a nuclear export signal (NIC-NES), restored autophagy and suppressor function in Notch1-/- Tregs. Furthermore, Notch1 deficiency in the Treg lineage resulted in immune hyperactivity, implicating Notch activity in Treg homeostasis. Notch1 integration with autophagy, revealed in these experiments, holds implications for Notch regulated cell-fate decisions governing differentiation. DOI: http://dx.doi.org/10.7554/eLife.14023.001 PMID:27267497

  16. Hyperphosphorylation regulates the activity of SREBP1 during mitosis.

    Science.gov (United States)

    Bengoechea-Alonso, Maria T; Punga, Tanel; Ericsson, Johan

    2005-08-16

    The sterol regulatory element-binding protein (SREBP) family of transcription factors controls the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We were, therefore, interested in the expression and activity of SREBPs during the cell cycle. We found that the expression of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in G2/M. In addition, the mature forms of SREBP1a and SREBP1c were hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In contrast, SREBP2 was not hyperphosphorylated in mitotic cells and was not recognized by the MPM-2 antibody. The MPM-2 epitope was mapped to the C terminus of mature SREBP1, and the mitosis-specific hyperphosphorylation of SREBP1 depended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in G2/M, and these effects depended on the C-terminal domain of the protein. In part, these effects could be explained by our observation that mature SREBP1 was stabilized in G2/M. In agreement with these observations, we found that the synthesis of cholesterol was increased in G2/M-arrested cells. Thus, our results demonstrate that the activity of mature SREBP1 is regulated by phosphorylation during the cell cycle, suggesting that SREBP1 may provide a link between lipid synthesis, proliferation, and cell growth.

  17. 22 CFR 143.2 - To what programs or activities do these regulations apply?

    Science.gov (United States)

    2010-04-01

    ... what programs or activities do these regulations apply? These regulations apply to each foreign affairs... 22 Foreign Relations 1 2010-04-01 2010-04-01 false To what programs or activities do these regulations apply? 143.2 Section 143.2 Foreign Relations DEPARTMENT OF STATE CIVIL RIGHTS NONDISCRIMINATION ON...

  18. Glycyrrhizin protects rat heart against ischemia-reperfusion injury through blockade of HMGB1-dependent phospho-JNK/Bax pathway

    Institute of Scientific and Technical Information of China (English)

    Chang-lin ZHAI; Mei-qi ZHANG; Yun ZHANG; Hong-xia XU; Jing-min WANG; Gui-peng AN; Yuan-yuan WANG; Li LI

    2012-01-01

    Aim: Glycyrrhizin (GL) has been found to inhibit extracellular HMGB1 cytokine's activity,and protect spinal cord,liver and brain against I/R-induced injury in experimental animals.The purpose of this study was to investigate the protective effect of GL in rat myocardial I/R-induced injury and to elucidate the underlying mechanisms.Methods: Male adult Sprague-Dawley rats underwent a 30-min left coronary artery occlusion followed by a 24-h reperfusion.The rats were treated with glycyrrhizin or glycyrrhizin plus recombinant HMGB1 after 30 min of ischemia and before reperfusion.Serum HMGB1,TNF-α and IL-6 levels,and hemodynamic parameters were measured at the onset and different time points of reperfusion.At the end of the experiment,the heart was excised,and the infarct size and histological changes were examined.The levels of Bcl2,Bax and cytochrome c,as well as phospho-ERK1/2,phospho-JNK and phospho-P38 in the heart tissue were evaluated using Western blot analysis,and myocardial caspase-3 activity was measured colorimetrically using BD pharmingen caspase 3 assay kit.Results: Intravenous administration of GL (10 mg/kg) significantly reduced the infarct size,but did not change the hemodynamic parameters at different time points during reperfusion.GL significantly decreased the levels of serum HMGB1,TNF-α and IL-6.GL changed the distribution of Bax and cytochrome c expression between the mitochondrial and cytosolic fractions in the heart tissue,resulting in inhibition of myocardial apoptosis.Moreover,expression of phospho-JNK,but not ERK1/2 and P38 was decreased by GL in the heart tissue.All of the effects produced by GL treatment were reversed by co-administration with the recombinant HMGB1 (100 μg).Intravenous administration of SP600125,a selective phospho-JNK inhibitor (0.5 mg/kg),attenuated HMGB1-dependent Bax translocation and the subsequent apoptosis.Conclusion: These results demonstrate that GL alleviates rat myocardial I/R-induced injury via directly

  19. The decrease in milk yield during once daily milking is due to regulation of synthetic activity rather than apoptosis of mammary epithelial cells in goats.

    Science.gov (United States)

    Ben Chedly, H; Lacasse, P; Marnet, P-G; Boutinaud, M

    2013-01-01

    Once daily milking (ODM) is a management practice that can improve working conditions and reduce production costs in dairy farming compared with twice daily milking (TDM). However, ODM is associated with a decrease in milk yield. Previous research indicated that disruption of tight junctions in the mammary gland may be one of the regulatory factors involved in the milk yield decrease observed during ODM. The aim of this study was to investigate the involvement of mammary epithelium disruption in the regulation of the activity and dynamics of mammary epithelial cells (MEC) during 5 weeks of ODM in goats. Twelve alpine goats (producing 3.67 ± 0.64 kg/day and 47 ± 1.6 days in milk) were assigned to two groups that were milked once or twice a day during 5 weeks and then switched back to TDM. Mammary biopsies were collected before and on days 2 and 16 of both ODM and TDM switchback periods. Milk purified epithelial cells were collected before and on days 1, 7, 21 and 28 during ODM as well on days 1 and 7 of the TDM switchback period. The mRNA levels of genes involved in the regulation of synthetic activity and apoptosis were analysed by RT-PCR in milk MEC and mammary biopsies. ODM decreased yields of milk (-23%), lactose (-23%) and casein (-16%). Lactose synthesis was regulated at the transcriptional level by downregulation of α-lactalbumin mRNA levels in both biopsy samples (-30%) and milk MEC (-74%). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling) staining of mammary gland biopsies did not show any increase in cell apoptosis after 2 and 16 days of ODM (0.8% and 1%, respectively) despite upregulation of Bax mRNA levels in milk MEC. This suggests that the decrease in milk yield observed during ODM is attributable to a decrease in synthetic activity rather than to induction of MEC cell death. ODM induced the disruption of tight junctions in the mammary gland only on the first day of the treatment as indicated by increased blood

  20. MG132 Inhibits Myocardial Ischemia-reperfusion Injury by Regulating Apoptotic Pathway

    Institute of Scientific and Technical Information of China (English)

    Dai Cuilian; Luo Kailiang; Chen Zhangrong

    2007-01-01

    Objectives To administrated proteasome inhibitor-MG-132 prior to reperfusion in rat myocardial ischemia-reperfusion model to determine whether MG-132 could reduce myocytic apoptosis. Methods and results MG-132 (0.75 mg/kg in 2 ml DMSO) injection 5 min prior to reperfusion resulted significant reduction of myocardial reperfusion injury. This effect was accompanied by reduced polymorphonuclear neutrophils(PMN) infiltration in myocardial region surrounding the myocardial infarct, reduced apoptosis in cardiac myocytes, reduced NF-κB activation, as determined by electron microscopy, histology, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick endlabeling (TUNEL) method, reverse transcription-polymerase chain reaction. Functional effects of MG-132 on PMN accumulation, activation of nuclear factor kappa B(p65 mRNA and protein levels ), and apoptosis were characterized in rat myocardial tissue. MG132 time-dependently inhibited myocardial p65 mRNA expression and reduced myocardial apoptotic index (AI) after reperfusion for 2 h, 6 h and 24 h ( P<0.01 ). Moreover, MG-132 time-dependently decreased Bax protein levels, while increased Bcl-2 protein levels in ischemic and reperfused myocardium ( P<0.05 ), its effect peaked after reperfusion for 24 h. Conclusions Our results demonstrate that MG-132 reduced myocardial reperfusion injury by inhibiting myosytic apoptotic cell death and blocking activation of NF-κB, down-regulating Bax expression and up-regulating Bcl-2 expression as well as elevating Bcl-2/Bax ratio.

  1. Doxycycline Protects Thymic Epithelial Cells from Mitomycin C-Mediated Apoptosis In Vitro via Trx2-NF-κB-Bcl-2/Bax Axis

    Directory of Open Access Journals (Sweden)

    Jun Wang

    2016-02-01

    Full Text Available Background/Aims: Age-associated and stress-induced involution of the thymus is accompanied by reduced numbers of thymic epithelial cells (TECs and severe reduction in peripheral T cell repertoire specificities. These events seriously affect immune function, but the mechanisms involved are unclear. Our preliminary findings showed that doxycycline (Dox could drive the proliferation of a TEC line (MTEC1 cells partially via the MAPK signaling pathway. Dox can also up-regulate IL-6 and GM-CSF expression via the NF-κB and MAPK/ERK pathways. Herein, we investigate the effects and mechanisms used by Dox that protect against mitomycin C (MMC-induced MTEC1 cell apoptosis. Methods: MTEC1 cells were treated with Dox, MMC, and Dox plus MMC for different amounts of time. The expression of Trx2, NF-κB, Bcl-2, and Bax proteins were then detected by western blotting. Results: Our findings show that Dox protects MTEC1 cells from MMC-induced apoptosis. Dox up-regulated the expression of Trx2 and promoted NF-κB phosphorylation. Meanwhile, Dox also increased the expression of Bcl-2, partially reduced the expression of Bax, and normalized the ratio of Bcl-2 to Bax. Conclusion: Dox exerts an anti-apoptosis function via the NF-κB-Bcl-2/Bax and Trx2-ASK1/JNK pathways in vitro. Therefore, Dox may represent a drug that could be used to attenuate thymic senescence, rescue thymic function, and promote T cell reconstitution.

  2. V-1 regulates capping protein activity in vivo.

    Science.gov (United States)

    Jung, Goeh; Alexander, Christopher J; Wu, Xufeng S; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A

    2016-10-25

    Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

  3. Phosphorylation regulates NCC stability and transporter activity in vivo.

    Science.gov (United States)

    Yang, Sung-Sen; Fang, Yu-Wei; Tseng, Min-Hua; Chu, Pei-Yi; Yu, I-Shing; Wu, Han-Chung; Lin, Shu-Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh-Feng; Sytwu, Huey-Kang; Lin, Shih-Hua

    2013-10-01

    A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

  4. Ubiquitin chain conformation regulates recognition and activity of interacting proteins.

    Science.gov (United States)

    Ye, Yu; Blaser, Georg; Horrocks, Mathew H; Ruedas-Rama, Maria J; Ibrahim, Shehu; Zhukov, Alexander A; Orte, Angel; Klenerman, David; Jackson, Sophie E; Komander, David

    2012-12-13

    Mechanisms of protein recognition have been extensively studied for single-domain proteins, but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins. Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48- and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63- and Met 1-linked diubiquitin adopt extended 'open' and more compact 'closed' conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48-diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.

  5. German National Galileo Public Regulated Service (PRS) Testing Activities

    Science.gov (United States)

    Habrich, Heinz; Söhne, Wolfgang

    2013-04-01

    The European Global Navigation System (GNSS) Galileo is going to be established in the near future. Currently, four satellites are in place forming the In-Orbit-Testing (IOT) phase. Within the next years, the constellation will be filled. Full Operational Capability (FOC) will be reached 2019. Beside the Open Service (OS) which is comparable to other OS of existing GNSS, e.g., GPS C/A, there is a so-called Public Regulated Service (PRS) included in the IOT satellites already. The PRS will have improved robustness, i.e. robust signals which will be resistant against involuntary interferences, jamming and spoofing. The PRS signal is encrypted and there will be a restricted access to authorized users, e.g. safety and emergency services, authorities with security task, critical infrastructure organizations etc. The access to the PRS which will be controlled through a special key management will be managed and supervised within the European Union (EU) Member States (MS) by national authorities, the Competent PRS Authority (CPA). But a set of Common Minimum Standards (CMS) will define the minimum requirements applicable to each PRS participant. Nevertheless, each MS is responsible for its national key management. This presentation will inform about the testing activities for Galileo PRS in Germany. The coarse concept for the testing is explained, the schedule is outlined. Finally, the paper will formulate some expectations to the Galileo PRS, e.g. for international cooperation.

  6. AMP-activated protein kinase (AMPK) activity negatively regulates chondrogenic differentiation.

    Science.gov (United States)

    Bandow, Kenjiro; Kusuyama, Joji; Kakimoto, Kyoko; Ohnishi, Tomokazu; Matsuguchi, Tetsuya

    2015-05-01

    Chondrocytes are derived from mesenchymal stem cells, and play an important role in cartilage formation. Sex determining region Y box (Sox) family transcription factors are essential for chondrogenic differentiation, whereas the intracellular signal pathways of Sox activation have not been clearly elucidated. AMP-activated protein kinase (AMPK) is a serine-threonine kinase generally regarded as a key regulator of cellular energy homeostasis. It is known that the catalytic alpha subunit of AMPK is activated by upstream AMPK kinases (AMPKKs) including liver kinase B1 (LKB1). We have previously reported that AMPK is a negative regulator of osteoblastic differentiation. Here, we have explored the role of AMPK in chondrogenic differentiation using in vitro culture models. The phosphorylation level of the catalytic AMPK alpha subunit significantly decreased during chondrogenic differentiation of primary chondrocyte precursors as well as ATDC-5, a well-characterized chondrogenic cell line. Treatment with metformin, an activator of AMPK, significantly reduced cartilage matrix formation and inhibited gene expression of sox6, sox9, col2a1 and aggrecan core protein (acp). Thus, chondrocyte differentiation is functionally associated with decreased AMPK activity. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Study on the Expression of Apoptosis - Controlled Gene Bcl - 2/Bax mRNA in the Uterine Leiomyoma%凋亡调控基因Bcl2/Bax mRNA在子宫平滑肌瘤中的表达

    Institute of Scientific and Technical Information of China (English)

    李卫平; 严隽鸿; 林其德; 裴军; 丁传伟

    2000-01-01

    Objective To investigate the expression of apoptosis - controlled gene Bcl - 2/BaxmRNA in leiomyoma and it's relationship with ER、PR. Methods To measure ER, PR volume (28cases) in leiomyoma and the ratio of Bcl - 2/Bax mRNA (21 cases) by immunochemistry and RT -PCR, respectively, and use the myometrium as control. Results The content of ER, PR and the ra-tio of Bcl- 2/Bax mRNA in leiomyoma were significantly higher than those in the myometrium (P< 0.05 and 0.01); there was positive correlation for ER, PR versus ratio of Bcl- 2/Bax mRNA in leiomy-oma Conclusion Apoptosic-controlled gene Bcl - 2/Bax may play a role in the pathogenesis ofleiomyoma; the stimalating effect of estrogen and progesterone combining with ER, PR on leiomyomamay be related with their regulation of the expression of Bcl - 2/Bax mRNA.%目的 探讨细胞凋亡调控基因Bcl-2/Bax mRNA在子宫肌瘤中的表达及与ER、PR的关系。方法应用免疫组化法测定子宫肌瘤内ER、PR的含量(28例)。用RT-PCR法测定肌瘤内Bcl-2/Bax mRNA(21例)。以肌瘤邻近的正常子宫平滑肌作对照。结果子宫肌瘤内ER、PR,Bcl-2/Bax比值显著高于正常子宫平滑肌(P<0.05和P<0.01);肌瘤ER、PR与Bcl-2/BaxmRNA比值呈正相关。结论细胞凋亡调控基因Bcl-2/Bax可能参与子宫肌瘤的发生;而雌、孕激素与ER、PR结合刺激肌瘤生长则可能与影响这一基因的表达有关。

  8. Cystatin F regulates proteinase activity in IL-2-activated natural killer cells.

    Science.gov (United States)

    Maher, Katarina; Konjar, Spela; Watts, Colin; Turk, Boris; Kopitar-Jerala, Natasa

    2014-01-01

    Cystatin F is a unique member of the cystatin family of cysteine protease inhibitors, which is synthesized as an inactive dimer and it is activated by N-terminal cleavage in the endolysosomes. It is expressed in the cells of the immune system: myeloid cells and the cells involved in target cell killing: natural killer (NK) cells and cytotoxic T cells (CTLs). Upon activation of the NK cells with interleukin 2 (IL-2), cystatin F was found upregulated and co-localized in cytotoxic granules with cathepsin C (CatC) and CatV. However, cystatin F inhibits the CatC in cells only when its N-terminal part is processed. Although cystatin F could inhibit both CatV and CatC, the IL-2 stimulation of the YT cells resulted in an increased CatV activity, while the CatC activity was unchanged. The incubation of IL-2 activated NK cells with a cysteine proteinase inhibitor E-64d increased the cystatin F dimer formation. Our results suggest that cystatin F not only inhibits CatV, but it is processed by the CatV in order to inhibit the CatC activity in cytotoxic granules. The regulation of the CatC activity in the cytotoxic granules of the NK cells by the cystatin F could be important for the processing and activation of granule-associated serine proteases - granzymes.

  9. THE EXPRESSION AND CLINICAL VALUE OF APOPTOSIS CONTROL GENE Bcl-2 AND Bax IN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jun; YAO Zhen-xiang; ZHANG Jing

    1999-01-01

    Objective: To study the expression and clinical value of apoptosis control gene bcl-2 and bax in breast cancer.Methods: Protein bax and bcl-2 in 41 breast cancers obtained from operations in our hospital in 1996 were detected using ABC immunohistochemical stain assay and compared with 10 cases with normal breast tissues.Results: The positive rate of bax in normal breast tissue was 90% and in breast cancer was 59%, with a significant statistical difference between them (P<0.05), but there was no statistical difference in bcl-2 protein expression. Among the 41 breast cancer, the group with lymph node metastasis (21 cases) had obviously low bax expression (43%) and high bcl-2 expression (76%), showing significant difference to the group without lymph node metastasis (P<0.05).Conclusion: The antiapoptosis function of bcl-2 was stronger than bax in breast cancer. Protein bax and bcl-2 assay may be useful in understanding the biological behaviors of breast cancer.

  10. Collagen I-induced dendritic cells activation is regulated by TNF- production through down-regulation of IRF4

    Indian Academy of Sciences (India)

    Barun Poudel; Hyeon-Hui Ki; Young-Mi Lee; Dae-Ki Kim

    2015-03-01

    Previously we have shown that collagen I enhances the maturation and function of dendritic cells (DCs). Inflammatory mediators such as tumour necrosis factor (TNF)-, interleukin (IL)-1 and lipopolysaccharide (LPS) are also known to activate DCs. Here we investigated the involvement of TNF- on the collagen I-induced DCs activation. TNF-a neutralization inhibited collagen I-induced IL-12 secretions by DCs. Additionally, we observed suppression of collagen I-induced costimulatory molecules expression along with down-regulation of genes involved in DCs activation pathway. Furthermore, TNF- inhibition upon collagen Istimulation up-regulated the expression of interferon regulatory transcription factor IRF4, when compared to collagen I only treated cells. Collectively, our data demonstrate that collagen I induce TNF- production, which is crucial for the activation and function of DCs, through down-regulation of IRF4, and implicates the importance in development of anti- TNF- therapeutics for several inflammatory diseases.

  11. Arabidopsis TTG2 regulates TRY expression through enhancement of activator complex-triggered activation.

    Science.gov (United States)

    Pesch, Martina; Dartan, Burcu; Birkenbihl, Rainer; Somssich, Imre E; Hülskamp, Martin

    2014-10-01

    Trichome patterning in Arabidopsis thaliana is regulated by a regulatory feedback loop of the trichome promoting factors TRANSPARENT TESTA GLABRA1 (TTG1), GLABRA3 (GL3)/ENHANCER OF GL3 (EGL3), and GL1 and a group of homologous R3MYB proteins that act as their inhibitors. Together, they regulate the temporal and spatial expression of GL2 and TTG2, which are considered to control trichome cell differentiation. In this work, we show that TTG2 is a specific activator of TRY (but not CPC or GL2). The WRKY protein TTG2 binds to W-boxes in a minimal promoter fragment of TRY, and these W-boxes are essential for rescue of the try mutant phenotype. We further show that TTG2 alone is not able to activate TRY expression, but rather drastically enhances the activation by TTG1 and GL3. As TTG2 physically interacts with TTG1 and because TTG2 can associate with GL3 through its interaction with TTG1, we propose that TTG2 enhances the activity of TTG1 and GL3 by forming a protein complex.

  12. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity

    Science.gov (United States)

    Burket, Jessica A.; Benson, Andrew D.; Tang, Amy H.; Deutsch, Stephen I.

    2017-01-01

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORCl in neurons (e.g., cerebellar Purkinje cells). mTORCl is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORCl, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORCl overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORCl activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are “drivers” of mTORCl activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders. PMID:25703582

  13. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity.

    Science.gov (United States)

    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2015-07-01

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders.

  14. Regulator of complement activation (RCA) gene cluster in Xenopus tropicalis.

    Science.gov (United States)

    Oshiumi, Hiroyuki; Suzuki, Yuzuru; Matsumoto, Misako; Seya, Tsukasa

    2009-05-01

    Genome and expressed sequence tag information of Xenopus tropicalis suggested that short-consensus repeat (SCR)-containing proteins are encoded by three genes that are mapped within a 300-kb downstream of PFKFB2, which is a marker gene for the regulator of complement activation (RCA) loci in human and chicken. Based on this observation, we cloned the three cDNAs of these proteins using 3'- or 5'-RACE technique. Since their primary structures and locations of the proximity to the PFKFB2 locus, we named them amphibian RCA protein (ARC) 1, 2, and 3. Expression in human HEK293 or CHO cells suggested that ARC1 is a soluble protein of Mr approximately 67 kDa, ARC2 is a membrane protein with Mr 44 kDa, and ARC3 a secretary protein with a putative transmembrane region. They were N-glycosylated during maturation. In human and chicken RCA clusters, the order in which genes for soluble, GPI-anchored, and membrane forms of SCR proteins are arranged is from the distant to proximity to the PFKFB2 gene. However, the amphibian ARC1, 2, and 3 resembled one another and did not reflect the same order found in human and chicken RCA genes. This may be due to self-duplication of ARCs to form a family, and it evolved after the amphibia separated from the ancestor of the amniotes, which possessed soluble, GPI-anchored, and membrane forms of SCR protein members. Taken together, frog possesses a RCA locus, but the constitution of the ARC proteins differs from that of the amniotes with a unique self-resemblance.

  15. Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize.

    Science.gov (United States)

    Schmelz, Eric A; Kaplan, Fatma; Huffaker, Alisa; Dafoe, Nicole J; Vaughan, Martha M; Ni, Xinzhi; Rocca, James R; Alborn, Hans T; Teal, Peter E

    2011-03-29

    Phytoalexins constitute a broad category of pathogen- and insect-inducible biochemicals that locally protect plant tissues. Because of their agronomic significance, maize and rice have been extensively investigated for their terpenoid-based defenses, which include insect-inducible monoterpene and sesquiterpene volatiles. Rice also produces a complex array of pathogen-inducible diterpenoid phytoalexins. Despite the demonstration of fungal-induced ent-kaur-15-ene production in maize over 30 y ago, the identity of functionally analogous maize diterpenoid phytoalexins has remained elusive. In response to stem attack by the European corn borer (Ostrinia nubilalis) and fungi, we observed the induced accumulation of six ent-kaurane-related diterpenoids, collectively termed kauralexins. Isolation and identification of the predominant Rhizopus microsporus-induced metabolites revealed ent-kaur-19-al-17-oic acid and the unique analog ent-kaur-15-en-19-al-17-oic acid, assigned as kauralexins A3 and B3, respectively. Encoding an ent-copalyl diphosphate synthase, fungal-induced An2 transcript accumulation precedes highly localized kauralexin production, which can eventually exceed 100 μg · g(-1) fresh weight. Pharmacological applications of jasmonic acid and ethylene also synergize the induced accumulation of kauralexins. Occurring at elevated levels in the scutella of all inbred lines examined, kauralexins appear ubiquitous in maize. At concentrations as low as 10 μg · mL(-1), kauralexin B3 significantly inhibited the growth of the opportunistic necrotroph R. microsporus and the causal agent of anthracnose stalk rot, Colletotrichum graminicola. Kauralexins also exhibited significant O. nubilalis antifeedant activity. Our work establishes the presence of diterpenoid defenses in maize and enables a more detailed analysis of their biosynthetic pathways, regulation, and crop defense function.

  16. Termination of the Activating NK Cell Immunological Synapse Is an Active and Regulated Process.

    Science.gov (United States)

    Netter, Petra; Anft, Moritz; Watzl, Carsten

    2017-08-23

    Cellular cytotoxicity is essential for the elimination of virus-infected and cancerous cells by NK cells. It requires a direct cellular contact through the establishment of an immunological synapse (IS) between the NK cell and the target cell. In this article, we show that not only the establishment of the IS, but also its maintenance is a highly regulated process. Ongoing receptor-proximal signaling events from activating NK cell receptors and actin dynamics were necessary to maintain a stable contact in an energy-dependent fashion, even after the IS was formed successfully. More importantly, the initiation of a contact to a new susceptible target cell resulted in accelerated detachment from an old target cell. We propose that the maintenance of an existing IS is a dynamic and regulated process to allow for effective serial killing of NK cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  17. Neurorescue activity, APP regulation and amyloid-beta peptide reduction by novel multi-functional brain permeable iron- chelating- antioxidants, M-30 and green tea polyphenol, EGCG.

    Science.gov (United States)

    Avramovich-Tirosh, Yael; Reznichenko, Lydia; Mit, Tamar; Zheng, Hailin; Fridkin, Mati; Weinreb, Orly; Mandel, Silvia; Youdim, Moussa B H

    2007-09-01

    Accumulation of iron at sites where neurons degenerate in Parkinson's disease (PD) and Alzheimer's disease (AD) is thought to have a major role in oxidative stress induced process of neurodegeneration. The novel non-toxic lipophilic brain- permeable iron chelators, VK-28 (5- [4- (2- hydroxyethyl) piperazine-1-ylmethyl]- quinoline- 8- ol) and its multi-functional derivative, M-30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxyquinoline), as well as the main polyphenol constituent of green tea (-)-epigallocatechin-3-gallate (EGCG), which possesses iron metal chelating, radical scavenging and neuroprotective properties, offer potential therapeutic benefits for these diseases. M-30 and EGCG decreased apoptosis of human SH-SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via multiple protection mechanisms including: reduction of the pro-apoptotic proteins, Bad and Bax, reduction of apoptosis-associated Ser139 phosphorylated H2A.X and inhibition of the cleavage and activation of caspase-3. M-30 and EGCG also promoted morphological changes, resulting in axonal growth-associated protein-43 (GAP-43) implicating neuronal differentiation. Both compounds significantly reduced the levels of cellular holo-amyloid precursor protein (APP) in SH-SY5Y cells. The ability of theses novel iron chelators and EGCG to regulate APP are in line with the presence of an iron-responsive element (IRE) in the 5'-untranslated region (5'UTR) of APP. Also, EGCG reduced the levels of toxic amyloid-beta peptides in CHO cells over-expressing the APP "Swedish" mutation. The diverse molecular mechanisms and cell signaling pathways participating in the neuroprotective/neurorescue and APP regulation/processing actions of M-30 and EGCG, make these multifunctional compounds potential neuroprotective drugs for the treatment of neurodegenerative diseases, such as PD, AD, Huntington's disease and amyotrophic lateral sclerosis.

  18. GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

    Science.gov (United States)

    Reshi, Latif; Wu, Horng-Cherng; Wu, Jen-Leih; Wang, Hao-Ven; Hong, Jiann-Ruey

    2016-04-01

    Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1 h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24 h (37 %) and 48 h post-transfection (PT) (49 %). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48 h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these

  19. Bim regulates B-cell receptor-mediated apoptosis in the presence of CD40 signaling in CD40-pre-activated splenic B cells differentiating into plasma cells.

    Science.gov (United States)

    Gao, Yuanyuan; Kazama, Hirotaka; Yonehara, Shin

    2012-05-01

    B-cell receptor (BCR)-mediated apoptosis is critical for B-cell development and homeostasis. CD40 signaling has been shown to protect immature or mature B cells from BCR-mediated apoptosis. In this study, to understand the fate of CD40-pre-activated splenic B cells stimulated by BCR engagement in the presence of CD40 signaling, murine splenic B cells were cultured with anti-Igκ and anti-CD40 antibodies after pre-activation with anti-CD40 antibody. We found that apoptosis was induced in the cultured B cells even in the presence of CD40 signaling during the 3-4 days cultivation. We detected up-regulation of Bim expression followed by Bax activation in this apoptotic process and cessation of the apoptosis in Bim-deficient B cells, indicating that Bim is a key regulator of the BCR-mediated apoptosis in the presence of CD40 signaling in CD40-pre-activated B cells. Importantly, this BCR-mediated apoptosis in CD40-pre-activated B cells was shown to be induced at the initiation of plasma cell differentiation at around the preplasmablast stage, and Bim-deficient B cells cultured under these conditions differentiated into plasma cells. Additionally, transforming growth factor-β was found to protect CD40-pre-activated B cells from BCR-mediated apoptosis in the presence of CD40 signaling. Our identified BCR-mediated apoptosis, which is unpreventable by CD40 signaling, suggests a potential mechanism that regulates the elimination of peripheral B cells, which should be derived from nonspecific T-dependent activation of bystander B cells and continuous stimulation with antigens including self-antigens in the presence of T cell help through CD40.

  20. Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

    Science.gov (United States)

    Gillissen, B; Richter, A; Richter, A; Overkamp, T; Essmann, F; Hemmati, P G; Preissner, R; Belka, C; Daniel, P T

    2013-05-23

    TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of th