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Sample records for kinase-1 ask-1 rescues

  1. Production of recombinant human apoptosis signal-regulating kinase 1 (ASK1) in Escherichia coli.

    PubMed

    Volynets, Galyna P; Gorbatiuk, Oksana B; Kukharenko, Oleksandr P; Usenko, Mariya O; Yarmoluk, Sergiy M

    2016-10-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. beta-Arrestins facilitate ubiquitin-dependent degradation of apoptosis signal-regulating kinase 1 (ASK1) and attenuate H2O2-induced apoptosis.

    PubMed

    Zhang, Zhengping; Hao, Jiaying; Zhao, Zhihui; Ben, Peiling; Fang, Fang; Shi, Lijun; Gao, Yanhong; Liu, Junhong; Wen, Chuanjun; Luo, Lan; Yin, Zhimin

    2009-07-01

    beta-Arrestins are ubiquitously expressed proteins that play important roles in receptor desensitization, endocytosis, proteosomal degradation, apoptosis and signaling. It has been reported that beta-Arrestin2 acts as a scaffold by directly interacting with the JNK3 isoform and recruiting MKK4 and the apoptosis-signaling kinase-1 (ASK1). Here, we report a novel function of beta-Arrestins in regulating H(2)O(2)-induced apoptosis. Our study demonstrates that beta-Arrestins physically associate with C-terminal domain of ASK1, and moreover, both over-expression and RNA interference (RNAi) experiments indicate that beta-Arrestins down-regulate ASK1 protein. In detail, beta-Arrestin-induced reduction of ASK1 protein is due to ubiquitination and proteasome-dependent degradation of ASK1 in response to association of beta-Arrestins and ASK1. Upon H(2)O(2) stimulation, the protein binding between beta-Arrestins and ASK1 increases and ASK1 degradation is expedited. In consequence, beta-Arrestins prevent ASK1-JNK signaling and as a result attenuate H(2)O(2)-induced apoptosis. Structurally, C-terminal domain of ASK1 is essential for beta-Arrestins and ASK1 association. We also found that CHIP is required for beta-Arrestins-induced ASK1 degradation, which suggested that beta-Arrestins function as a scaffold of ASK1 and CHIP, leading to CHIP-mediated ASK1 degradation. All these findings indicate that beta-Arrestins play a negative regulatory role in H(2)O(2)-induced apoptosis signaling through associating with ASK1 and CHIP and facilitating ASK1 degradation, which provides a new insight for analyzing the effects of beta-Arrestins on protecting cells from oxidative stress-induced apoptosis.

  3. A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway.

    PubMed

    Kim, So Yong; Kim, Tae Jin; Lee, Ki-Young

    2008-06-11

    We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.

  4. SOCS-1 rescues IL-1β-mediated suppression of epithelial sodium channel in mouse lung epithelial cells via ASK-1

    PubMed Central

    Galam, Lakshmi; Soundararajan, Ramani; Breitzig, Mason; Rajan, Ashna; Yeruva, Rajashekar Reddy; Czachor, Alexander; Harris, Francine; Lockey, Richard F; Kolliputi, Narasaiah

    2016-01-01

    Background Acute lung injury (ALI) is characterized by alveolar damage, increased levels of pro-inflammatory cytokines and impaired alveolar fluid clearance. Recently, we showed that the deletion of Apoptosis signal-regulating kinase 1 (ASK1) protects against hyperoxia-induced acute lung injury (HALI) by suppressing IL-1β and TNF-α. Previously, our data revealed that the suppressor of cytokine signaling-1 (SOCS-1) overexpression restores alveolar fluid clearance in HALI by inhibiting ASK-1 and suppressing IL-1β levels. Furthermore, IL-1β is known to inhibit the expression of epithelial sodium channel α-subunit (ENaC) via a p38 MAPK signaling pathway. Objective To determine whether SOCS-1 overexpression in MLE-12 cells would protect against IL-1β-mediated depletion of αENaC by suppressing ASK-1 expression. Methods We co-transfected MLE-12 cells with SOCS-1 overexpressing plasmid with or without IL-1β in the presence or absence of sodium channel inhibitor, amiloride. We measured potential difference, transepithelial current, resistance, and sodium uptake levels across MLE-12 cells. We studied the effect of ASK-1 depletion, as well as ASK-1 and SOCS-1 overexpression on αENaC expression. Results SOCS-1 overexpression sufficiently restored transepithelial current and resistance in MLE-12 cells treated with either IL-1β or amiloride. The αENaC mRNA levels and sodium transport were increased in SOCS-1 overexpressing MLE-12 cells exposed to IL-1β. Depletion of ASK-1 in MLE-12 cells increased αENaC mRNA levels. Interestingly, SOCS-1 overexpression restored αENaC expression in MLE-12 cells in the presence of ASK-1 overexpression. Conclusion Collectively, these findings suggest that SOCS-1 may exert its protective effect by rescuing αENaC expression via suppression of ASK-1. PMID:27058411

  5. Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase*

    PubMed Central

    Subramanyam, Muthangi; Takahashi, Nobuyuki; Hasegawa, Yuichi; Mohri, Tatsuma; Okada, Yasunobu

    2010-01-01

    Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-α, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1. PMID:20048146

  6. Apoptosis Signal-regulating Kinase 1 (ASK1)-p38 Pathway-dependent Cytoplasmic Translocation of the Orphan Nuclear Receptor NR4A2 Is Required for Oxidative Stress-induced Necrosis.

    PubMed

    Watanabe, Takeshi; Sekine, Shiori; Naguro, Isao; Sekine, Yusuke; Ichijo, Hidenori

    2015-04-24

    p38 mitogen-activated protein kinases (MAPKs) play important roles in various cellular stress responses, including cell death, which is roughly categorized into apoptosis and necrosis. Although p38 signaling has been extensively studied, the molecular mechanisms of p38-mediated cell death are unclear. ASK1 is a stress-responsive MAP3K that acts as an upstream kinase of p38 and is activated by various stresses, such as oxidative stress. Here, we show that NR4A2, a member of the NR4A nuclear receptor family, acts as a necrosis promoter downstream of ASK1-p38 pathway during oxidative stress. Although NR4A2 is well known as a nucleus-localized transcription factor, we found that it is translocated into the cytosol after phosphorylation by p38. Because the phosphorylation site mutants of NR4A2 cannot rescue the cell death-promoting activity, ASK1-p38 pathway-dependent phosphorylation and subsequent cytoplasmic translocation of NR4A2 may be required for oxidative stress-induced cell death. In addition, NR4A2-mediated cell death does not depend on caspases and receptor-interacting protein 1 (RIP1)-RIP3 complex, suggesting that NR4A2 promotes an RIP kinase-independent necrotic type of cell death. Our findings may enable a more precise understanding of molecular mechanisms that regulate oxidative stress-induced and p38-mediated necrosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3

    PubMed Central

    Zhang, Rong; He, Xiangrong; Liu, Weimin; Lu, Meng; Hsieh, Jer-Tsong; Min, Wang

    2003-01-01

    TNF-α activates ASK1 in part by dissociating 14-3-3 from apoptosis signal–regulating kinase 1 (ASK1). In the present study, we identified a novel Ras GTPase-activating protein (Ras-GAP) as an ASK1-interacting protein (AIP1). AIP1 binds to the C-terminal domain of ASK1 via a lysine-rich cluster within the N-terminal C2 domain. AIP1 exists in a closed form through an intramolecular interaction between the N-terminus and the C-terminus, and TNF-α induces unfolding of AIP1 leading to association of AIP1 with ASK1. Thus, the N-terminus of AIP1 containing the C2 and GAP domains constitutively binds to ASK1 and facilitates the release of 14-3-3 from ASK1. In contrast to 14-3-3, AIP1 binds preferentially to dephosphorylated ASK1. Recruited AIP1 enhances ASK1-induced JNK activation, and the ASK1 binding and the GAP activity of AIP1 are critical for AIP1-enhanced ASK1 activation. Furthermore, TNF-induced ASK1/JNK activation is significantly blunted in cells where AIP1 is knocked down by RNA interference. These data suggest that AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of inhibitor 14-3-3 from ASK1, a novel mechanism by which TNF-α activates ASK1. PMID:12813029

  8. Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

    PubMed

    Onda, Kenji; Tong, Stephen; Beard, Sally; Binder, Natalie; Muto, Masanaga; Senadheera, Sevvandi N; Parry, Laura; Dilworth, Mark; Renshall, Lewis; Brownfoot, Fiona; Hastie, Roxanne; Tuohey, Laura; Palmer, Kirsten; Hirano, Toshihiko; Ikawa, Masahito; Kaitu'u-Lino, Tu'uhevaha; Hannan, Natalie J

    2017-03-01

    Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-α-induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

  9. Erythropoietin Rescues Primary Rat Cortical Neurons by Altering the Nrf2:Bach1 Ratio: Roles of Extracellular Signal-Regulated Kinase 1/2.

    PubMed

    Zhang, Li-Min; Zhang, Dong-Xue; Zhao, Xiao-Chun; Sun, Wenbo

    2017-01-12

    While inhalation anesthetics are indispensable, and generally considered safe and effective, there is growing concern about the selective neurotoxicity of these agents, especially sevoflurane. Erythropoetin (EPO)-induced protection against sevoflurane-induced neuronal death is an effective intervention, but the underlying mechanism is poorly understood. Extracellular signal-related kinases (Erk) 1/2 plays a pivotal role in cell growth and proliferation. Alteration of the nuclear factor erythroid 2-related factor (Nrf2)/BTB-to-CNC homology 1 (Bach1) ratio by Erk1/2 ameliorates the oxidative stress which occurs in human macrophages. Primary cortical neuron cultures exposed to sevoflurane were assessed for Nrf2, Bach1, total Erk1/2, and phosphorylated Erk1/2 with the following: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; propidium iodide uptake; lactate dehydrogenase; malondialdehyde (MDA); superoxide dismutase (SOD); and Western blot. Sevoflurane exposure increased cell death, injury, and MDA (n = 9, P < 0.05), but decreased cell viability and the Nrf2:Bach1 ratio (n = 9, P < 0.05) and down-regulated SOD (n = 9, P < 0.05), while EPO partially rescued the neurotoxicity induced by sevoflurane (n = 9, P < 0.05). Inhibition of Erk1/2 phosphorylation via PD98059 reversed the protective effect of EPO (n = 9, P < 0.05). Thus, protection of EPO markedly attenuated death of neurons exposed to sevoflurane by altering the Nrf2:Bach1 ratio mediated by phosphorylation and activation of Erk1/2.

  10. The effect of ASK1 on vascular permeability and edema formation in cerebral ischemia.

    PubMed

    Song, Juhyun; Cheon, So Yeong; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2015-01-21

    Apoptosis signal-regulating kinase-1 (ASK1) is the mitogen-activated protein kinase kinase kinase (MAPKKK) and participates in the various central nervous system (CNS) signaling pathways. In cerebral ischemia, vascular permeability in the brain is an important issue because regulation failure of it results in edema formation and blood-brain barrier (BBB) disruption. To determine the role of ASK1 on vascular permeability and edema formation following cerebral ischemia, we first investigated ASK1-related gene expression using microarray analyses of ischemic brain tissue. We then measured protein levels of ASK1 and vascular endothelial growth factor (VEGF) in brain endothelial cells after hypoxia injury. We also examined protein expression of ASK1 and VEGF, edema formation, and morphological alteration through cresyl violet staining in ischemic brain tissue using ASK1-small interference RNA (ASK1-siRNA). Finally, immunohistochemistry was performed to examine VEGF and aquaporin-1 (AQP-1) expression in ischemic brain injury. Based on our findings, we propose that ASK1 is a regulating factor of vascular permeability and edema formation in cerebral ischemia.

  11. NQDI 1, an inhibitor of ASK1 attenuates acute ischemic renal injury by modulating oxidative stress and cell death.

    PubMed

    El Eter, Eman

    2013-09-01

    Apoptosis signal-regulating kinase 1 (ASK1) is among the signaling events that lead to postischemic cell death. Inhibition of ASK1 pathway protected hearts from ischemic damage. The present study evaluated the renal protective effects of NQDI 1, an inhibitor of ASK1, in an animal model of acute ischemic renal failure. Male Wistar rats were subjected to right nephrectomy and clamping of left renal pedicle for 45 min, or sham operation. The administration of NQDI 1 attenuated renal dysfunction and histological changes characteristic for renal ischemia/reperfusion injury (IRI). Apoptosis of renal tissues, as detected by TUNEL staining, was also reduced together with p53 protein expression, and renal levels of MDA and SOD with NQDI 1 administration and BCL2 was up regulated. In conclusion, inhibition of ASK1 is of therapeutic potential against acute ischemic renal injury. Its protective effects are mediated via inhibition of apoptosis and oxidative stress.

  12. ASK1 controls spindle orientation and positioning by phosphorylating EB1 and stabilizing astral microtubules

    PubMed Central

    Luo, Youguang; Ran, Jie; Xie, Songbo; Yang, Yunfan; Chen, Jie; Li, Shanshan; Shui, Wenqing; Li, Dengwen; Liu, Min; Zhou, Jun

    2016-01-01

    Orientation and positioning of the mitotic spindle are involved in dictating cell division axis and cleavage site, and play important roles in cell fate determination and tissue morphogenesis. However, how spindle movement is controlled to achieve a defined alignment within the dividing cell is not fully understood. Here, we describe an unexpected role for apoptosis signal-regulating kinase 1 (ASK1) in regulating spindle behavior. We find that ASK1 is required for proper mitotic progression and daughter cell adhesion to the substratum. ASK1 interacts with end-binding protein 1 (EB1) and phosphorylates EB1 at serine 40, threonine 154 and threonine 206, enhancing its binding to the plus ends of astral microtubules. Consequently, astral microtubules are stabilized and therefore capable of mediating spindle interaction with the cell cortex, a requirement for spindle movement. These findings reveal a previously undiscovered function of ASK1 in cell division by regulating spindle orientation and positioning, and point to the importance of protein phosphorylation in the regulation of spindle behavior. PMID:27721984

  13. LIM-kinase1.

    PubMed

    Stanyon, C A; Bernard, O

    1999-01-01

    LIM-kinase1 (LIMK1) is a serine-only protein kinase that contains LIM and PDZ protein-protein interaction domains which is highly expressed in neurons. Overexpression of LIMK1 in cultured cells results in accumulation of filamentous (F-) actin. LIMK1 phosphorylates cofilin, an actin depolymerisation factor, which is then unable to bind and depolymerise F-actin. Rac-GTP enhances phosphorylation of LIMK1 and cofilin, which leads to accumulation of F-actin, while Rac-GDP and PMA reduce these effects. LIMK1 is therefore a key component of a signal transduction network that connects extracellular stimuli to changes in cytoskeletal structure. Control of cell morphology and mobility via LIMK1 activity may provide novel approaches to cancer therapy.

  14. AIP1 recruits phosphatase PP2A to ASK1 in tumor necrosis factor-induced ASK1-JNK activation.

    PubMed

    Min, Wang; Lin, Yan; Tang, Shibo; Yu, Luyang; Zhang, Haifeng; Wan, Ting; Luhn, Tricia; Fu, Haian; Chen, Hong

    2008-04-11

    Previously we have shown that AIP1 (apoptosis signal-regulating kinase [ASK]1-interacting protein 1), a novel member of the Ras-GAP protein family, facilitates dephosphorylation of ASK1 at pSer967 and subsequently 14-3-3 release from ASK1, leading to enhanced ASK1-JNK signaling. However, the phosphatase(s) responsible for ASK1 dephosphorylation at pSer967 has not been identified. In the present study, we identified protein phosphatase (PP)2A as a potential phosphatase in vascular endothelial cells (ECs). Tumor necrosis factor (TNF)-induced dephosphorylation of ASK1 pSer967 in ECs was blocked by PP2A inhibitor okadaic acid. Overexpression of PP2A catalytic subunit induced dephosphorylation of ASK1 pSer967 and activation of c-Jun N-terminal kinase (JNK). In contrast, a catalytic inactive form of PP2A or PP2A small interfering RNA blunted TNF-induced dephosphorylation of ASK1 pSer967 and activation of JNK without effects on NF-kappaB activation. Whereas AIP1, via its C2 domain, binds to ASK1, PP2A binds to the GAP domain of AIP1. Endogenous AIP1-PP2A complex can be detected in the resting state, and TNF induces a complex formation of AIP1-PP2A with ASK1. Furthermore, TNF-induced association of PP2A with ASK1 was diminished in AIP1-knockdown ECs, suggesting a critical role of AIP1 in recruiting PP2A to ASK1. TNF-signaling molecules TRAF2 and RIP1, known to be in complex with AIP1 and activate AIP1 by phosphorylating AIP1 at Ser604, are critical for TNF-induced ASK1 dephosphorylation. Finally, PP2A and AIP1 cooperatively induce activation of ASK1-JNK signaling and EC apoptosis, as demonstrated by both overexpression and small interfering RNA knockdown approaches. Taken together, our data support a critical role of PP2A-AIP1 complex in TNF-induced activation of ASK1-JNK apoptotic signaling.

  15. Ask1 gene deletion blocks maternal diabetes-induced endoplasmic reticulum stress in the developing embryo by disrupting the unfolded protein response signalosome.

    PubMed

    Wang, Fang; Wu, Yanqing; Gu, Hui; Reece, E Albert; Fang, Shengyun; Gabbay-Benziv, Rinat; Aberdeen, Graham; Yang, Peixin

    2015-03-01

    Apoptosis signal-regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1α (IRE1α), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1α signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1α. Ask1 deletion suppressed diabetes-induced IRE1α endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1α signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose- or tunicamycin-induced IRE1α activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis.

  16. Ask1 Gene Deletion Blocks Maternal Diabetes–Induced Endoplasmic Reticulum Stress in the Developing Embryo by Disrupting the Unfolded Protein Response Signalosome

    PubMed Central

    Wang, Fang; Wu, Yanqing; Gu, Hui; Reece, E. Albert; Fang, Shengyun; Gabbay-Benziv, Rinat; Aberdeen, Graham

    2015-01-01

    Apoptosis signal–regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1α (IRE1α), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1α signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1α. Ask1 deletion suppressed diabetes-induced IRE1α endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1α signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose– or tunicamycin-induced IRE1α activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis. PMID:25249581

  17. 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

    SciTech Connect

    Fu, Meili; Wan, Fuqiang; Li, Zhengling; Zhang, Fenghua

    2016-03-04

    The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation–inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D, a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.

  18. MiR-17 Downregulation by High Glucose Stabilizes Thioredoxin-Interacting Protein and Removes Thioredoxin Inhibition on ASK1 Leading to Apoptosis.

    PubMed

    Dong, Daoyin; Fu, Noah; Yang, Peixin

    2016-03-01

    Pregestational diabetes significantly increases the risk of neural tube defects (NTDs). Maternal diabetes activates an Apoptosis Signal-regulating Kinase 1 (ASK1)-initiated pathway, which triggers neural stem cell apoptosis of the developing neuroepithelium leading to NTD formation. How high glucose of diabetes activates ASK1 is still unclear. In this study, we investigated the mechanism underlying high glucose-induced ASK1 activation. High glucose suppressed miR-17 expression, which led to an increase in its target gene Txnip (Thioredoxin-interacting protein). High glucose-increased Txnip enhanced its binding to the ASK1 inhibitor, thioredoxin (Trx), and thereby sequestered Trx from the Trx-ASK1 complex. High glucose-induced ASK1 activation and consequent apoptosis were abrogated by either the miR-17 mimic or Txnip siRNA knockdown. In contrast, the miR-17 inhibitor or Txnip ectopic overexpression mimicked the stimulative effect of high glucose on ASK1 and apoptosis. Thus, our study demonstrated that miR-17 repression mediates the pro-apoptotic effect of high glucose, and revealed a new mechanism underlying ASK1 activation, in which decreased miR-17 removes Trx inhibition on ASK1 through Txnip. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. ASK1 mediates the teratogenicity of diabetes in the developing heart by inducing ER stress and inhibiting critical factors essential for cardiac development.

    PubMed

    Wang, Fang; Wu, Yanqing; Quon, Michael J; Li, Xuezheng; Yang, Peixin

    2015-09-01

    Maternal diabetes in mice induces heart defects similar to those observed in human diabetic pregnancies. Diabetes enhances apoptosis and suppresses cell proliferation in the developing heart, yet the underlying mechanism remains elusive. Apoptosis signal-regulating kinase 1 (ASK1) activates the proapoptotic c-Jun NH2-terminal kinase 1/2 (JNK1/2) leading to apoptosis, suggesting a possible role of ASK1 in diabetes-induced heart defects. We aimed to investigate whether ASK1 is activated in the heart and whether deleting the Ask1 gene blocks diabetes-induced adverse events and heart defect formation. The ASK1-JNK1/2 pathway was activated by diabetes. Deleting Ask1 gene significantly reduced the rate of heart defects, including ventricular septal defects (VSDs) and persistent truncus arteriosus (PTA). Additionally, Ask1 deletion diminished diabetes-induced JNK1/2 phosphorylation and its downstream transcription factors and endoplasmic reticulum (ER) stress markers. Consistent with this, caspase activation and apoptosis were blunted. Ask1 deletion blocked the increase in cell cycle inhibitors (p21 and p27) and the decrease in cyclin D1 and D3 and reversed diabetes-repressed cell proliferation. Ask1 deletion also restored the expression of BMP4, NKX2.5, and GATA5, Smad1/5/8 phosphorylation, whose mutations or deletion result in reduced cell proliferation, VSD, and PTA formation. We conclude that ASK1 may mediate the teratogenicity of diabetes through activating the JNK1/2-ER stress pathway and inhibiting cell cycle progression, thereby impeding the cardiogenesis pathways essential for ventricular septation and outflow tract development.

  20. ASK1 is involved in cognitive impairment caused by long-term high-fat diet feeding in mice

    PubMed Central

    Toyama, Kensuke; Koibuchi, Nobutaka; Hasegawa, Yu; Uekawa, Ken; Yasuda, Osamu; Sueta, Daisuke; Nakagawa, Takashi; Ma, Mingjie; Kusaka, Hiroaki; Lin, Bowen; Ogawa, Hisao; Ichijo, Hidenori; Kim-Mitsuyama, Shokei

    2015-01-01

    Although high-fat diet intake is known to cause obesity and diabetes, the effect of high-fat diet itself on cognitive function remains to be clarified. We have previously shown that apoptosis signal-regulating kinase 1 (ASK1) is responsible for cognitive impairment caused by chronic cerebral hypoperfusion. The present work, by using ASK1 deficient mice, was undertaken to explore the influence of chronic high-fat diet intake on cognitive function and the role of ASK1. Cognitive function in wild-type mice fed high-fat diet from 2 to 24 months of age was significantly impaired compared to those fed control diet, which was associated with the significant white matter lesions, reduction of hippocampal capillary density, and decrement of hippocampal neuronal cell. However, ASK1 deficiency abolished the development of cognitive impairment and cerebral injury caused by high-fat diet. Our results provided the evidence that high-fat diet itself causes cognitive impairment and ASK1 participates in such cognitive impairment. PMID:26044555

  1. Adenosine 5′-monophosphate blocks acetaminophen toxicity by increasing ubiquitination-mediated ASK1 degradation

    PubMed Central

    Sun, Qi; Xu, Xi; Kong, Yi; Zhang, Jianfa

    2017-01-01

    Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the world. Hepatic c-jun NH2-terminal protein kinase (JNK) activation is thought to be a consequence of oxidative stress produced during APAP metabolism. Activation of JNK signals causes hepatocellular damage with necrotic and apoptotic cell death. Here we found that APAP caused a feedback increase in plasma adenosine 5′-monophsphate (5′-AMP). We demonstrated that co-administration of APAP and 5′-AMP significantly ameliorated APAP-induced hepatotoxicity in mice, without influences on APAP metabolism and its analgesic function. The mechanism of protection by 5′-AMP was through inhibiting APAP-induced activation of JNK, and attenuating downstream c-jun and c-fos gene expression. This was triggered by attenuating apoptosis signal-regulated kinase 1(ASK1) methylation and increasing ubiquitination-mediated ASK1 protein degradation. Our findings indicate that replacing the current APAP with a safe and functional APAP/5′-AMP formulation could prevent APAP-induced hepatotoxicity. PMID:28031524

  2. ASK1 modulates the expression of microRNA Let7A in microglia under high glucose in vitro condition

    PubMed Central

    Song, Juhyun; Lee, Jong Eun

    2015-01-01

    Hyperglycemia results in oxidative stress and leads to neuronal apoptosis in the brain. Diabetes studies show that microglia participate in the progression of neuropathogenesis through their involvement in inflammation in vivo and in vitro. In high-glucose-induced inflammation, apoptosis signal regulating kinase 1 (ASK1) triggers the release of apoptosis cytokines and apoptotic gene expression. MicroRNA-Let7A (miR-Let7A) is reported to be a regulator of inflammation. In the present study, we investigated whether miR-Let7A regulates the function of microglia by controlling ASK1 in response to high-glucose-induced oxidative stress. We performed reverse transcription (RT) polymerase chain reaction, Taqman assay, real-time polymerase chain reaction, and immunocytochemistry to confirm the alteration of microglia function. Our results show that miR-Let7A is associated with the activation of ASK1 and the expression of anti-inflammatory cytokine (interleukin (IL)-10) and Mycs (c-Myc and N-Myc). Thus, the relationship between Let-7A and ASK1 could be a novel target for enhancing the beneficial function of microglia in central nervous system (CNS) disorders. PMID:26041997

  3. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

    SciTech Connect

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K.; Ahmed, Salahuddin

    2015-09-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. - Highlights: • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA). • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS. • ASK1 may be used as a potential therapeutic target in RA. • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS. • Thymoquinone might serve as a potential small

  4. Novel anti-apoptotic mechanism of A20 through targeting ASK1 to suppress TNF-induced JNK activation.

    PubMed

    Won, M; Park, K A; Byun, H S; Sohn, K-C; Kim, Y-R; Jeon, J; Hong, J H; Park, J; Seok, J H; Kim, J M; Yoon, W-H; Jang, I-S; Shen, H M; Liu, Z G; Hur, G M

    2010-12-01

    The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-κB (NF-κB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-κB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-κB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.

  5. Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1.

    PubMed

    Dorion, Sonia; Lambert, Herman; Landry, Jacques

    2002-08-23

    Despite the importance of the stress-activated protein kinase pathways in cell death and survival, it is unclear how stressful stimuli lead to their activation. In the case of heat shock, the existence of a specific mechanism of activation has been evidenced, but the molecular nature of this pathway is undefined. Here, we found that Ask1 (apoptosis signal-regulating kinase 1), an upstream activator of the stress-activated protein kinase p38 during exposure to oxidative stress and other stressful stimuli, was also activated by heat shock. Ask1 activity was required for p38 activation since overexpression of a kinase dead mutant of Ask1, Ask1(K709M), inhibited heat shock-induced p38 activation. The activation of Ask1 by oxidative stress involves the oxidation of thioredoxin, an endogenous inhibitor of Ask1. A different activation mechanism takes place during heat shock. In contrast to p38 induction by H(2)O(2), induction by heat shock was not antagonized by pretreatment with the antioxidant N-acetyl-l-cysteine or by overexpressing thioredoxin and was not accompanied by the dissociation of thioredoxin from Ask1. Instead, heat shock caused the dissociation of glutathione S-transferase Mu1-1 (GSTM1-1) from Ask1 and overexpression of GSTM1-1-inhibited induction of p38 by heat shock. We concluded that because of an alternative regulation by the two distinct repressors thioredoxin and GSTM1-1, Ask1 constitutes the converging point of the heat shock and oxidative stress-sensing pathways that lead to p38 activation.

  6. Maternal hyperglycemia activates an ASK1-FoxO3a-caspase 8 pathway that leads to embryonic neural tube defects.

    PubMed

    Yang, Peixin; Li, Xuezheng; Xu, Cheng; Eckert, Richard L; Reece, E Albert; Zielke, Horst Ronald; Wang, Fang

    2013-08-27

    Neural tube defects result from failure to completely close neural tubes during development. Maternal diabetes is a substantial risk factor for neural tube defects, and available evidence suggests that the mechanism that links hyperglycemia to neural tube defects involves oxidative stress and apoptosis. We demonstrated that maternal hyperglycemia correlated with activation of the apoptosis signal-regulating kinase 1 (ASK1) in the developing neural tube, and Ask1 gene deletion was associated with reduced neuroepithelial cell apoptosis and development of neural tube defects. ASK1 activation stimulated the activity of the transcription factor FoxO3a, which increased the abundance of the apoptosis-promoting adaptor protein TRADD, leading to activation of caspase 8. Hyperglycemia-induced apoptosis and the development of neural tube defects were reduced with genetic ablation of either FoxO3a or Casp8 or inhibition of ASK1 by thioredoxin. Examination of human neural tissues affected by neural tube defects revealed increased activation or abundance of ASK1, FoxO3a, TRADD, and caspase 8. Thus, activation of an ASK1-FoxO3a-TRADD-caspase 8 pathway participates in the development of neural tube defects, which could be prevented by inhibiting intermediates in this cascade.

  7. Cathepsin B deficiency attenuates cardiac remodeling in response to pressure overload via TNF-α/ASK1/JNK pathway.

    PubMed

    Wu, Qing-Qing; Xu, Man; Yuan, Yuan; Li, Fang-Fang; Yang, Zheng; Liu, Yuan; Zhou, Meng-Qiao; Bian, Zhou-Yan; Deng, Wei; Gao, Lu; Li, Hongliang; Tang, Qi-Zhu

    2015-05-01

    Cathepsin B (CTSB), a member of the lysosomal cathepsin family that is expressed in both murine and human hearts, was previously shown to participate in apoptosis, autophagy, and the progression of certain types of cancers. Recently, CTSB has been linked to myocardial infarction. Given that cathepsin L, another member of the lysosomal cathepsin family, ameliorates pathological cardiac hypertrophy, we hypothesized that CTSB plays a role in pressure overload-induced cardiac remodeling. Here we report that CTSB was upregulated in cardiomyocytes in response to hypertrophic stimuli both in vivo and in vitro. Moreover, knockout of CTSB attenuated pressure overload-induced cardiac hypertrophy, fibrosis, dysfunction, and apoptosis. Furthermore, the aortic banding-induced activation of TNF-α, apoptosis signal-regulating kinase 1 (ASK1), c-Jun NH2-terminal kinases (JNK), c-Jun, and release of cytochrome c was blunted by CTSB deficiency, which was further confirmed in in vitro studies induced by angiotensin II. In cardiomyocytes pretreatment with SP600125, a JNK inhibitor, suppressed the cardiomyocytes hypertrophy by inhibiting the ASK1/JNK pathway. Altogether, these data indicate that the CTSB protein functions as a necessary modulator of hypertrophic response by regulating TNF-α/ASK1/JNK signaling pathway involved in cardiac remodeling. Copyright © 2015 the American Physiological Society.

  8. Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation.

    PubMed

    Umar, Sadiq; Hedaya, Omar; Singh, Anil K; Ahmed, Salahuddin

    2015-09-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1-5μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.

  9. Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival.

    PubMed

    Eaton, G J; Zhang, Q-S; Diallo, C; Matsuzawa, A; Ichijo, H; Steinbeck, M J; Freeman, T A

    2014-11-13

    Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has

  10. Inhibition of apoptosis signal-regulating kinase 1 enhances endochondral bone formation by increasing chondrocyte survival

    PubMed Central

    Eaton, G J; Zhang, Q-S; Diallo, C; Matsuzawa, A; Ichijo, H; Steinbeck, M J; Freeman, T A

    2014-01-01

    Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has

  11. Apoptosis signal-regulating kinase 1 exhibits oncogenic activity in pancreatic cancer

    PubMed Central

    Hao, Ziwei; Yang, Yang; Xie, Songbo; Li, Dengwen; Liu, Min; Zhou, Jun

    2016-01-01

    Pancreatic cancer has an extremely grim prognosis, with an overall 5-year survival rate less than 5%, as a result of its rapid metastasis and late diagnosis. To combat this disease, it is crucial to better understand the molecular mechanisms that contribute to its pathogenesis. Herein, we report that apoptosis signal-regulating kinase 1 (ASK1) is overexpressed in pancreatic cancer tissues and that its expression correlates with the histological grade of pancreatic cancer. The expression of ASK1 is also elevated in pancreatic cancer cell lines at both protein and mRNA levels. In addition, ASK1 promotes the proliferation and stimulates the tumorigenic capacity of pancreatic cancer cells. These functions of ASK1 are abrogated by pharmacological inhibition of its kinase activity or by introduction of a kinase-dead mutation, suggesting that the kinase activity of ASK1 is required for its role in pancreatic cancer. However, the alteration of ASK1 expression or activity does not significantly affect the migration or invasion of pancreatic cancer cells. Collectively, these findings reveal a critical role for ASK1 in the development of pancreatic cancer and have important implications for the diagnosis and treatment of this malignancy. PMID:27655673

  12. Serine 58 of 14-3-3zeta is a molecular switch regulating ASK1 and oxidant stress-induced cell death.

    PubMed

    Zhou, Jibin; Shao, Zhili; Kerkela, Risto; Ichijo, Hidenori; Muslin, Anthony J; Pombo, Celia; Force, Thomas

    2009-08-01

    Oxidant stress is a ubiquitous stressor with negative impacts on multiple cell types. ASK1 is a central mediator of oxidant injury, but while mechanisms of its inhibition, such as sequestration by 14-3-3 proteins and thioredoxin, have been identified, mechanisms of activation have remained obscure and the signaling pathways regulating this are not clear. Here, we report that phosphorylation of 14-3-3zeta at serine 58 (S58) is dynamically regulated in the cell and that the phosphorylation status of S58 is a critical factor regulating oxidant stress-induced cell death. Phosphorylation of S58 releases ASK1 from 14-3-3zeta, and ASK1 then activates stress-activated protein kinases, leading to cell death. While several members of the mammalian sterile 20 (Mst) family of kinases can phosphorylate S58 when overexpressed, we identify Ste20/oxidant stress response kinase 1 (SOK-1), an Mst family member known to be activated by oxidant stress, as a central endogenous regulator of S58 phosphorylation and thereby of ASK1-mediated cell death. Our findings identify a novel pathway that regulates ASK1 activation and oxidant stress-induced cell death.

  13. Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

    PubMed

    Yuan, Feng-Hua; Chen, Yong-Gui; Zhang, Ze-Zhi; Yue, Hai-Tao; Bi, Hai-Tao; Yuan, Kai; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-03-01

    Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.

  14. Endogenous nitric oxide induces activation of apoptosis signal-regulating kinase 1 via S-nitrosylation in rat hippocampus during cerebral ischemia-reperfusion.

    PubMed

    Liu, D-H; Yuan, F-G; Hu, S-Q; Diao, F; Wu, Y-P; Zong, Y-Y; Song, T; Li, C; Zhang, G-Y

    2013-01-15

    Apoptosis signal-regulating kinase 1 (ASK1) is a general mediator of cell death in response to a variety of stimuli, including reactive oxygen species, tumor necrosis factor α, lipopolysaccharide, endoplasmic reticulum stress, calcium influx and ischemia. Here we reported ASK1 was activated by nitric oxide (NO) through S-nitrosylation during cerebral ischemia-reperfusion. The reagents that abrogate neuronal nitric oxide synthase (nNOS) activity such as nNOS inhibitor 7NI and N-methyl-D-aspartate receptor antagonist MK801 prevented ASK1 activation via decreasing ASK1 S-nitrosylation. In HEK293 cells, over-expressed ASK1 could be S-nitrosylated by both exogenous and endogenous NO and Cys869 was identified as the site of ASK1 S-nitrosylation. S-nitrosylation increased the level of ASK1 phosphorylation at Thr845, which represents ASK1 activation. Our results further confirmed that S-nitrosylation led to the increment of ASK1 dimerization. S-nitrosylation of ASK1 also activated the downstream JNK signaling and JNK-mediated nucleic pathway. The exogenous NO (SNP and GSNO) reversed the effect of endogenous NO by suppressing S-nitrosylation of ASK1 and exerted neuroprotection during ischemia-reperfusion. These results suggest that inhibiting ASK1 S-nitrosylation may be a novel approach for stroke therapy. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  15. Apoptosis signal-regulating kinase 1 and cyclin D1 compose a positive feedback loop contributing to tumor growth in gastric cancer

    PubMed Central

    Hayakawa, Yoku; Hirata, Yoshihiro; Nakagawa, Hayato; Sakamoto, Kei; Hikiba, Yohko; Kinoshita, Hiroto; Nakata, Wachiko; Takahashi, Ryota; Tateishi, Keisuke; Tada, Motohisa; Akanuma, Masao; Yoshida, Haruhiko; Takeda, Kohsuke; Ichijo, Hidenori; Omata, Masao; Maeda, Shin; Koike, Kazuhiko

    2011-01-01

    Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular functions and are highly active in many types of human cancers. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream MAPK involved in apoptosis, inflammation, and carcinogenesis. This study investigated the role of ASK1 in the development of gastric cancer. In human gastric cancer specimens, we observed increased ASK1 expression, compared to nontumor epithelium. Using a chemically induced murine gastric tumorigenesis model, we observed increased tumor ASK1 expression, and ASK1 knockout mice had both fewer and smaller tumors than wild-type (WT) mice. ASK1 siRNA inhibited cell proliferation through the accumulation of cells in G1 phase of the cell cycle, and reduced cyclin D1 expression in gastric cancer cells, whereas these effects were uncommon in other cancer cells. ASK1 overexpression induced the transcription of cyclin D1, through AP-1 activation, and ASK1 levels were regulated by cyclin D1, via the Rb–E2F pathway. Exogenous ASK1 induced cyclin D1 expression, followed by elevated expression of endogenous ASK1. These results indicate an autoregulatory mechanism of ASK1 in the development of gastric cancer. Targeting this positive feedback loop, ASK1 may present a potential therapeutic target for the treatment of advanced gastric cancer. PMID:21187402

  16. Induction of apoptosis by quercetin is mediated through AMPKalpha1/ASK1/p38 pathway.

    PubMed

    Lee, Yun-Kyoung; Hwang, Jin-Taek; Kwon, Dae Young; Surh, Young-Joon; Park, Ock Jin

    2010-06-28

    Effective strategies for cancer prevention and treatment can be identified by understanding the mechanism of apoptotic pathways. In this study, we investigated the regulatory mechanism of quercetin-induced apoptosis through apoptosis signal-regulating kinase (ASK)-1 and mitogen-activated protein kinase pathways. Our results showed that quercetin increased apoptotic cell death through reactive oxygen species (ROS) generation and was responsible for ASK1 activation. Increasing ASK1 activity was accompanied by p38 activation. Interestingly, AMP-activated protein kinase (AMPK) seemed to be a critical controller of quercetin-regulated ASK1/p38 activation. Blocking AMPKalpha1 activity using Compound C, a synthetic inhibitor or siRNA showed that quercetin-activated ASK1 could not stimulate p38 activity. Thus, we suggested that quercetin-exerted apoptotic effects involve ROS/AMPKalpha1/ASK1/p38 signaling pathway, and AMPKalpha1 is a necessary element for apoptotic event induced by ASK1.

  17. Cardiac hypertrophy is associated with altered thioredoxin and ASK-1 signaling in a mouse model of menopause.

    PubMed

    Ebrahimian, Talin; Sairam, M Ram; Schiffrin, Ernesto L; Touyz, Rhian M

    2008-10-01

    Oxidative stress is implicated in menopause-associated hypertension and cardiovascular disease. The role of antioxidants in this process is unclear. We questioned whether the downregulation of thioredoxin (TRX) is associated with oxidative stress and the development of hypertension and target-organ damage (cardiac hypertrophy) in a menopause model. TRX is an endogenous antioxidant that also interacts with signaling molecules, such as apoptosis signal-regulated kinase 1 (ASK-1), independently of its antioxidant function. Aged female wild-type (WT) and follitropin receptor knockout (FORKO) mice (20-24 wk), with hormonal imbalances, were studied. Mice were infused with ANG II (400 ng x kg(-1) x min(-1); 14 days). Systolic blood pressure was increased by ANG II in WT (166+/-8 vs. 121+/-5 mmHg) and FORKO (176+/-7 vs. 115+/-5 mmHg; P<0.0001; n=9/group) mice. In ANG II-infused FORKO mice, cardiac mass was increased by 42% (P<0.001). This was associated with increased collagen content and augmented ERK1/2 phosphorylation (2-fold). Cardiac TRX expression and activity were decreased by ANG II in FORKO but not in WT (P<0.01) mice. ASK-1 expression, cleaved caspase III content, and Bax/Bcl-2 content were increased in ANG II-infused FORKO (P<0.05). ANG II had no effect on cardiac NAD(P)H oxidase activity or on O(2)(*-) levels in WT or FORKO. Cardiac ANG II type 1 receptor expression was similar in FORKO and WT. These findings indicate that in female FORKO, ANG II-induced cardiac hypertrophy and fibrosis are associated with the TRX downregulation and upregulation of ASK-1/caspase signaling. Our data suggest that in a model of menopause, protective actions of TRX may be blunted, which could contribute to cardiac remodeling independently of oxidative stress and hypertension.

  18. Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain.

    PubMed

    Deak, M; Casamayor, A; Currie, R A; Downes, C P; Alessi, D R

    1999-05-28

    A plant homologue of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3-phosphoinositide-dependent protein kinase-1. Like the mammalian 3-phosphoinositide-dependent protein kinase-1, Arabidopsis 3-phosphoinositide-dependent protein kinase-1 and rice 3-phosphoinositide-dependent protein kinase-1 possess a kinase domain at N-termini and a pleckstrin homology domain at their C-termini. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3-phosphoinositide-dependent protein kinase-1 homologues. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is able to activate human protein kinase B alpha (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3-phosphoinositides.

  19. Lys29-linkage of ASK1 by Skp1−Cullin 1−Fbxo21 ubiquitin ligase complex is required for antiviral innate response

    PubMed Central

    Yu, Zhou; Chen, Taoyong; Li, Xuelian; Yang, Mingjin; Tang, Songqing; Zhu, Xuhui; Gu, Yan; Su, Xiaoping; Xia, Meng; Li, Weihua; Zhang, Xuemin; Wang, Qingqing; Cao, Xuetao; Wang, Jianli

    2016-01-01

    Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity. However, key regulators of ubiquitination during innate response and roles of new types of ubiquitination (apart from Lys48- and Lys63-linkage) in control of innate signaling have not been clearly understood. Here we report that F-box only protein Fbxo21, a functionally unknown component of SCF (Skp1–Cul1–F-box protein) complex, facilitates Lys29-linkage and activation of ASK1 (apoptosis signal-regulating kinase 1), and promotes type I interferon production upon viral infection. Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1, attenuates c-Jun N-terminal kinase (JNK) and p38 signaling pathway, and decreases the production of proinflammatory cytokines and type I interferon, resulting in reduced antiviral innate response and enhanced virus replication. Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response, providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response. DOI: http://dx.doi.org/10.7554/eLife.14087.001 PMID:27063938

  20. Rescuing the duty to rescue.

    PubMed

    Rulli, Tina; Millum, Joseph

    2016-04-01

    Clinicians and health researchers frequently encounter opportunities to rescue people. Rescue cases can generate a moral duty to aid those in peril. As such, bioethicists have leveraged a duty to rescue for a variety of purposes. Yet, despite its broad application, the duty to rescue is underanalysed. In this paper, we assess the state of theorising about the duty to rescue. There are large gaps in bioethicists' understanding of the force, scope and justification of the two most cited duties to rescue--the individual duty of easy rescue and the institutional rule of rescue. We argue that the duty of easy rescue faces unresolved challenges regarding its force and scope, and the rule of rescue is indefensible. If the duty to rescue is to help solve ethical problems, these theoretical gaps must be addressed. We identify two further conceptions of the duty to rescue that have received less attention--an institutional duty of easy rescue and the professional duty to rescue. Both provide guidance in addressing force and scope concerns and, thereby, traction in answering the outstanding problems with the duty to rescue. We conclude by proposing research priorities for developing accounts of duties to rescue in bioethics. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  1. Knockdown of apoptosis signal-regulating kinase 1 affects ischaemia-induced astrocyte activation and glial scar formation.

    PubMed

    Cheon, So Yeong; Cho, Kyoung Joo; Song, Juhyun; Kim, Gyung Whan

    2016-04-01

    Reactive astrocytes play an essential role in determining the tissue response to ischaemia. Formation of a glial scar can block the neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important; however, the mediator of this process has not been fully elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is an early responder to oxidative stress, and plays a pivotal role in the intracellular signalling pathway of apoptosis, inflammation, and differentiation. To confirm whether ASK1 mediates astrocyte activation and leads to glial scar formation after cerebral ischaemia, we conducted in vivo and in vitro experiments. C57BL/6 mice were subjected to occlusion of the middle cerebral artery, and astrocyte cultures were exposed to oxygen-glucose deprivation. After silencing of ASK1 , astrocyte-associated genes were downregulated, as seen with the use of microarrays. The glial fibrillary acidic protein (GFAP) level was decreased, and correlated with the reduction in the ASK1 level. In astrocytes, reduction in the ASK1 level decreased the activity of the p38 pathway, and the levels of transcription factors for GFAP and GFAP transcripts after hypoxia. In the chronic phase, ASK1 depletion reduced glial scar formation and conserved neuronal structure, which may lead to better functional recovery. These data suggest that ASK1 may be an important mediator of ischaemia-induced astrocyte activation and scar formation, and could provide a potential therapeutic target for treatment after ischaemic stroke.

  2. Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE1[OPEN

    PubMed Central

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; Chen, Jin-Gui

    2015-01-01

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. Here, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. Taken together, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability. PMID:25489024

  3. ACTIVATION OF ASK-1 AND DOWNSTREAM MAP KINASES IN CYTOCHROME P4502E1 POTENTIATED TUMOR NECROSIS FACTOR ALPHA LIVER INJURY

    PubMed Central

    Wu, Defeng; Cederbaum, Arthur

    2010-01-01

    Cytochrome P4502E1 (CYP2E1) potentiated TNFα-toxicity by a mechanism involving increased oxidative stress and activation of JNK and p38 MAPKs. The current study evaluated what are the upstream mediators of this MAPK activation with a special focus on studying whether Apoptosis Signal Regulating Kinase-1 (ASK-1) is activated in the CYP2E1-TNFα hepatotoxic model. Wild type and CYP2E1−/− mice were treated with pyrazole (PY) for three days to induce CYP2E1 and challenged with TNFα on day three. Liver injury occurred between 8–12h after TNFα addition only to the wild type PY-treated mice. Oxidative stress was elevated in the PY mice at 4h, a time prior to the liver injury. ASK-1 was dissociated from the thioredoxin-ASK1 complex, and was activated at 4h after addition of TNFα to PY mice. This was followed by activation of MKK3/MKK6 and MKK4/MKK7 at 4–8 or 12h and then JNK/p38 MAPK at 8 to 12h. MAPK phosphatase-1 was decreased 12 to 24h after TNFα addition. This may promote a sustained activation of JNK. Bax was elevated while Bcl-2 and cFLIPS/L were lowered at 4h after addition of TNFα. These changes were followed by increases in caspase 8 and 3 activities and apoptosis. None of the above changes were found when TNFα was added to PY-treated CYP2E1−/− mice. These studies show that TNFα increases oxidative stress in mice with elevated CYP2E1 with subsequent activation of ASK-1 via a mechanism involving thioredoxin/ASK-1 dissociation, followed by activation of downstream MAPKK and MAPK. We speculate that similar interactions between CYP2E1 and TNFα may be important for alcohol-induced liver injury. PMID:20438834

  4. ASK1/JNK-mediated TAp63 activation controls the cell survival signal of baicalein-treated EBV-transformed B cells.

    PubMed

    Park, Ga Bin; Kim, Yeong Seok; Lee, Hyun-Kyung; Yang, Jae Wook; Kim, Daejin; Hur, Dae Young

    2016-01-01

    Transcriptionally active p63 (TAp63) promotes cell cycle arrest, senescence, and apoptosis in several cancer cells. Migration inhibitory factor (MIF)/CD74 regulates B-cell survival through nuclear factor (NF)-κB-dependent TAp63 expression. In this study, we investigated how the level of TAp63 expression influences the induction of apoptosis in baicalein-treated EBV-transformed B cells. Baicalein induced the expression of TAp63 and apoptosis signal-regulating kinase 1 (ASK1), as well as cytotoxicity, by disrupting the mitochondrial membrane and inhibiting the activation of phosphoinositide 3-kinase (PI3K)/Akt and NF-κB. Genetic knockdown of TAp63 or ASK1 by small interfering RNA resulted in protection from apoptosis accompanied by the recovery of CD74, CD44, α4 integrin, Bcl-2, and NF-κB activation. Baicalein-induced reactive oxygen species activated the ASK1/JNK pathway with subsequent expression of TAp63. Pre-engagement with MIF/CD74 maintained the expression of CD74, CD44, and α4 integrin, as well as Syk/Src-mediated PI3K/Akt activation, in baicalein-treated EBV-transformed B cells. Meanwhile, ASK1/JNK-dependent TAp63 expression was efficiently suppressed after pre-treatment with MIF. Our results suggest that baicalein-mediated ASK1/JNK activation regulates the mitochondria-dependent apoptosis pathway through the up-regulation of TAp63 and down-regulation of NF-κB and CD74/CD44 in B-cell malignancies.

  5. ER stress and ASK1-JNK activation contribute to oridonin-induced apoptosis and growth inhibition in cultured human hepatoblastoma HuH-6 cells.

    PubMed

    Cai, Duo-te; Jin, Hua; Xiong, Qi-Xing; Liu, Wei-Guang; Gao, Zhi-gang; Gu, Gui-xiong; Qiu, Yu-hui

    2013-07-01

    Oridonin, the main active component of Rabdosia rubescens, has antitumor activities in experimental and clinical settings. The aims of the current study were to explore the anticancer abilities of oridonin in hepatoblastoma (HB) HuH-6 cells and to investigate the underlying mechanisms. We found that oridonin inhibited HuH-6 cell in vitro growth in a dose- and time-dependent manner. Further, oridonin induced HuH-6 cell apoptosis and G2/M cell cycle arrest. Upon studying the mechanism, we found that oridonin treatment caused endoplasmic reticulum (ER) stress activation. Meanwhile, ER stress inhibitor salubrinal- or inositol-requiring enzyme 1 (IRE-1) shRNA silencing inhibited oridonin's anti-HuH-6 effects, while ER stress inducers thapsigargin (Tg) and tunicamycin (Tm) mimicked oridonin's actions on HuH-6 cells. Oridonin also activated apoptosis signal regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling in cultured HuH-6 cells, which was inhibited by IRE-1 silencing. Importantly, the JNK inhibitors suppressed oridonin-induced growth inhibition and apoptosis in HuH-6 cells. In conclusion, our results suggest that oridonin induces growth inhibition and apoptosis in cultured HuH-6 cells involving ER stress and ASK1/JNK signaling pathways, which enhances our understanding of the molecular mechanisms of oridonin in HB management.

  6. ASK1-dependent endothelial cell activation is critical in ovarian cancer growth and metastasis

    PubMed Central

    Yin, Mingzhu; Zhou, Huanjiao Jenny; Zhang, Jiqin; Lin, Caixia; Li, Hongmei; Li, Xia; Li, Yonghao; Zhang, Haifeng; Breckenridge, David G.; Ji, Weidong

    2017-01-01

    We have recently reported that tumor-associated macrophages (TAMs) promote early transcoelomic metastasis of ovarian cancer by facilitating TAM–ovarian cancer cell spheroid formation. ASK1 is known to be important for macrophage activation and inflammation-mediated tumorigenesis. In the present study, we show that ASK1 deficiency attenuates TAM-spheroid formation and ovarian cancer progression in an orthotopic ovarian cancer model. Interestingly, ASK1 in stroma, but not in TAMs, is critical for peritoneal tumor growth of ovarian cancer. Moreover, overexpression of an ASK1 inhibitory protein (suppressor of cytokine signaling-1; SOCS1) in vascular endothelium attenuates vascular permeability, TAM infiltration, and ovarian cancer growth. Mechanistically, we show that ASK1 mediates degradation of endothelial junction protein VE-cadherin via a lysosomal pathway to promote macrophage transmigration. Importantly, a pharmacological ASK1 inhibitor prevents tumor-induced vascular leakage, macrophage infiltration, and tumor growth in two mouse models. Since transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our study provides ASK1 as a therapeutic target for the treatment of ovarian cancer and other transcoelomic metastasis cancers. PMID:28931753

  7. Paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in human neuroblastoma SH-SY5Y cells.

    PubMed

    Yang, Wonsuk; Tiffany-Castiglioni, Evelyn; Koh, Hyun Chul; Son, Il-Hong

    2009-12-15

    Epidemiologic and laboratory studies suggest that paraquat can be an environmental etiologic factor in Parkinson's disease (PD). One mechanism by which paraquat may mediate cell death of dopaminergic neurons is by inducing endoplasmic reticulum (ER) stress, as suggested in a recent report. In this study, we further investigated this linkage by examining ER stress cascades. To this aim, human neuroblastoma cells (SH-SY5Y cells) were treated with paraquat and the signaling cascades through which ER stress results in apoptosis were examined. Then, it was examined whether ER stress is produced by paraquat. Paraquat increased ER stress biomarker proteins, glucose-regulated protein 78 (GRP78), ER degradation-enhancing alpha-mannosidae-like protein (EDEM), and C/EBP homologous protein (CHOP). Then, it was investigated which ER stress cascades are affected by paraquat. Paraquat activated inositol-requiring enzyme 1 (IRE1), apoptosis signal regulating kinase 1 (ASK1), and c-jun kinase (JNK). Also, paraquat activated calpain and caspase 3, but did not affect the levels of intracellular calcium and the activity of caspase 12. Finally, apoptotic DNA damage by paraquat was investigated and this damage was attenuated by salubrinal (ER stress inhibitor), thioredoxin (ASK1 inhibitor) and SP600125 (JNK inhibitor). Therefore, current data indicate that paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in SY5Y cells.

  8. Cytoprotective mechanisms of DJ-1 against oxidative stress through modulating ERK1/2 and ASK1 signal transduction.

    PubMed

    Oh, Stephanie E; Mouradian, M Maral

    2017-09-18

    DJ-1 is a highly conserved multifunctional protein linked to both neurodegeneration and neoplasia. Among its various activities is an antioxidant property leading to cytoprotection under oxidative stress conditions. This is associated with the ability to modulate signal transduction events that determine how the cell regulates normal processes such as growth, senescence, apoptosis, and autophagy in order to adapt to environmental stimuli and stresses. Alterations in DJ-1 expression or function can disrupt homeostatic signaling networks and initiate cascades that play a role in the pathogenesis of conditions such as Parkinson's disease and cancer. DJ-1 plays a major role in various signaling pathways. Related to its anti-oxidant properties, it mediates cell survival and proliferation by activating the extracellular signal-regulated kinase (ERK1/2) pathway and attenuates cell death signaling by inhibiting apoptosis signal-regulating kinase 1 (ASK1) activation. Here, we review the ways through which DJ-1 regulates these pathways, focusing on how its regulation of signal transduction contributes to cellular homeostasis and the pathologic states that result from their dysregulation. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury

    SciTech Connect

    Xie, Yuchao; Ramachandran, Anup; Breckenridge, David G.; Liles, John T.; Lebofsky, Margitta; Farhood, Anwar; Jaeschke, Hartmut

    2015-07-01

    Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24 h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5 h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients. - Highlights: • Two ASK1 inhibitors protected against acetaminophen-induced liver injury. • The ASK1 inhibitors protect when used as pre- or post-treatment. • Protection by ASK1 inhibitor is

  10. Cellular Iron Depletion Stimulates the JNK and p38 MAPK Signaling Transduction Pathways, Dissociation of ASK1-Thioredoxin, and Activation of ASK1*

    PubMed Central

    Yu, Yu; Richardson, Des R.

    2011-01-01

    The role of signaling pathways in the regulation of cellular iron metabolism is becoming increasingly recognized. Iron chelation is used for the treatment of iron overload but also as a potential strategy for cancer therapy, because iron depletion results in cell cycle arrest and apoptosis. This study examined potential signaling pathways affected by iron depletion induced by desferrioxamine (DFO) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Both chelators affected multiple molecules in the mitogen-activated protein kinase (MAPK) pathway, including a number of dual specificity phosphatases that directly de-phosphorylate MAPKs. Examination of the phosphorylation of major MAPKs revealed that DFO and Dp44mT markedly increased phosphorylation of stress-activated protein kinases, JNK and p38, without significantly affecting the extracellular signal-regulated kinase (ERK). Redox-inactive DFO-iron complexes did not affect phosphorylation of JNK or p38, whereas the redox-active Dp44mT-iron complex significantly increased the phosphorylation of these kinases similarly to Dp44mT alone. Iron or N-acetylcysteine supplementation reversed Dp44mT-induced up-regulation of phospho-JNK, but only iron was able to reverse the effect of DFO on JNK. Both iron chelators significantly reduced ASK1-thioredoxin complex formation, resulting in the increased phosphorylation of ASK1, which activates the JNK and p38 pathways. Thus, dissociation of ASK1 could serve as an important signal for the phosphorylation of JNK and p38 activation observed after iron chelation. Phosphorylation of JNK and p38 likely play an important role in mediating the cell cycle arrest and apoptosis induced by iron depletion. PMID:21378396

  11. Targeting checkpoint kinase 1 in cancer therapeutics.

    PubMed

    Tse, Archie N; Carvajal, Richard; Schwartz, Gary K

    2007-04-01

    Progression through the cell cycle is monitored by surveillance mechanisms known as cell cycle checkpoints. Our knowledge of the biochemical nature of checkpoint regulation during an unperturbed cell cycle and following DNA damage has expanded tremendously over the past decade. We now know that dysfunction in cell cycle checkpoints leads to genomic instability and contributes to tumor progression, and most agents used for cancer therapy, such as cytotoxic chemotherapy and ionizing radiation, also activate cell cycle checkpoints. Understanding how checkpoints are regulated is therefore important from the points of view of both tumorigenesis and cancer treatment. In this review, we present an overview of the molecular hierarchy of the checkpoint signaling network and the emerging role of checkpoint targets, especially checkpoint kinase 1, in cancer therapy. Further, we discuss the results of recent clinical trials involving the nonspecific checkpoint kinase 1 inhibitor, UCN-01, and the challenges we face with this new therapeutic approach.

  12. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  13. Ablation of the Proapoptotic Genes Chop or Ask1 Does Not Prevent or Delay Loss of Visual Function in a P23H Transgenic Mouse Model of Retinitis Pigmentosa

    PubMed Central

    Adekeye, Adeseye; Haeri, Mohammad; Solessio, Eduardo; Knox, Barry E.

    2014-01-01

    The P23H mutation in rhodopsin (RhoP23H) is a prevalent cause of autosomal dominant retinitis pigmentosa. We examined the role of the ER stress proteins, Chop and Ask1, in regulating the death of rod photoreceptors in a mouse line harboring the RhoP23H rhodopsin transgene (GHL+). We used knockout mice models to determine whether Chop and Ask1 regulate rod survival or retinal degeneration. Electrophysiological recordings showed similar retinal responses and sensitivities for GHL+, GHL+/Chop−/− and GHL+/Ask1−/− animals between 4–28 weeks, by which time all three mouse lines exhibited severe loss of retinal function. Histologically, ablation of Chop and Ask1 did not rescue photoreceptor loss in young animals. However, in older mice, a regional protective effect was observed in the central retina of GHL+/Chop−/− and GHL+/Ask1−/−, a region that was severely degenerated in GHL+ mice. Our results show that in the presence of the RhoP23H transgene, the rate of decline in retinal sensitivity is similar in Chop or Ask1 ablated and wild-type retinas, suggesting that these proteins do not play a major role during the acute phase of photoreceptor loss in GHL+ mice. Instead they may be involved in regulating secondary pathological responses such as inflammation that are upregulated during later stages of disease progression. PMID:24523853

  14. Space Rescue

    NASA Technical Reports Server (NTRS)

    Muratore, John F.

    2007-01-01

    Space Rescue has been a topic of speculation for a wide community of people for decades. Astronauts, aerospace engineers, diplomats, medical and rescue professionals, inventors and science fiction writers have all speculated on this problem. Martin Caidin's 1964 novel Marooned dealt with the problems of rescuing a crew stranded in low earth orbit. Legend at the Johnson Space Center says that Caidin's portrayal of a Russian attempt to save the American crew played a pivotal role in convincing the Russians to join the real joint Apollo-Soyuz mission. Space Rescue has been a staple in science fiction television and movies portrayed in programs such as Star Trek, Stargate-SG1 and Space 1999 and movies such as Mission To Mars and Red Planet. As dramatic and as difficult as rescue appears in fictional accounts, in the real world it has even greater drama and greater difficulty. Space rescue is still in its infancy as a discipline and the purpose of this chapter is to describe the issues associated with space rescue and the work done so far in this field. For the purposes of this chapter, the term space rescue will refer to any system which allows for rescue or escape of personnel from situations which endanger human life in a spaceflight operation. This will span the period from crew ingress prior to flight through crew egress postlanding. For the purposes of this chapter, the term primary system will refer to the spacecraft system that a crew is either attempting to escape from or from which an attempt is being made to rescue the crew.

  15. ASK1 Inhibitor Halts Progression of Diabetic Nephropathy in Nos3-Deficient Mice.

    PubMed

    Tesch, Greg H; Ma, Frank Y; Han, Yingjie; Liles, John T; Breckenridge, David G; Nikolic-Paterson, David J

    2015-11-01

    p38 mitogen-activated protein kinase (MAPK) signaling promotes diabetic kidney injury. Apoptosis signal-regulating kinase (ASK)1 is one of the upstream kinases in the p38 MAPK-signaling pathway, which is activated by inflammation and oxidative stress, suggesting a possible role for ASK1 in diabetic nephropathy. In this study, we examined whether a selective ASK1 inhibitor can prevent the induction and progression of diabetic nephropathy in mice. Diabetes was induced in hypertensive endothelial nitric oxide synthase (Nos3)-deficient mice by five low-dose streptozotocin (STZ) injections. Groups of diabetic Nos3(-/-) mice received ASK1 inhibitor (GS-444217 delivered in chow) as an early intervention (2-8 weeks after STZ) or late intervention (weeks 8-15 after STZ). Control diabetic and nondiabetic Nos3(-/-) mice received normal chow. Treatment with GS-444217 abrogated p38 MAPK activation in diabetic kidneys but had no effect upon hypertension in Nos3(-/-) mice. Early intervention with GS-444217 significantly inhibited diabetic glomerulosclerosis and reduced renal dysfunction but had no effect on the development of albuminuria. Late intervention with GS-444217 improved renal function and halted the progression of glomerulosclerosis, renal inflammation, and tubular injury despite having no effect on established albuminuria. In conclusion, this study identifies ASK1 as a new therapeutic target in diabetic nephropathy to reduce renal inflammation and fibrosis independent of blood pressure control. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  16. Thioredoxin-2 inhibits mitochondrial reactive oxygen species generation and apoptosis stress kinase-1 activity to maintain cardiac function.

    PubMed

    Huang, Qunhua; Zhou, Huanjiao Jenny; Zhang, Haifeng; Huang, Yan; Hinojosa-Kirschenbaum, Ford; Fan, Peidong; Yao, Lina; Belardinelli, Luiz; Tellides, George; Giordano, Frank J; Budas, Grant R; Min, Wang

    2015-03-24

    Thioredoxin 2 (Trx2) is a key mitochondrial protein that regulates cellular redox and survival by suppressing mitochondrial reactive oxygen species generation and by inhibiting apoptosis stress kinase-1 (ASK1)-dependent apoptotic signaling. To date, the role of the mitochondrial Trx2 system in heart failure pathogenesis has not been investigated. Western blot and histological analysis revealed that Trx2 protein expression levels were reduced in hearts from patients with dilated cardiomyopathy, with a concomitant increase in ASK1 phosphorylation/activity. Cardiac-specific Trx2 knockout mice develop spontaneous dilated cardiomyopathy at 1 month of age with increased heart size, reduced ventricular wall thickness, and a progressive decline in left ventricular contractile function, resulting in mortality due to heart failure by ≈4 months of age. The progressive decline in cardiac function observed in cardiac-specific Trx2 knockout mice was accompanied by the disruption of mitochondrial ultrastructure, mitochondrial membrane depolarization, increased mitochondrial reactive oxygen species generation, and reduced ATP production, correlating with increased ASK1 signaling and increased cardiomyocyte apoptosis. Chronic administration of a highly selective ASK1 inhibitor improved cardiac phenotype and reduced maladaptive left ventricular remodeling with significant reductions in oxidative stress, apoptosis, fibrosis, and cardiac failure. Cellular data from Trx2-deficient cardiomyocytes demonstrated that ASK1 inhibition reduced apoptosis and reduced mitochondrial reactive oxygen species generation. Our data support an essential role for mitochondrial Trx2 in preserving cardiac function by suppressing mitochondrial reactive oxygen species production and ASK1-dependent apoptosis. Inhibition of ASK1 represents a promising therapeutic strategy for the treatment of dilated cardiomyopathy and heart failure. © 2015 American Heart Association, Inc.

  17. Amplitude distribution of ionization jerks in ionization-chamber ASK-1 according long-term measurements

    NASA Astrophysics Data System (ADS)

    Timofeev, Vladislav

    2016-07-01

    As part of the Yakut complex systems by measuring the intensity of cosmic rays has a unique device spherical - ionization chamber ASK-1 with a lead screen thickness of 12 cm. The camera allows you to explore the physical characteristics of the so-called "ionization jerks " - sharp increases ionization current caused by the passage through the device much ionizing particles of cosmic origin. Due to a large increase in current nuclear cascade "showers", formed mainly by particles of cosmic rays in the camera screen. Over the entire period of observation (50 years old) camera ASK-1 was registered 59125 aftershocks. Their nature and properties still does not sufficiently studied, especially in medium and large amplitudes.

  18. ASK1 and MAP2K6 as modifiers of age at onset in Huntington's disease.

    PubMed

    Arning, Larissa; Monté, Didier; Hansen, Wiebke; Wieczorek, Stefan; Jagiello, Peter; Akkad, Denis A; Andrich, Jürgen; Kraus, Peter H; Saft, Carsten; Epplen, Jörg T

    2008-04-01

    Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disease associated with abnormal expansions of a stretch of perfect CAG repeats in the HD gene. The number of repeat units is predictive for the age at onset (AO) of neurological symptoms. Part of the remaining variation in AO is attributed to modifier genes. In this study, genes involved in apoptosis were investigated as candidates for modulating AO in HD. A panel of 304 candidate genes was screened for allelic associations with motor AO via linked micro-satellite markers by pooling the DNAs of HD individuals from opposite ends of the AO distribution. After genotyping promising markers from the pooling experiment individually, markers revealed consolidated evidence for association in a candidate region comprising the genes MAP3K5 (ASK1)/PEX7 at 6q23.3 and in the gene MAP2K6 at 17q24.3. Fine-mapping of these candidate regions in a cohort of 250 Caucasian HD patients using single nucleotide polymorphism (SNP) markers delimitated the precise locations of association. Certain variations in an ASK1-PEX7 haplotype block explain 2.6% of additional variance in AO in our HD cohort. In males, 4.9% additional variance could be attributed to MAP2K6 genotype variations. Altogether, ASK1-PEX7 haplotypes and MAP2K2 genotype variations explain 6.3% additional variance in AO for HD. We hypothesise that sequence variations of ASK1 and MAP2K6 lead to partially sex-specific changes in the levels and/or phosphorylation states of p38 and p38-regulated proteins that might contribute to the observed delaying effects in the AO of HD.

  19. Kaempferol Attenuates Cardiac Hypertrophy via Regulation of ASK1/MAPK Signaling Pathway and Oxidative Stress.

    PubMed

    Feng, Hong; Cao, Jianlei; Zhang, Guangyu; Wang, Yanggan

    2017-02-20

    Kaempferol has been demonstrated to provide benefits for the treatment of atherosclerosis, coronary heart disease, hyperlipidemia, and diabetes through its antioxidant and anti-inflammatory properties. However, its role in cardiac hypertrophy remains to be elucidated. The aim of our study was to investigate the effects of kaempferol on cardiac hypertrophy and the underlying mechanism. Mice subjected to aorta banding were treated with or without kaempferol (100 mg/kg/d, p. o.) for 6 weeks. Echocardiography was performed to evaluate cardiac function. Mice hearts were collected for pathological observation and molecular mechanism investigation. H9c2 cardiomyocytes were stimulated with or without phenylephrine for in vitro study. Kaempferol significantly attenuated cardiac hypertrophy induced by aorta banding as evidenced by decreased cardiomyocyte areas and interstitial fibrosis, accompanied with improved cardiac functions and decreased apoptosis. The ASK1/MAPK signaling pathways (JNK1/2 and p38) were markedly activated in the aorta banding mouse heart but inhibited by kaempferol treatment. In in vitro experiments, kaempferol also inhibited the activity of ASK1/JNK1/2/p38 signaling pathway and the enlargement of H9c2 cardiomyocytes. Furthermore, our study revealed that kaempferol could protect the mouse heart and H9c2 cells from pathological oxidative stress. Our investigation indicated that treatment with kaempferol protects against cardiac hypertrophy, and its cardioprotection may be partially explained by the inhibition of the ASK1/MAPK signaling pathway and the regulation of oxidative stress.

  20. CIIA prevents SOD1(G93A)-induced cytotoxicity by blocking ASK1-mediated signaling

    PubMed Central

    Lee, Jae Keun; Hwang, Sang Gil; Shin, Jin Hee; Shim, Jaekyung; Choi, Eui-Ju

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease with higher selectivity in degeneration of motor neurons. However, the molecular mechanism by which the ALS-linked mutants of human superoxide dismutase 1 (SOD1) gene induce neurotoxicity remains obscure yet. Here, we show that depletion of CIIA expression by RNA interference (RNAi) promoted cytotoxicity caused by ALS-linked G93A mutant of the SOD1 gene. The RNAi-mediated knockdown of CIIA also enhanced the SOD1(G93A)-induced interaction between ASK1 and TRAF2 as well as ASK1 activity. Furthermore, endogenous silencing of CIIA by RNAi augmented the effects of SOD1(G93A) on reduction of mitochondria membrane potential (Δψm), release of cytochrome c into the cytoplasm, and caspase activation. Together, our results suggest that CIIA negatively modulates ASK1-mediated cytotoxic signaling processes in a SOD1(G93A)-expressing cellular model of ALS. PMID:25018698

  1. Nephroprotective Effects of N-Acetylcysteine Amide against Contrast-Induced Nephropathy through Upregulating Thioredoxin-1, Inhibiting ASK1/p38MAPK Pathway, and Suppressing Oxidative Stress and Apoptosis in Rats.

    PubMed

    Gong, Xuezhong; Duan, Yiru; Zheng, Junli; Wang, Yiquan; Wang, Guohua; Norgren, Svante; Hei, Tom K

    2016-01-01

    Contrast-induced nephropathy (CIN) is a leading cause of hospital-acquired acute kidney injury (AKI) due to apoptosis induced in renal tubular cells. Our previous study demonstrated the novel N-acetylcysteine amide (NACA); the amide form of N-acetyl cysteine (NAC) prevented renal tubular cells from contrast-induced apoptosis through inhibiting p38 MAPK pathway in vitro. In the present study, we aimed to compare the efficacies of NACA and NAC in preventing CIN in a well-established rat model and investigate whether thioredoxin-1 (Trx1) and apoptosis signal-regulating kinase 1 (ASK1) act as the potential activator for p38 MAPK. NACA significantly attenuated elevations of serum creatinine, blood urea nitrogen, and biomarkers of AKI. At equimolar concentration, NACA was more effective than NAC in reducing histological changes of renal tubular injuries. NACA attenuated activation of p38 MAPK signal, reduced oxidative stress, and diminished apoptosis. Furthermore, we demonstrated that contrast exposure resulted in Trx1 downregulation and increased ASK1/p38 MAPK phosphorylation, which could be reversed by NACA and NAC. To our knowledge, this is the first report that Trx1 and ASK1 are involved in CIN. Our study highlights a renal protective role of NACA against CIN through modulating Trx1 and ASK1/p38 MAPK pathway to result in the inhibition of apoptosis among renal cells.

  2. Nephroprotective Effects of N-Acetylcysteine Amide against Contrast-Induced Nephropathy through Upregulating Thioredoxin-1, Inhibiting ASK1/p38MAPK Pathway, and Suppressing Oxidative Stress and Apoptosis in Rats

    PubMed Central

    Duan, Yiru; Zheng, Junli; Wang, Yiquan; Wang, Guohua; Norgren, Svante; Hei, Tom K.

    2016-01-01

    Contrast-induced nephropathy (CIN) is a leading cause of hospital-acquired acute kidney injury (AKI) due to apoptosis induced in renal tubular cells. Our previous study demonstrated the novel N-acetylcysteine amide (NACA); the amide form of N-acetyl cysteine (NAC) prevented renal tubular cells from contrast-induced apoptosis through inhibiting p38 MAPK pathway in vitro. In the present study, we aimed to compare the efficacies of NACA and NAC in preventing CIN in a well-established rat model and investigate whether thioredoxin-1 (Trx1) and apoptosis signal-regulating kinase 1 (ASK1) act as the potential activator for p38 MAPK. NACA significantly attenuated elevations of serum creatinine, blood urea nitrogen, and biomarkers of AKI. At equimolar concentration, NACA was more effective than NAC in reducing histological changes of renal tubular injuries. NACA attenuated activation of p38 MAPK signal, reduced oxidative stress, and diminished apoptosis. Furthermore, we demonstrated that contrast exposure resulted in Trx1 downregulation and increased ASK1/p38 MAPK phosphorylation, which could be reversed by NACA and NAC. To our knowledge, this is the first report that Trx1 and ASK1 are involved in CIN. Our study highlights a renal protective role of NACA against CIN through modulating Trx1 and ASK1/p38 MAPK pathway to result in the inhibition of apoptosis among renal cells. PMID:28105252

  3. Regulation of Microglia and Macrophage Polarization via Apoptosis Signal-Regulating Kinase 1 Silencing after Ischemic/Hypoxic Injury.

    PubMed

    Cheon, So Yeong; Kim, Eun Jung; Kim, Jeong Min; Kam, Eun Hee; Ko, Byung Woong; Koo, Bon-Nyeo

    2017-01-01

    Inflammation is implicated in ischemic stroke and is involved in abnormal homeostasis. Activation of the immune system leads to breakdown of the blood-brain barrier and, thereby, infiltration of immune cells into the brain. Upon cerebral ischemia, infiltrated macrophages and microglia (resident CNS immune cell) are activated, change their phenotype to M1 or M2 based on the microenvironment, migrate toward damaged tissue, and are involved in repair or damage. Those of M1 phenotype release pro-inflammatory mediators, which are associated with tissue damage, while those of M2 phenotype release anti-inflammatory mediators, which are related to tissue recovery. Moreover, late inflammation continually stimulates immune cell infiltration and leads to brain infarction. Therefore, regulation of M1/M2 phenotypes under persistent inflammatory conditions after cerebral ischemia is important for brain repair. Herein, we focus on apoptosis signal-regulating kinase 1 (ASK1), which is involved in apoptotic cell death, brain infarction, and production of inflammatory mediators after cerebral ischemia. We hypothesized that ASK1 is involved in the polarization of M1/M2 phenotype and the function of microglia and macrophage during the late stage of ischemia/hypoxia. We investigated the effects of ASK1 in mice subjected to middle cerebral artery occlusion and on BV2 microglia and RAW264.7 macrophage cell lines subjected to oxygen-glucose deprivation. Our results showed that ASK1 silencing effectively reduced Iba-1 or CD11b-positive cells in ischemic areas, suppressed pro-inflammatory cytokines, and increased anti-inflammatory mediator levels at 7 days after cerebral ischemia. In cultured microglia and macrophages, ASK1 inhibition, induced by NQDI-1 drug, decreased the expression and release of M1-associated factors and increased those of M2-associated factors after hypoxia/reperfusion (H/R). At the gene level, ASK1 inhibition suppressed M1-associated genes and augmented M2-associated

  4. Targeting Sphingosine Kinase-1 To Inhibit Melanoma

    PubMed Central

    Madhunapantula, SubbaRao V.; Hengst, Jeremy; Gowda, Raghavendra; Fox, Todd E.; Yun, Jong K; Robertson, Gavin P.

    2012-01-01

    SUMMARY Resistance to therapies develops rapidly for melanoma leading to more aggressive disease. Therefore, agents are needed that specifically inhibit proteins or pathways controlling the development of this disease, which can be combined, dependent on genes deregulated in a particular patient’s tumors. This study shows that elevated sphingosine-1-phosphate (S-1-P) levels resulting from increased activity of sphingosine kinase-1 (SPHK1) occur in advanced melanomas. Targeting SPHK1 using siRNA decreased anchorage dependent and independent growth as well as sensitized melanoma cells to apoptosis inducing agents. Pharmacological SPHK1 inhibitors SKI-I but not SKI-II decreased S-1-P content, elevated ceramide levels, caused a G2-M block and induced apoptotic cell death in melanomas. Targeting SPHK1 using siRNA or the pharmacological agent called SKI-I, decreased the levels of pAKT. Furthermore, SKI-I inhibited the expression of CYCLIN D1 protein and increased the activity of caspase-3/7, which in turn led to the degradation of PARP. In animals, SKI-I but not SKI-II retarded melanoma growth by 25-40%. Thus, targeting SPHK1 using siRNAs or SKI-I has therapeutic potential for melanoma treatment either alone or in combination with other targeted agents. PMID:22236408

  5. Calcium/Ask1/MKK7/JNK2/c-Src signalling cascade mediates disruption of intestinal epithelial tight junctions by dextran sulfate sodium.

    PubMed

    Samak, Geetha; Chaudhry, Kamaljit K; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

    2015-02-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-Jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca(2+) concentration, and depletion of intracellular Ca(2+) by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM) or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of apoptosis signal-regulated kinase 1 (Ask1) or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased tyrosine phosphorylation of occludin, zonula occludens-1 (ZO-1), E-cadherin and β-catenin. SP600125 abrogated DSS-induced tyrosine phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto-phosphorylation of c-Src. The present study demonstrates that Ca(2+)/Ask1/MKK7/JNK2/cSrc signalling cascade mediates DSS-induced tight

  6. Heavy Rescue - Course Outline.

    DTIC Science & Technology

    1980-11-01

    used during heavy rescue operations . Methods and procedures for utilizing heavy rescue equipment. 4 Methods of developing improvised rescue equipment...utilizing available materials. A simulated exercise utilizing various rescue operations and techniques. Methods and procedures for the maintenance and...HEAVY RESCUE CONSIDERATIONS LEVEL I PERFORMANCE GOALS: 1 Hour GIVEN: 1. Summary of blocked access considerations during heavy rescue operations 2

  7. The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis.

    PubMed

    Zhao, D; Yu, Q; Chen, M; Ma, H

    2001-07-01

    The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.

  8. Resveratrol dimers are novel sphingosine kinase 1 inhibitors and affect sphingosine kinase 1 expression and cancer cell growth and survival

    PubMed Central

    Lim, Keng Gat; Gray, Alexander I; Pyne, Susan; Pyne, Nigel J

    2012-01-01

    BACKGROUND AND PURPOSE Sphingosine kinase 1 catalyses formation of the bioactive lipid, sphingosine 1-phosphate, which protects cancer cells from apoptosis. Therefore, sphingosine kinase 1 is a novel target for intervention with anti-cancer agents. We have assessed the effect of the anti-cancer agent, resveratrol and its dimers (ampelopsin A and balanocarpol) on sphingosine kinase 1 activity and on survival of MCF-7 breast cancer cells. EXPERIMENTAL APPROACH Ampelopsin A and balanocarpol were purified from Hopea dryobalanoides and their effect on sphingosine kinase 1 activity and expression, [3H] thymidine incorporation, ERK-1/2 phosphorylation and PARP activity assessed in MCF-7 cells. KEY RESULTS Resveratrol, ampelopsin A and balanocarpol were novel inhibitors of sphingosine kinase 1 activity. Balanocarpol was a mixed inhibitor (with sphingosine) of sphingosine kinase 1 with a Kic= 90 ± 10 µM and a Kiu of ∼500 µM. Balanocarpol and ampelopsin A also induced down-regulation of sphingosine kinase 1 expression and reduced DNA synthesis, while balanocarpol stimulated PARP cleavage in MCF-7 breast cancer cells. Resveratrol was a competitive inhibitor (with sphingosine) of sphingosine kinase 1 with a Kic= 160 ± 40 µM, reduced sphingosine kinase 1 expression and induced PARP cleavage in MCF-7 cells. CONCLUSIONS AND IMPLICATIONS Each molecule of balanocarpol may bind at least two sphingosine kinase 1 catalytic molecules to reduce the activity of each simultaneously. These findings suggest that resveratrol, ampelopsin A and balanocarpol could perturb sphingosine kinase 1-mediated signalling and this might explain their activity against MCF-7 breast cancer cells. LINKED ARTICLE This article is commented on by Hergst and Yun, pp. 1603–1604 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01898.x PMID:22251058

  9. Glutathione S-transferase class μ regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries.

    PubMed

    Bhattacharya, Poulomi; Madden, Jill A; Sen, Nivedita; Hoyer, Patricia B; Keating, Aileen F

    2013-02-15

    4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2-8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1.

  10. Rescue Equipment

    NASA Technical Reports Server (NTRS)

    1995-01-01

    The Lifeshear cutter, a rescue tool for freeing accident victims from wreckage, was developed under the Clinton Administration's Technology Reinvestment Program. Prior cutting equipment was cumbersome and expensive; the new cutter is 50 percent lighter and 70 percent cheaper. The cutter is pyrotechnically-actuated, using a miniature version of the power cartridges used for separation devices on the Space Shuttle and other NASA spacecraft. Hi-Shear Technology Corporation developed the cutter with the Jet Propulsion Laboratory and input from the City of Torrance (California) Fire Department.

  11. The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

    PubMed

    Zhao, D; Yang, M; Solava, J; Ma, H

    1999-09-01

    Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo. Copyright 1999 Wiley-Liss, Inc.

  12. Psoralen Inhibited Apoptosis of Osteoporotic Osteoblasts by Modulating IRE1-ASK1-JNK Pathway

    PubMed Central

    Chen, Shuqing; Wang, Yongqian; Yang, Yubin; Xiang, Ting; Liu, Jiahui

    2017-01-01

    Osteoporosis is a common disease causing fracture in older populations. Abnormal apoptosis of osteoblasts contributes to the genesis of osteoporosis. Inhibiting apoptosis of osteoblasts provides a promising strategy to prevent osteoporosis. The proliferation of osteoblasts isolated from osteoporotic patients or healthy subjects was determined by MTT assay. Apoptosis was determined by Annexin V/PI assay. Protein expression was measured by western blot. The proliferation of osteoblasts isolated from osteoporotic patients was inhibited and the apoptosis level of these cells was higher than the osteoblasts from healthy subjects. Incubation with psoralen or estradiol significantly enhanced the proliferation and decreased the apoptosis level of osteoporotic osteoblasts. Western blot demonstrated that psoralen or estradiol treatment downregulated the expression of IRE1, p-ASK, p-JNK, and Bax. Meanwhile, expression of Bcl-2 was upregulated. Pretreatment by IRE1 agonist tunicamycin or JNK agonist anisomycin attenuated the effect of psoralen on osteoporotic osteoblasts. Psoralen inhibited apoptosis of osteoporotic osteoblasts by regulating IRE1-ASK1-JNK pathway.

  13. Rescue Manual. Module 9.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The ninth of 10 modules contains 7 chapters: (1) ice characteristics; (2) river characteristics and tactics for rescue; (3) water rescue techniques; (4) water rescue/recovery operations; (5) dive operations; (6) water rescue equipment; and…

  14. Rescue Manual. Module 8.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The eighth of 10 modules contains 6 chapters: (1) trench rescue; (2) shoring and tunneling techniques; (3) farm accident rescue; (4) wilderness search and rescue; (5) aircraft rescue; and (6) helicopter information. Key points, an…

  15. Photoreceptor rescue.

    PubMed

    Luthert, P J; Chong, N H

    1998-01-01

    Photoreceptor cell death is the final, irreversible event in many blinding diseases including retinitis pigmentosa, age-related macular disease and retinal detachment. This paper examines the potential strategies for preventing photoreceptor cell death in the context of current understanding of the mechanisms of cell death. There is evidence to suggest that photoreceptor cells are inherently vulnerable, apoptosis is the final common pathway of photoreceptor cell loss, and other retinal cells play an important role in the survival of rods and cones. Furthermore, the rationale of using neurotrophic factors as therapeutic agents in retinal degeneration is discussed in detail. Photoreceptor rescue by manipulation of genes involved in apoptosis and some pharmacological agents is also described.

  16. Regular Article: Interaction of ASK1 and the β-Amyloid Precursor Protein in a Stress-Signaling Complex

    PubMed Central

    Galvan, Veronica; Banwait, Surita; Spilman, Patricia; Gorostiza, Olivia F.; Peel, Alyson; Crippen, Danielle; Sidhu, Gurleen; Ichijo, Hidenori; Bredesen, Dale E.

    2007-01-01

    The amyloid precursor protein (APP) is a type I transmembrane protein translocated to neuronal terminals, whose function is still unknown. The C-terminus of APP mediates its interaction with cellular adaptor and signaling proteins, some of which signal to the stress-activated protein kinase (SAPK) pathway. Here we show that ASK1, a MAPKKK that activates two SAPKs, c-Jun N-terminal-kinase (JNK) and p38, is present in a complex containing APP, phospho-MKK6, JIP1 and JNK1. In primary neurons deprived of growth factors, as well as in brains of (FAD)APP-transgenic mice, ASK1 was upregulated in neuronal projections, where it interacted with APP. In non-transgenic brains, ASK1 and APP associated mainly in the ER. Our results indicate that recruitment of ASK1 to stress-signaling complexes assembled with APP may be triggered and enhanced by cellular stress. Thus, ASK1 may be the apical MAPKKK in a signaling complex assembled with APP as a response to stress. PMID:17719230

  17. Interaction of ASK1 and the beta-amyloid precursor protein in a stress-signaling complex.

    PubMed

    Galvan, Veronica; Banwait, Surita; Spilman, Patricia; Gorostiza, Olivia F; Peel, Alyson; Ataie, Marina; Crippen, Danielle; Huang, Wei; Sidhu, Gurleen; Ichijo, Hidenori; Bredesen, Dale E

    2007-10-01

    The amyloid precursor protein (APP) is a type I transmembrane protein translocated to neuronal terminals, whose function is still unknown. The C-terminus of APP mediates its interaction with cellular adaptor and signaling proteins, some of which signal to the stress-activated protein kinase (SAPK) pathway. Here we show that ASK1, a MAPKKK that activates two SAPKs, c-Jun N-terminal-kinase (JNK) and p38, is present in a complex containing APP, phospho-MKK6, JIP1 and JNK1. In primary neurons deprived of growth factors, as well as in brains of (FAD)APP-transgenic mice, ASK1 was upregulated in neuronal projections, where it interacted with APP. In non-transgenic brains, ASK1 and APP associated mainly in the ER. Our results indicate that recruitment of ASK1 to stress-signaling complexes assembled with APP may be triggered and enhanced by cellular stress. Thus, ASK1 may be the apical MAPKKK in a signaling complex assembled with APP as a response to stress.

  18. Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

    SciTech Connect

    Bhattacharya, Poulomi; Madden, Jill A.; Sen, Nivedita; Hoyer, Patricia B.; Keating, Aileen F.

    2013-02-15

    4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2–8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P < 0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P < 0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P < 0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P < 0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. - Highlights: ► GSTM protein increases in response to ovarian VCD exposure. ► VCD increases Ask1 mRNA at the onset of follicle loss. ► Ovarian GSTM binds more ASK1 protein during VCD-induced ovotoxicity. ► PI3K regulates ovarian GSTM protein.

  19. Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury.

    PubMed

    Branco, Vasco; Coppo, Lucia; Solá, Susana; Lu, Jun; Rodrigues, Cecília M P; Holmgren, Arne; Carvalho, Cristina

    2017-10-01

    Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5μM of inorganic mercury (Hg(2+)), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells

  20. Lack of Casein Kinase 1 Delta Promotes Genomic Instability - The Accumulation of DNA Damage and Down-Regulation of Checkpoint Kinase 1

    PubMed Central

    Greer, Yoshimi Endo; Gao, Bo; Yang, Yingzi; Nussenzweig, Andre; Rubin, Jeffrey S.

    2017-01-01

    Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from Csnk1d null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEFCsnk1d null). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEFCsnk1d null cells. These cells often contain micronuclei, an indicator of genomic instability. Similarly, abrogation of CK1δ expression in control MEFs stimulated micronuclei formation after doxorubicin treatment, suggesting that CK1δ loss increases vulnerability to genotoxic stress. Cellular levels of total and activated checkpoint kinase 1 (Chk1), which functions in the DNA damage response and mitotic checkpoints, and its downstream effector, Cdc2/CDK1 kinase, were often decreased in MEFCsnk1d null cells as well as in control MEFs transfected with CK1δ siRNA. Hydroxyurea-induced Chk1 activation, as measured by Ser345 phosphorylation, and nuclear localization also were impaired in MEF cells following siRNA knockdown of CK1δ. Similar results were observed in the MCF7 human breast cancer cell line. The decreases in phosphorylated Chk1 were rescued by concomitant expression of siRNA-resistant CK1δ. Experiments with cycloheximide demonstrated that the stability of Chk1 protein was diminished in cells subjected to CK1δ knockdown. Together, these findings suggest that CK1δ contributes to the efficient repair of DNA damage and the proper functioning of mitotic checkpoints by maintaining appropriate levels of Chk1. PMID:28125685

  1. Rescue Manual. Module 6.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The sixth of 10 modules contains 4 chapters: (1) industrial rescue; (2) rescue from a confined space; (3) extrication from heavy equipment; and (4) rescue operations involving elevators. Key points, an introduction, and conclusion accompany…

  2. Zinc finger protein ZPR9 functions as an activator of AMPK-related serine/threonine kinase MPK38/MELK involved in ASK1/TGF-β/p53 signaling pathways

    PubMed Central

    Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

    2017-01-01

    Murine protein serine-threonine kinase 38 (MPK38), an AMP‐activated protein kinase (AMPK)-related kinase, has been implicated in the induction of apoptosis signal-regulating kinase 1 (ASK1)-, transforming growth factor-β (TGF‐β)-, and p53-mediated activity involved in metabolic homeostasis. Here, zinc finger protein ZPR9 was found to be an activator of MPK38. The association of MPK38 and ZPR9 was mediated by cysteine residues present in each of these two proteins, Cys269 and Cys286 of MPK38 and Cys305 and Cys308 of ZPR9. MPK38 phosphorylated ZPR9 at Thr252. Wild‐type ZPR9, but not the ZPR9 mutant T252A, enhanced ASK1, TGF‐β, and p53 function by stabilizing MPK38. The requirement of ZPR9 Thr252 phosphorylation was validated using CRISPR/Cas9-mediated ZPR9 (T252A) knockin cell lines. The knockdown of endogenous ZPR9 showed an opposite trend, resulting in the inhibition of MPK38‐dependent ASK1, TGF‐β, and p53 function. This effect was also demonstrated in mouse embryonic fibroblast (MEF) cells that were haploinsufficient (+/−) for ZPR9, NIH 3T3 cells with inducible knockdown of ZPR9, and CRISPR/Cas9-mediated ZPR9 knockout cells. Furthermore, high-fat diet (HFD)-fed mice displayed reduced MPK38 kinase activity and ZPR9 expression compared to that in mice on control chow, suggesting that ZPR9 acts as a physiological activator of MPK38 that may participate in obesity. PMID:28195154

  3. Unwinding the Wnt action of casein kinase 1.

    PubMed

    Yim, Daniel G R; Virshup, David M

    2013-06-01

    The casein kinase 1 (CK1) family, a major intracellular serine/threonine kinase, is implicated in multiple pathways; however, understanding its regulation has proven challenging. A recent study published in Science identifying allosteric activation of CK1 by the DEAD-box RNA helicase DDX3 expands our understanding of the control of this abundant kinase family.

  4. The Two Faces of Receptor Interacting Protein Kinase-1

    PubMed Central

    Weinlich, Ricardo; Green, Douglas R.

    2014-01-01

    Receptor Interacting Protein Kinase-1 (RIPK1), a key player in inflammation and cell death, assumes opposite functions depending on the cellular context and its posttranslational modifications. Genetic evidence supported by biochemical and cellular biology approaches shed light on the circumstances in which RIPK1 promotes or inhibits these processes. PMID:25459879

  5. Heat-shock protein 70 modulates apoptosis signal-regulating kinase 1 in stressed hepatocytes of Mugil cephalus.

    PubMed

    Padmini, Ekambaram; Tharani, Jayachandran

    2014-10-01

    Oxidative stress causes damage at the cellular level and activates a number of signaling pathways. Heat-shock proteins (HSPs) play an important role in repair and protective mechanisms under cell response to stress conditions. HSP70 has been shown to act as an inhibitor of apoptosis. Apoptosis signal-regulating kinase-1 (ASK1) activity is regulated at multiple levels, one of which is through inhibition by cytosolic chaperons HSP70. The current study was aimed to investigate the alteration in signaling molecules that allow the fish to survive under stressed natural field conditions. The study also investigates the variation in biomolecular composition of hepatocytes by using Fourier transform infrared spectroscopy. The impact of stress on hepatocytes was assessed by measuring the level of lipid peroxides (LPO), catalase activity (CAT) and assessing the changes in hepatocytes of Mugil cephalus inhabiting Kovalam and Ennore estuaries. The expression of HSP70 and ASK1 were analyzed by immunoblot analysis and ELISA, respectively. The spectral analysis showed variations in biomolecular composition of hepatocytes at a wave number region of 4,000-400 cm(-1). There was significant decrease of CAT activity (p < 0.01) (25 %) with significant increase of LPO (p < 0.001) (35 %) and HSP70 (p < 0.001) and insignificant increase of ASK1 (p < 0.05) (16 %) in fish hepatocytes inhabiting Ennore estuary than Kovalam estuary. In conclusion, the present study suggests that the survival of fish in the Ennore estuary under stressed condition may be due to the upregulation of HSP70 that mediates the altered signal pathway which promotes cellular resistance against apoptosis.

  6. Rescue Manual. Module 1.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The first of 10 modules contains 9 chapters: (1) introduction; (2) occupational stresses in rescue operations; (3) size-up; (4) critique; (5) reports and recordkeeping; (6) tools and equipment for rescue operations; (7) planning for…

  7. Rescue Manual. Module 4.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The fourth of 10 modules contains 8 chapters: (1) construction and characteristics of rescue rope; (2) knots, bends, and hitches; (3) critical angles; (4) raising systems; (5) rigging; (6) using the brake-bar rack for rope rescue; (7) rope…

  8. Rescue Manual. Module 3.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The third of 10 modules contains 4 chapters: (1) forcible entry; (2) structure search and rescue; (3) rescue operations involving electricity; and (4) cutting torches. Key points, an introduction, and conclusion accompany substantive…

  9. Rescue Skills and Techniques.

    ERIC Educational Resources Information Center

    Defense Civil Preparedness Agency (DOD), Washington, DC.

    The guide has been prepared for use as a textbook in rescue training courses at DCPA (Defense Civil Preparedness Agency) approved training schools and is to be used in rescue training programs of State and local governments. The document explains the various types of rescue missions, command structure, the personnel of the operating unit,…

  10. S6 kinase 1 is required for rapamycin-sensitive liver proliferation after mouse hepatectomy.

    PubMed

    Espeillac, Catherine; Mitchell, Claudia; Celton-Morizur, Séverine; Chauvin, Céline; Koka, Vonda; Gillet, Cynthia; Albrecht, Jeffrey H; Desdouets, Chantal; Pende, Mario

    2011-07-01

    Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.

  11. Inhibition of serum and glucocorticoid regulated kinase-1 as novel therapy for cardiac arrhythmia disorders.

    PubMed

    Bezzerides, Vassilios J; Zhang, Aifeng; Xiao, Ling; Simonson, Bridget; Khedkar, Santosh A; Baba, Shiro; Ottaviano, Filomena; Lynch, Stacey; Hessler, Katherine; Rigby, Alan C; Milan, David; Das, Saumya; Rosenzweig, Anthony

    2017-03-23

    Alterations in sodium flux (INa) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. We have recently demonstrated a critical role for the regulation of the voltage-gated sodium channel NaV1.5 in the heart by the serum and glucocorticoid regulated kinase-1 (SGK1). Activation of SGK1 in the heart causes a marked increase in both the peak and late sodium currents leading to prolongation of the action potential duration and an increased propensity to arrhythmia. Here we show that SGK1 directly regulates NaV1.5 channel function, and genetic inhibition of SGK1 in a zebrafish model of inherited long QT syndrome rescues the long QT phenotype. Using computer-aided drug discovery coupled with in vitro kinase assays, we identified a novel class of SGK1 inhibitors. Our lead SGK1 inhibitor (5377051) selectively inhibits SGK1 in cultured cardiomyocytes, and inhibits phosphorylation of an SGK1-specific target as well as proliferation in the prostate cancer cell line, LNCaP. Finally, 5377051 can reverse SGK1's effects on NaV1.5 and shorten the action potential duration in induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a patient with a gain-of-function mutation in Nav 1.5 (Long QT3 syndrome). Our data suggests that SGK1 inhibitors warrant further investigation in the treatment of cardiac arrhythmias.

  12. Palmitoylation of LIM Kinase-1 ensures spine-specific actin polymerization and morphological plasticity

    PubMed Central

    George, Joju; Soares, Cary; Montersino, Audrey; Beique, Jean-Claude; Thomas, Gareth M

    2015-01-01

    Precise regulation of the dendritic spine actin cytoskeleton is critical for neurodevelopment and neuronal plasticity, but how neurons spatially control actin dynamics is not well defined. Here, we identify direct palmitoylation of the actin regulator LIM kinase-1 (LIMK1) as a novel mechanism to control spine-specific actin dynamics. A conserved palmitoyl-motif is necessary and sufficient to target LIMK1 to spines and to anchor LIMK1 in spines. ShRNA knockdown/rescue experiments reveal that LIMK1 palmitoylation is essential for normal spine actin polymerization, for spine-specific structural plasticity and for long-term spine stability. Palmitoylation is critical for LIMK1 function because this modification not only controls LIMK1 targeting, but is also essential for LIMK1 activation by its membrane-localized upstream activator PAK. These novel roles for palmitoylation in the spatial control of actin dynamics and kinase signaling provide new insights into structural plasticity mechanisms and strengthen links between dendritic spine impairments and neuropathological conditions. DOI: http://dx.doi.org/10.7554/eLife.06327.001 PMID:25884247

  13. Enhanced Rescue Lift Capability

    NASA Technical Reports Server (NTRS)

    Young, Larry A.

    2007-01-01

    The evolving and ever-increasing demands of emergency response and disaster relief support provided by rotorcraft dictate, among other things, the development of enhanced rescue lift capability for these platforms. This preliminary analysis is first-order in nature but provides considerable insight into some of the challenges inherent in trying to effect rescue using a unique form of robotic rescue device deployed and operated from rotary-wing aerial platforms.

  14. A novel synthetic analogue of ω-3 17,18-epoxy-eicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells.

    PubMed

    Dyari, Herryawan Ryadi Eziwar; Rawling, Tristan; Chen, Yongjuan; Sudarmana, William; Bourget, Kirsi; Dwyer, Julie M; Allison, Sarah E; Murray, Michael

    2017-08-10

    A saturated analog of the cytochrome P450-mediated ω-3-17,18-epoxide of ω-3-eicosapentaenoic acid (C20E) activated apoptosis in human triple-negative MDA-MB-231 breast cancer cells. This study evaluated the apoptotic mechanism of C20E. Increased cytosolic cytochrome c expression and altered expression of pro- and antiapoptotic B-cell lymphoma-2 proteins indicated activation of the mitochondrial pathway. Caspase-3 activation by C20E was prevented by pharmacological inhibition and silencing of the JNK and p38 MAP kinases (MAPK), upstream MAPK kinases MKK4 and MKK7, and the upstream MAPK kinase kinase apoptosis signal-regulating kinase 1 (ASK1). Silencing of the death receptor TNF receptor 1 (TNFR1), but not Fas, DR4, or DR5, and the adapters TRADD and TNF receptor-associated factor 2, but not Fas-associated death domain, prevented C20E-mediated apoptosis. B-cell lymphoma-2 homology 3-interacting domain death agonist (Bid) cleavage by JNK/p38 MAPK linked the extrinsic and mitochondrial pathways of apoptosis. In further studies, an antibody against the extracellular domain of TNFR1 prevented apoptosis by TNF-α but not C20E. These findings suggest that C20E acts intracellularly at TNFR1 to activate ASK1-MKK4/7-JNK/p38 MAPK signaling and to promote Bid-dependent mitochondrial disruption and apoptosis. In in vivo studies, tumors isolated from C20E-treated nu/nu mice carrying MDA-MB-231 xenografts showed increased TUNEL staining and decreased Ki67 staining, reflecting increased apoptosis and decreased proliferation, respectively. ω-3-Epoxy fatty acids like C20E could be incorporated into treatments for triple-negative breast cancers.-Dyari, H. R. E., Rawling, T., Chen, Y., Sudarmana, W., Bourget, K., Dwyer, J. M., Allison, S. E., Murray, M. A novel synthetic analogue of ω-3 17,18-epoxy-eicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells. © FASEB.

  15. Rescue Manual. Module 10.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The 10th of 10 modules contains a 16-page glossary of rescue terms and 3 appendices: (1) 4 computer programs and 32 other technical assistance materials available for hazardous materials; (2) hazardous materials resources--60 phone numbers,…

  16. Rescue Manual. Module 2.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The second of 10 modules contains 5 chapters: (1) patient care and handling techniques; (2) rescue carries and drags; (3) emergency vehicle operations; (4) self-contained breathing apparatus; and (5) protective clothing. Key points, an…

  17. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    SciTech Connect

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  18. Myeloid cell death associated with Toll-like receptor 7/8-mediated inflammatory response. Implication of ASK1, HIF-1 alpha, IL-1 beta and TNF-alpha.

    PubMed

    Nicholas, Sally A; Oniku, Abraham E; Sumbayev, Vadim V

    2010-01-01

    Programmed cell death or apoptosis is an important part of the host innate immune defence, especially against ssRNA viruses (influenza virus, HIV-1, ebola virus, hepatitis C virus and many others). Viral ssRNA is recognised by endosomal Toll-like receptors 7 and 8 (TLR7/8) which induce further stages of immune defence against these pathogens. Some of the immune cells die because of inflammatory stress allowing for the selection of those cells which are resistant to stress-induced apoptosis and which are used in further stages of the host immune response. On the other hand, apoptosis could be used as an instrument to suppress the function of activated inflammatory cells. However, the mechanisms underlying death of the inflammatory cells associated with stress induced by ligands of TLR7/8 remain unclear. In this study we have found that programmed death of human myeloid cells from different cell lines associated with ligand-induced TLR7/8-mediated inflammatory stress depends on activation of apoptosis signal-regulating kinase 1 (ASK1). This enzyme is, however, not required for the production of pro-inflammatory cytokines - TNF-α and IL-1β. We have found that released IL-1β and TNF-α are involved in apoptosis of myeloid cells associated with TLR7/8-mediated inflammatory stress. The pro-apoptotic effect of released TNF-α in this case is much lower compared to that of IL-1β.

  19. Countries renew rescue agreement

    NASA Astrophysics Data System (ADS)

    Bush, Susan M.

    To insure long-term continuity for the international satellite search and rescue system, COSPAS/SARSAT, an intergovernmental agreement binding the four sponsoring nations to cooperate was signed July 1 in Paris. According to Russell Vollmers of the National Oceanic and Atmospheric Administration, the agreement is binding for 15 years, with an automatic extension.The system marked the fifth anniversary of its first rescue last year, when on September 10, 1982, three persons were rescued. Begun in the 1970s by NASA as an experiment, COSPAS/SARSAT (a Russian-English acronym) is now a cooperative project among the United States, Canada, France, and the Soviet Union. Its goal is to reduce the time required to rescue air and maritime distress victims and also to locate victims who otherwise may not be found, thus using the satellite system as a life-saving device.

  20. Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

    DOE PAGES

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; ...

    2014-12-08

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. In this paper, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1more » acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. In conclusion, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.« less

  1. Arabidopsis receptor of activated C kinase1 phosphorylation by WITH NO LYSINE8 KINASE.

    PubMed

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; Jones, Alan M; Chen, Jin-Gui

    2015-02-01

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. Here, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1A(S122A/T162A), but not the phosphomimetic form, RACK1A(S122D/T162E), rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1A(S122D/T162E) accumulated at similar levels as those of RACK1(S122A/T162A). However, although the steady-state level of the RACK1A(S122A/T162A) protein was similar to wild-type RACK1A protein, the RACK1A(S122D/T162E) protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. Taken together, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.

  2. Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

    SciTech Connect

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; Jones, Alan M.; Chen, Jin-Gui

    2014-12-08

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. In this paper, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. In conclusion, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.

  3. Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis

    PubMed Central

    Lou, Yiyun; Zhang, Fan; Luo, Yuqin; Wang, Liya; Huang, Shisi; Jin, Fan

    2016-01-01

    The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na+ homeostasis. Here, we focus particularly on recent findings of SGK1’s involvement in Na+ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na+ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure. PMID:27517916

  4. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    PubMed

    Makarchikov, Alexander F; Wins, Pierre; Janssen, Edwin; Wieringa, Bé; Grisar, Thierry; Bettendorff, Lucien

    2002-10-21

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

  5. Microchip Immunoaffinity Electrophoresis of Antibody-Thymidine Kinase 1 Complex

    PubMed Central

    Pagaduan, Jayson V.; Ramsden, Madison; O’Neill, Kim; Woolley, Adam T.

    2015-01-01

    Thymidine kinase-1 (TK1) is an important cancer biomarker whose serum levels are elevated in early cancer development. We developed a microchip electrophoresis immunoaffinity assay to measure recombinant purified TK1 (pTK1) using an antibody that binds to human TK1. We fabricated poly(methyl methacrylate) microfluidic devices to test the feasibility of detecting antibody (Ab)-pTK1 immune complexes as a step towards TK1 analysis in clinical serum samples. We were able to separate immune complexes from unbound antibodies using 0.5X phosphate buffer saline (pH 7.4) containing 0.01% Tween-20, with 1% w/v methylcellulose that acts as a dynamic surface coating and sieving matrix. Separation of the antibody and Ab-pTK1 complex was observed within a 5 mm effective separation length. This method of detecting pTK1 is easy to perform, requires only a 10 μL sample volume, and takes just 1 minute for separation. PMID:25486911

  6. Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking.

    PubMed

    Lima, Santiago; Milstien, Sheldon; Spiegel, Sarah

    2017-02-24

    The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

    PubMed

    Qian, Xu; Li, Xinjian; Cai, Qingsong; Zhang, Chuanbao; Yu, Qiujing; Jiang, Yuhui; Lee, Jong-Ho; Hawke, David; Wang, Yugang; Xia, Yan; Zheng, Yanhua; Jiang, Bing-Hua; Liu, David X; Jiang, Tao; Lu, Zhimin

    2017-03-02

    Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

  8. Carbamoylating Activity Associated with the Activation of the Antitumor Agent Laromustine Inhibits Angiogenesis by Inducing ASK1-Dependent Endothelial Cell Death

    PubMed Central

    Praggastis, Alexandra; Li, Yonghao; Zhou, Huanjiao Jenny; He, Yun; Ghazvinian, Roxanne; Cincotta, Dylan J.; Rice, Kevin P.; Min, Wang

    2014-01-01

    The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis. PMID:25068797

  9. TNF-α Suppresses Prolyl-4-Hydroxylase α1 Expression via the ASK1–JNK–NonO Pathway

    PubMed Central

    Zhang, Cheng; Zhang, Ming-Xiang; Shen, Ying H.; Burks, Jared K.; Zhang, Yun; Wang, Jian; LeMaire, Scott A.; Yoshimura, Koichi; Aoki, Hiroki; Coselli, Joseph S.; Wang, Xing Li

    2008-01-01

    Background Inflammation is known to contribute to the pathogenesis of vascular diseases in which arterial wall extracellular matrix (ECM) homeostasis is disrupted. Tumor necrosis factor-α (TNF-α), a pivotal cytokine that regulates ECM metabolism by increasing degradation and decreasing production of arterial collagens, is associated with vulnerable plaques and aortic aneurysms. Methods and Results In the current study, we showed that, when administered in doses of 1 to 100 ng/mL, TNF-α dose-dependently downregulated the expression of prolyl-4-hydroxylase αI [P4Hα (I)]—the rate-limiting subunit for the P4H enzyme essential for procollagen hydroxylation, secretion, and deposition in primary human aortic smooth muscle cells (HASMCs). Using a progressive deletion cloning approach, we characterized the TNF-α–responsive element (TaRE) in the human P4Hα (I) promoter and found that a negative regulatory region at the position of −32 to +18bp is responsible for ≈80% of TNF-α–mediated suppression. Using oligonucleotide-based transcription factor pull-down method in which proteins were resolved in 1-D gel electrophoresis and identified using LC-MS/MS, we identified the NonO protein binds this region. When NonO expression silenced with specific siRNA, we found that 70% of the TNF-α–mediated P4Hα suppression was abolished, which appeared to be mediated by the ASK1-JNK pathway. Conclusions Our findings define a novel molecular pathway for inflammation associated extracellular matrix dysregulation, which may account for atherosclerotic plaque rupture and aortic aneurysm formation. Further understanding of this pathway may facilitate development of novel therapeutics for vascular diseases. PMID:17478756

  10. Sphingosine kinase 1 is overexpressed and promotes adrenocortical carcinoma progression

    PubMed Central

    Huang, Jiwei; Kong, Wen; Xue, Wei; Zhu, Yu; Zhang, Jin; Huang, Yiran

    2016-01-01

    Adrenocortical carcinoma (ACC) is a rare endocrine tumor with a very poor prognosis. Sphingosine kinase 1 (SphK1), an oncogenic kinase, has previously been found to be upregulated in various cancers. However, the role of the SphK1 in ACC has not been investigated. In this study, SphK1 mRNA and protein expression levels as well as clinicopathological significance were evaluated in ACC samples. In vitro siRNA knockdown of SphK1 in two ACC cell lines (H295R and SW13) was used to determine its effect on cellular proliferation and invasion. In addition, we further evaluated the effect of SphK1 antagonist fingolimod (FTY720) in ACC in vitro and in vivo, as a single agent or in combination with mitotane, and attempted to explore its anticarcinogenic mechanisms. Our results show a significant over-expression of SphK1 mRNA and protein expression in the carcinomas compared with adenomas (P < 0.01 for all comparisons). Functionally, konckdown of SphK1 gene expression in ACC cell lines significantly decreased cell proliferation and invasion. FTY720 could result in a decreased cell proliferation and induction of apoptosis, and the combination of mitotane and FTY720 resulted in a greater anti-proliferative effect over single agent treatment in SW13 cells. Furthermore, FTY720 could markedly inhibit tumor growth in ACC xenografts. SphK1 expression is functionally associated to cellular proliferation, apoptosis, invasion and mitotane sensitivity of ACC. Our data suggest that SphK1 might be a potential therapeutic target for the treatment of ACC. PMID:26673009

  11. Airborne rescue system

    NASA Technical Reports Server (NTRS)

    Haslim, Leonard A. (Inventor)

    1991-01-01

    The airborne rescue system includes a boom with telescoping members for extending a line and collar to a rescue victim. The boom extends beyond the tip of the helicopter rotor so that the victim may avoid the rotor downwash. The rescue line is played out and reeled in by winch. The line is temporarily retained under the boom. When the boom is extended, the rescue line passes through clips. When the victim dons the collar and the tension in the line reaches a predetermined level, the clips open and release the line from the boom. Then the rescue line can form a straight line between the victim and the winch, and the victim can be lifted to the helicopter. A translator is utilized to push out or pull in the telescoping members. The translator comprises a tape and a rope. Inside the telescoping members the tape is curled around the rope and the tape has a tube-like configuration. The tape and rope are provided from supply spools.

  12. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida participate in a training exercise at the Shuttle Landing Facility. Firefighters, wearing protective gear, use hoses to put out a fire burning close to a mock-up of a small plane. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  13. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida participate in a training exercise at the Shuttle Landing Facility. Firefighters, wearing protective gear, use hoses to put out a fire burning near the mock-up of a small plane. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  14. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida participate in a training exercise at the Shuttle Landing Facility. Firefighters use a fire truck to put out a fire burning close to a mock-up of a small plane and a truck. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  15. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida participate in a training exercise at the Shuttle Landing Facility. Firefighters, wearing protective gear, use hoses to put out a fire burning on a mock-up of a small plane. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  16. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - During a training exercise, Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida use hoses to put out a fire burning on a mock-up of a small plane at the Shuttle Landing Facility. They are wearing protective gear for the training exercise. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  17. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida participate in a training exercise at the Shuttle Landing Facility. Firefighters use fire trucks and hoses to extinguish flames burning on and around a mock-up of a small plane. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  18. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida participate in a training exercise at the Shuttle Landing Facility. Firefighters, wearing protective gear, use hoses to put out a fire burning near the mock-up of a small plane as another firefighter checks inside the plane. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  19. Fire Rescue Exercise

    NASA Image and Video Library

    2014-03-06

    CAPE CANAVERAL, Fla. - Special Rescue Operations firefighters with NASA Fire Rescue Services in the Protective Services Office at NASA’s Kennedy Space Center in Florida prepare to participate in a training exercise at the Shuttle Landing Facility. A small fire is burning near a mock-up of a plane during the training exercise. Kennedy’s firefighters recently achieved Pro Board Certification in aerial fire truck operations and completed vehicle extrication training using the Jaws of Life. The Protective Services Office is one step closer to achieving certification in vehicle machinery extrication and other rescue skills. Kennedy’s firefighters are with G4S Government Solutions Inc., on the Kennedy Protective Services Contract. Photo credit: NASA/Kim Shiflett

  20. FPipTB, a benzimidazole derivative, induces chondrosarcoma cell apoptosis via endoplasmic reticulum stress and apoptosis signal-regulating kinase 1.

    PubMed

    Liu, Ju-Fang; Chang, Chih-Shiang; Fong, Yi-Chin; Kuo, Sheng-Chu; Tang, Chih-Hsin

    2012-04-01

    Chondrosarcoma is the second most common primary bone tumor and it responds poorly to both chemotherapy and radiation treatment. In this study, we investigated the anticancer effects of a new benzimidazole derivative, 2-(furanyl)-5-(piperidinyl)- (3,4,5-trimethoxybenzyl) benzimidazole (FPipTB) in human chondrosarcoma cells. FPipTB-induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. Furthermore, it triggered endoplasmic reticulum (ER) stress, which was characterized by changes in cytosolic calcium levels. Treatment of chondrosarcoma cells with FPipTB was associated with increased intracellular levels of ASK1, p38, p53, and Bax, followed by release of cytochrome c from mitochondria and activation of caspases. It is also known that ER stress activates apoptosis signal-regulating kinase 1 (ASK1), which mediates activation of JNK and p38 pathways. We also found that FPipTB-induced p38 and p53 phosphorylation and upregulated Bax expression. To study the mechanism of Bax upregulation, we determined that Bax promoter activity was increased in FPipTB-treated cells, leading to an increase in intracellular levels of Bax. In addition, cell treated with Ca(2+) chelator or p38 inhibitor showed reduced transcriptional activity. The results further suggest that FPipTB triggered ER stress, as indicated by changes in cytosolic calcium levels and activated the ASK1-MKK3/6-p38-p53-Bax pathway, causing chondrosarcoma cell death. Importantly, animal studies revealed a dramatic 40% reduction in tumor volume after 21 d of treatment. Thus, FPipTB may be a novel anticancer agent for the treatment of chondrosarcoma.

  1. Identification of novel polo-like kinase 1 inhibitors by a hybrid virtual screening.

    PubMed

    Lu, Shuai; Sun, Shan-Liang; Liu, Hai-Chun; Chen, Ya-Dong; Yuan, Hao-Liang; Gao, Yi-Ping; Yang, Pei; Lu, Tao

    2012-08-01

    Polo-like kinase 1 is an important and attractive oncological target that plays a key role in mitosis and cytokinesis. A combined pharmacophore- and docking-based virtual screening was performed to identify novel polo-like kinase 1 inhibitors. A total of 34 hit compounds were selected and tested in vitro, and some compounds showed inhibition of polo-like kinase 1 and human tumor cell growth. The most potent compound (66) inhibited polo-like kinase 1 with an IC(50) value of 6.99 μm. The docked binding models of two hit compounds were discussed in detail. These compounds contained novel chemical scaffolds and may be used as foundations for the development of novel classes of polo-like kinase 1 inhibitors.

  2. [Effects of flavone from leaves of Diospyros kaki on expression of apoptosis signal-regulating kinase 1 and rat vascular smooth muscle cells proliferation by tumor necrosis factor alpha in vitro].

    PubMed

    Ouyancg, Ping; Zhang, Bin; Bei, Wei-jian; Lai, Wen-yan; Peng, Wen-lie; Xu, Ding-li

    2007-07-01

    To observe the effects of flavone from leaves of Diospyros kaki on expression of apoptosis signal-regulating kinase 1 (ASK1) and rat vascular smooth muscle cells (VSMCs) proliferation by tumor necrosis factor alpha in vitro. Rat aortic VSMCs were cultured in vitro and treated with tumor necrosis factor alpha (TNF-alpha) and flavone from leaves of Diospyros kaki, respectively, and were observed in comparison with the control group. The ratio of cell proliferation was determined by non-radioactive MTS/PES as-say. The expression of ASK1 protein was evaluated by the immunoblotting technique using anti-ASKL antibody. The ratio of cell proliferation was 0.817 +/- 0.074 in the control group, and was 1.865 +/- 0.093 in TNF-alpha20 ng/ml group. It was shown that TNF-alpha significantly induced rat VSMCs proliferation (P < 0.05). The ratio of cell proliferatioh was 0.905 +/- 0.044 in flavone from leaves of Diospyros kaki group corresponding to concentration of 50 microg/ml. It was shown that flavone from leaves of Diospyros kaki alone had no effect on rat VSMCs proliferation (p > 0.05). With TNF-alpha stimulation, flavone from leaves of Diospyros kaki significantly inhibited rat expression of ASK1 protein enhanced by TNF-alpha was significantly inhibited rat-VSMNCs proliferation (1.247 +/- 0.061 vs. 1.865 +/- 0.093, p < 0.05). The expression of ASK1 protein enhanced by TNF-alpha was significantly inhibited by flavone from leaves of Diospyros kaki in VSMCs. Flavone from leaves of Diospyros kaki can significantly inhibit expression of ASK1 protein stimulated by TNF-alpha of rat VSMcs in vitro.

  3. Close Call: Unwanted Rescue.

    ERIC Educational Resources Information Center

    Journal of Adventure Education and Outdoor Leadership, 1991

    1991-01-01

    Describes incident where group engaged in training exercise was almost "rescued" by Coast Guard, although Coast Guard had been alerted that training exercise would be taking place. On another occasion Coast Guard did not react to actual report, thinking it was training group. Group was studying grey seal breeding colonies in…

  4. Rescue Manual. Module 5.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The fifth of 10 modules contains information on hazardous materials. Key points, an introduction, and conclusion accompany substantive material in this module. In addition, the module contains a Department of Transportation guide chart on…

  5. Rescue Manual. Module 7.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. Instructional Materials Lab.

    This learner manual for rescuers covers the current techniques or practices required in the rescue service. The seventh of 10 modules contains information on extrication from vehicles. Key points, an introduction, and conclusion accompany substantive material in this module. In addition, suggested tools and equipment for extrication procedures are…

  6. Close Call: Unwanted Rescue.

    ERIC Educational Resources Information Center

    Journal of Adventure Education and Outdoor Leadership, 1991

    1991-01-01

    Describes incident where group engaged in training exercise was almost "rescued" by Coast Guard, although Coast Guard had been alerted that training exercise would be taking place. On another occasion Coast Guard did not react to actual report, thinking it was training group. Group was studying grey seal breeding colonies in…

  7. Operation Rescue. Final Report.

    ERIC Educational Resources Information Center

    McKee, Neena; Crawford, Georgette

    The Operation Rescue project was designed to develop a classroom setting for the delivery of comprehensive educational services to "at risk" young adolescents. The classroom was established as part of the pre-existing Jonesboro Alternative School, and it utilized the basic academic and social program developed by this entity over 12…

  8. Combat Rescue Helicopter (CRH)

    DTIC Science & Technology

    2015-12-01

    Selected Acquisition Report (SAR) RCS: DD-A&T(Q&A)823-479 Combat Rescue Helicopter (CRH) As of FY 2017 President’s Budget Defense Acquisition...Research, Development, Test, and Evaluation SAR - Selected Acquisition Report SCP - Service Cost Position TBD - To Be Determined TY - Then Year UCR

  9. Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm

    PubMed Central

    Song, Yuyu; Nagy, Maria; Ni, Weiming; Tyagi, Navneet K.; Fenton, Wayne A.; López-Giráldez, Francesc; Overton, John D.; Horwich, Arthur L.; Brady, Scott T.

    2013-01-01

    Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity. PMID:23509252

  10. Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

    PubMed

    Song, Yuyu; Nagy, Maria; Ni, Weiming; Tyagi, Navneet K; Fenton, Wayne A; López-Giráldez, Francesc; Overton, John D; Horwich, Arthur L; Brady, Scott T

    2013-04-02

    Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

  11. Microtubule catastrophe and rescue.

    PubMed

    Gardner, Melissa K; Zanic, Marija; Howard, Jonathon

    2013-02-01

    Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed 'dynamic instability'. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule 'catastrophe'), and then by switching back from shrinkage to growth (called microtubule 'rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Microtubule Catastrophe and Rescue

    PubMed Central

    Gardner, Melissa K.; Zanic, Marija; Howard, Jonathon

    2012-01-01

    Microtubules are long cylindrical polymers composed of tubulin subunits. In cells, microtubules play an essential role in architecture and motility. For example, microtubules give shape to cells, serve as intracellular transport tracks, and act as key elements in important cellular structures such as axonemes and mitotic spindles. To accomplish these varied functions, networks of microtubules in cells are very dynamic, continuously remodeling through stochastic length fluctuations at the ends of individual microtubules. The dynamic behavior at the end of an individual microtubule is termed “dynamic instability”. This behavior manifests itself by periods of persistent microtubule growth interrupted by occasional switching to rapid shrinkage (called microtubule `catastrophe'), and then by switching back from shrinkage to growth (called microtubule `rescue'). In this review, we summarize recent findings which provide new insights into the mechanisms of microtubule catastrophe and rescue, and discuss the impact of these findings in regards to the role of microtubule dynamics inside of cells. PMID:23092753

  13. Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1

    PubMed Central

    Weijman, Johannes F.; Kumar, Abhishek; Jamieson, Sam A.; King, Chontelle M.; Caradoc-Davies, Tom T.; Ledgerwood, Elizabeth C.; Murphy, James M.

    2017-01-01

    Apoptosis signal-regulating kinases (ASK1–3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling. PMID:28242696

  14. Collectivizing rescue obligations in bioethics.

    PubMed

    Garrett, Jeremy R

    2015-01-01

    Bioethicists invoke a duty to rescue in a wide range of cases. Indeed, arguably, there exists an entire medical paradigm whereby vast numbers of medical encounters are treated as rescue cases. The intuitive power of the rescue paradigm is considerable, but much of this power stems from the problematic way that rescue cases are conceptualized-namely, as random, unanticipated, unavoidable, interpersonal events for which context is irrelevant and beneficence is the paramount value. In this article, I critique the basic assumptions of the rescue paradigm, reframe the ethical landscape in which rescue obligations are understood, and defend the necessity and value of a wider social and institutional view. Along the way, I move back and forth between ethical theory and a concrete case where the duty to rescue has been problematically applied: the purported duty to regularly return incidental findings and individual research results in genomic and genetic research.

  15. Inflatable rescue device

    NASA Technical Reports Server (NTRS)

    Swan, Scott A. (Inventor)

    1995-01-01

    This invention discloses, in one aspect, a personal rescue device for use in outer space which has an inflatable flexible tube with a shaper apparatus herein. Gas under pressure flows through the shaper apparatus and into the flexible tube. The flexible tube is mounted to the shaper so that as it inflates it expands and deploys lengthwise away from the shaper. In one embodiment a housing contains the shaper and the flexible tube and the housing is designed to facilitate movement of the expanding tube from the housing so the expanding tube does not bunch up in the housing.

  16. [Implementation of modern rescue medicine].

    PubMed

    Hou, S K; Fan, H J; Ding, H; Dong, W L

    2016-01-01

    Catastrophic disasters occur frequently in recent years, resulting in a large number of casualties. Thus, more and more scholars begin to focus on a newly emerged displine-rescue medicine. This paper introduces the status quo of rescue medicine and expounds the practical experience related to rescue medicine as practiced by author's unit, in order to provide a reference for the establishment of the discipline and its future development.

  17. [Importance of helicopter rescue].

    PubMed

    Hofer, G; Voelckel, W G

    2014-03-01

    Helicopter emergency medical service (HEMS) have become a main part of prehospital emergency medical services over the last 40 years. Recently, an ongoing discussion about financial shortage and personal shortcomings question the role of cost-intensive air rescue. Thus, the value of HEMS must be examined and discussed appropriately. Since the number of physician-staffed ground ambulances may decrease due to the limited availability of qualified physicians, HEMS may fill the gap. In addition patient transfer to specialized hospitals will require an increasing number of air transports in order to minimize prehospital time. The higher risk ratio for HEMS missions when compared with ground rescue requires a rigorous quality management system. When it comes to missions in remote and exposed areas, the scope of medical treatment must be adjusted to the individual situation. Medical competence is key in order to balance guideline compliant or maximal care versus optimal care characterized as a mission-specific, individualized emergency care concept. Although, medical decision making and treatment is typically based on the best scientific evidence, personal skills, competence, and the mission scenario will determine the scope of interventions suitable to improve outcome. Thus, the profile of requirements for the HEMS medical crew is high.

  18. Molecular Mechanisms of IRE1α-ASK1 Pathway Reactions to Unfolded Protein Response in DRN Neurons of Post-Traumatic Stress Disorder Rats.

    PubMed

    Kong, Fanzhen; Han, Fang; Xu, Yanhao; Shi, Yuxiu

    2017-04-01

    The goal of this study was to further elucidate the molecular mechanisms of post-traumatic stress disorder (PTSD) pathogenesis and to provide experimental evidence for new drug targets for effective PTSD treatment. Expression changes of IRE1α, ASK1, and other downstream molecules of the IRE1α-ASK1 endoplasmic reticulum stress (ERS) signaling pathway were investigated. JNK, P38, CHOP, Bcl-2, and Bax were analyzed at both protein and mRNA levels of dorsal raphe nucleus (DRN) neurons of PTSD rats. The rat PTSD model was established via the single-prolonged stress (SPS) method. Animals were randomly divided into five groups: a normal control group, a 1-day SPS group, a 4-days SPS group, a 7-day SPS group, and a 14-day SPS group. Spatial memory and learning ability of rats were evaluated subsequent to SPS using the Morris water maze test. Changes of IRE1α expression in the control and SPS groups were detected via immunohistochemistry (IHC). Protein and mRNA expressions of IRE1α, ASK1, JNK, P38, CHOP, Bcl-2, and Bax in the control and SPS groups were detected via Western blot and RT-PCR, respectively. The Morris water maze test revealed significantly longer average escape latencies in all SPS groups compared to the control group. In the spatial probe test, the percentage of time spent in the target quadrant was significantly lower in the SPS groups compared to control. IHC revealed increased positive expression of IRE1α subsequent to SPS challenge, reaching maximal levels on days four and seven (P < 0.01), while significantly decreasing on day 14 (P < 0.01). Western blot and RT-PCR revealed that protein and mRNA expressions of IRE1α, ASK1, JNK, CHOP, and P38 were significantly increased compared to control, peaking on days one, four, and seven post-SPS before returning to previous levels. Compared to control, expressions of Bcl-2 and Bax presented an initial increasing tendency followed by a decrease. A peak of Bcl-2 expression appeared early on day one

  19. 46 CFR 108.560 - Rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Rescue boats. 108.560 Section 108.560 Shipping COAST... Lifesaving Equipment § 108.560 Rescue boats. Each unit must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156. A lifeboat is accepted as a rescue boat if it also...

  20. 46 CFR 108.560 - Rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Rescue boats. 108.560 Section 108.560 Shipping COAST... Lifesaving Equipment § 108.560 Rescue boats. Each unit must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156. A lifeboat is accepted as a rescue boat if it also...

  1. 46 CFR 108.560 - Rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Rescue boats. 108.560 Section 108.560 Shipping COAST... Lifesaving Equipment § 108.560 Rescue boats. Each unit must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156. A lifeboat is accepted as a rescue boat if it also...

  2. 46 CFR 108.560 - Rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Rescue boats. 108.560 Section 108.560 Shipping COAST... Lifesaving Equipment § 108.560 Rescue boats. Each unit must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156. A lifeboat is accepted as a rescue boat if it also...

  3. The immunostimulating activity of quercetin 3-O-xyloside in murine macrophages via activation of the ASK1/MAPK/NF-κB signaling pathway.

    PubMed

    Lee, Jisun; Choi, Ji Won; Sohng, Jae Kyung; Pandey, Ramesh Prasad; Park, Yong Il

    2016-02-01

    Quercetin is a natural plant flavonoid that has been reported to possess a wide range of beneficial health effects, including anti-cancer and anti-inflammatory activities. Glycosylation of natural flavonoids with various sugar moieties can affect their physical, chemical, and biological properties. In this study, quercetin 3-O-xyloside (Quer-xyl) was enzymatically synthesized, and the immunomodulatory activities of quercetin and Quer-xyl were evaluated and compared. The results showed that Quer-xyl more effectively induced the secretion of TNF-α and IL-6 than quercetin by 2.5 and 1.5-fold, respectively. Quer-xyl dose-dependently induced the inducible nitric oxide synthase (iNOS) expression and increased the production of nitric oxide (NO) 1.3-fold more than quercetin. Quer-xyl also increased the phosphorylation of ASK1 and MAPKs (JNK and p38). Treatment with NQDI-1 (an inhibitor of ASK1) significantly attenuated the Quer-xyl-induced up-regulation of TNF-α secretion. The activation and subsequent nuclear translocation of NF-κB were substantially enhanced upon treatment with Quer-xyl (2.5-20 μM), while NQDI-1 treatment blocked the nuclear translocation of NF-κB. These results demonstrated that Quer-xyl can enhance the early innate immunity more effectively than quercetin by activating macrophages to secrete TNF-α and IL-6 through up-regulation of the redox-dependent ASK1/MAPK/NF-κB signaling pathway, suggesting for the first time that Quer-xyl may represent a new immunostimulator.

  4. Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1.

    PubMed

    Vila, J; Payne, D M; Zioncheck, T F; Harrison, M L; Itarte, E; Weber, M J

    1990-05-07

    Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.

  5. Locate and rescue system components

    NASA Technical Reports Server (NTRS)

    1976-01-01

    Two types of search and rescue systems not involving satellites are studied; one using a network radio communications link and the other a characteristic beacon signal. Line of slight limitation of VHF radio and beacon signals limit the range (approximately 25 miles) between the origin of the distress signal and the mobile rescue unit.

  6. Psychological Factors in Wilderness Rescue.

    ERIC Educational Resources Information Center

    Ogilvie, Bruce C.

    This presentation provides wilderness rescue workers with an overview of the psychological reactions of victims of accidents and natural disasters and suggested responses for rescuers and caregivers. A personal account of rescue and death in a drowning accident illustrates how the rescuer can also be traumatized by such an incident and may suffer…

  7. How competition affects evolutionary rescue

    PubMed Central

    Osmond, Matthew Miles; de Mazancourt, Claire

    2013-01-01

    Populations facing novel environments can persist by adapting. In nature, the ability to adapt and persist will depend on interactions between coexisting individuals. Here we use an adaptive dynamic model to assess how the potential for evolutionary rescue is affected by intra- and interspecific competition. Intraspecific competition (negative density-dependence) lowers abundance, which decreases the supply rate of beneficial mutations, hindering evolutionary rescue. On the other hand, interspecific competition can aid evolutionary rescue when it speeds adaptation by increasing the strength of selection. Our results clarify this point and give an additional requirement: competition must increase selection pressure enough to overcome the negative effect of reduced abundance. We therefore expect evolutionary rescue to be most likely in communities which facilitate rapid niche displacement. Our model, which aligns to previous quantitative and population genetic models in the absence of competition, provides a first analysis of when competitors should help or hinder evolutionary rescue. PMID:23209167

  8. Evolutionary rescue beyond the models

    PubMed Central

    Gomulkiewicz, Richard; Shaw, Ruth G.

    2013-01-01

    Laboratory model systems and mathematical models have shed considerable light on the fundamental properties and processes of evolutionary rescue. But it remains to determine the extent to which these model-based findings can help biologists predict when evolution will fail or succeed in rescuing natural populations that are facing novel conditions that threaten their persistence. In this article, we present a prospectus for transferring our basic understanding of evolutionary rescue to wild and other non-laboratory populations. Current experimental and theoretical results emphasize how the interplay between inheritance processes and absolute fitness in changed environments drive population dynamics and determine prospects of extinction. We discuss the challenge of inferring these elements of the evolutionary rescue process in field and natural settings. Addressing this challenge will contribute to a more comprehensive understanding of population persistence that combines processes of evolutionary rescue with developmental and ecological mechanisms. PMID:23209173

  9. A Novel Chromone Derivative with Anti-Inflammatory Property via Inhibition of ROS-Dependent Activation of TRAF6-ASK1-p38 Pathway

    PubMed Central

    Liu, Hailiang; Xu, Rui; Feng, Lili; Guo, Wenjie; Cao, Ning; Qian, Cheng; Teng, Peng; Wang, Lu; Wu, Xuefeng; Sun, Yang; Li, Jianxin; Shen, Yan; Xu, Qiang

    2012-01-01

    The p38 MAPK signaling pathway plays a pivotal role in inflammation. Targeting p38 MAPK may be a potential strategy for the treatment of inflammatory diseases. In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK. Moreover, DCO-6 specifically inhibited TLR4-dependent p38 activation without directly inhibiting its kinase activity. LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex. Administering DCO-6 significantly protected mice from LPS-induced septic shock in parallel with the inhibition of p38 activation and ROS production. Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway. Blockade of the upstream events required for p38 MAPK action by DCO-6 may provide a new therapeutic option in the treatment of inflammatory diseases. PMID:22720096

  10. Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A ▿

    PubMed Central

    Chen, Yung-Chia; Su, Wen-Chi; Huang, Jing-Ying; Chao, Ti-Chun; Jeng, King-Song; Machida, Keigo; Lai, Michael M. C.

    2010-01-01

    Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirus-based RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy. PMID:20534861

  11. Casein Kinase 1 Suppresses Activation of REST in Insulted Hippocampal Neurons and Halts Ischemia-Induced Neuronal Death

    PubMed Central

    Kaneko, Naoki; Hwang, Jee-Yeon; Gertner, Michael; Pontarelli, Fabrizio

    2014-01-01

    Repressor Element-1 (RE1) Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF) is a gene-silencing factor that is widely expressed during embryogenesis and plays a strategic role in neuronal differentiation. Recent studies indicate that REST can be activated in differentiated neurons during a critical window of time in postnatal development and in adult neurons in response to neuronal insults such as seizures and ischemia. However, the mechanism by which REST is regulated in neurons is as yet unknown. Here, we show that REST is controlled at the level of protein stability via β-TrCP-dependent, ubiquitin-based proteasomal degradation in differentiated neurons under physiological conditions and identify Casein Kinase 1 (CK1) as an upstream effector that bidirectionally regulates REST cellular abundance. CK1 associates with and phosphorylates REST at two neighboring, but distinct, motifs within the C terminus of REST critical for binding of β-TrCP and targeting of REST for proteasomal degradation. We further show that global ischemia in rats in vivo triggers a decrease in CK1 and an increase in REST in selectively vulnerable hippocampal CA1 neurons. Administration of the CK1 activator pyrvinium pamoate by in vivo injection immediately after ischemia restores CK1 activity, suppresses REST expression, and rescues neurons destined to die. Our results identify a novel and previously unappreciated role for CK1 as a brake on REST stability and abundance in adult neurons and reveal that loss of CK1 is causally related to ischemia-induced neuronal death. These findings point to CK1 as a potential therapeutic target for the amelioration of hippocampal injury and cognitive deficits associated with global ischemia. PMID:24760862

  12. MiR-506 suppresses liver cancer angiogenesis through targeting sphingosine kinase 1 (SPHK1) mRNA.

    PubMed

    Lu, Zhanping; Zhang, Weiying; Gao, Shan; Jiang, Qiulei; Xiao, Zelin; Ye, Lihong; Zhang, Xiaodong

    MicroRNAs acting as oncogenes or tumor suppressor genes play crucial roles in human cancers. Sphingosine kinase 1 (SPHK1) and its metabolite sphingosine 1-phosphate (S1P) contribute to tumor angiogenesis. We have reported that the down-regulation of miR-506 targeting YAP mRNA results in the hepatocarcinogenesis. In the present study, we report a novel function of miR-506, which suppresses tumor angiogenesis through targeting SPHK1 mRNA in liver cancer. Bioinformatics analysis showed that miR-506 might target 3'-untranslated region (3'UTR) of SPHK1 mRNA. Then, we validated that by luciferase reporter gene assays. MiR-506 was able to reduce the expression of SPHK1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis in hepatoma HepG2 cells. Functionally, human umbilical vein endothelial cell (HUVEC) tube formation assays demonstrated that the forced miR-506 expression remarkably inhibited the production of S1P in the supernatant of hepatoma cells. The supernatant resulted in the inhibition of tumor angiogenesis. Interestingly, the supernatant with overexpression of SPHK1 could rescue the inhibition of angiogenesis of liver cancer mediated by miR-506. Anti-miR-506 increased the production of S1P in the supernatant of hepatoma cells, but the supernatant with silencing of SPHK1 abolished anti-miR-506-induced acceleration of tumor angiogenesis. Clinically, we observed that the levels of miR-506 were negatively related to those of SPHK1 mRNA in liver cancer tissues. Thus, we conclude that miR-506 depresses the angiogenesis of liver cancer through targeting 3'UTR of SPHK1 mRNA. Our finding provides new insights into the mechanism of tumor angiogenesis.

  13. Inhibiting Polo-like kinase 1 causes growth reduction and apoptosis in pediatric acute lymphoblastic leukemia cells.

    PubMed

    Hartsink-Segers, Stefanie A; Exalto, Carla; Allen, Matthew; Williamson, Daniel; Clifford, Steven C; Horstmann, Martin; Caron, Huib N; Pieters, Rob; Den Boer, Monique L

    2013-10-01

    This study investigated Polo-like kinase 1, a mitotic regulator often over-expressed in solid tumors and adult hematopoietic malignancies, as a potential new target in the treatment of pediatric acute lymphoblastic leukemia. Polo-like kinase 1 protein and Thr210 phosphorylation levels were higher in pediatric acute lymphoblastic leukemia (n=172) than in normal bone marrow mononuclear cells (n=10) (P<0.0001). High Polo-like kinase 1 protein phosphorylation, but not expression, was associated with a lower probability of event-free survival (P=0.042) and was a borderline significant prognostic factor (P=0.065) in a multivariate analysis including age and initial white blood cell count. Polo-like kinase 1 was necessary for leukemic cell survival, since short hairpin-mediated Polo-like kinase 1 knockdown in acute lymphoblastic leukemia cell lines inhibited cell proliferation by G2/M cell cycle arrest and induced apoptosis through caspase-3 and poly (ADP-ribose) polymerase cleavage. Primary patient cells with a high Polo-like kinase 1 protein expression were sensitive to the Polo-like kinase 1-specific inhibitor NMS-P937 in vitro, whereas cells with a low expression and normal bone marrow cells were resistant. This sensitivity was likely not caused by Polo-like kinase 1 mutations, since only one new mutation (Ser335Arg) was found by 454-sequencing of 38 pediatric acute lymphoblastic leukemia cases. This mutation did not affect Polo-like kinase 1 expression or NMS-P937 sensitivity. Together, these results indicate a pivotal role for Polo-like kinase 1 in pediatric acute lymphoblastic leukemia and show potential for Polo-like kinase 1-inhibiting drugs as an addition to current treatment strategies for cases expressing high Polo-like kinase 1 levels.

  14. Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy

    USDA-ARS?s Scientific Manuscript database

    Sarcocystis neurona is the most frequent cause of Equine Protozoal Myeloencephalitis (EPM), a debilitating neurologic disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma...

  15. Electronic search and rescue aids

    NASA Technical Reports Server (NTRS)

    Trudell, B. J.

    1980-01-01

    There are two elements to the basic electronic search and rescue problem: a means for immediately alerting potential rescuers and an effective method to guide the rescue forces to the scene of the emergency. An Emergency Locator Transmitter (ELT) used by aircraft or an Emergency Position Indicating Radio Beacon (EPIRB) used by maritime vessels has the capability of providing for both an immediate alert and a homing signal to assist rescue forces in locating the site of the distress. This paper describes the development of ELT/EPIRB systems. Emphasis is placed on the SARSAT project, the COSPAS/SARSAT project, and an experimental 406 MHz ELT/EPIRB system.

  16. Electronic search and rescue aids

    NASA Technical Reports Server (NTRS)

    Trudell, B. J.

    1980-01-01

    There are two elements to the basic electronic search and rescue problem: a means for immediately alerting potential rescuers and an effective method to guide the rescue forces to the scene of the emergency. An Emergency Locator Transmitter (ELT) used by aircraft or an Emergency Position Indicating Radio Beacon (EPIRB) used by maritime vessels has the capability of providing for both an immediate alert and a homing signal to assist rescue forces in locating the site of the distress. This paper describes the development of ELT/EPIRB systems. Emphasis is placed on the SARSAT project, the COSPAS/SARSAT project, and an experimental 406 MHz ELT/EPIRB system.

  17. Calcium-Ask1-MKK7-JNK2-c-Src Signaling Cascade Mediates Disruption of Intestinal Epithelial Tight Junctions by Dextran Sulfate Sodium

    PubMed Central

    Samak, Geetha; Chaudhry, Kamaljit K.; Gangwar, Ruchika; Narayanan, Damodaran; Jaggar, Jonathan H.; Rao, RadhaKrishna

    2015-01-01

    Disruption of intestinal epithelial tight junctions is an important event in the pathogenesis of ulcerative colitis. Dextran sodium sulfate (DSS) induces colitis in mice with the symptoms similar to ulcerative colitis. However, the mechanism of DSS-induced colitis is unknown. We investigated the mechanism of DSS-induced disruption of intestinal epithelial tight junctions and barrier dysfunction in Caco-2 cell monolayers in vitro and mouse colon in vivo. DSS treatment resulted in disruption of tight junctions, adherens junctions and actin cytoskeleton leading to barrier dysfunction in Caco-2 cell monolayers. DSS induced a rapid activation of c-jun N-terminal kinase (JNK), and the inhibition or knockdown of JNK2 attenuated DSS-induced tight junction disruption and barrier dysfunction. In mice, DSS administration for 4 days caused redistribution of tight junction and adherens junction proteins from the epithelial junctions, which was blocked by JNK inhibitor. In Caco-2 cell monolayers, DSS increased intracellular Ca2+ concentration, and depletion of intracellular Ca2+ by BAPTA or thapsigargin attenuated DSS-induced JNK activation, tight junction disruption and barrier dysfunction. Knockdown of Ask1 or MKK7 blocked DSS-induced tight junction disruption and barrier dysfunction. DSS activated c-Src by a Ca2+ and JNK-dependent mechanism. Inhibition of Src kinase activity or knockdown of c-Src blocked DSS-induced tight junction disruption and barrier dysfunction. DSS increased Tyr-phosphorylation of occludin, ZO-1, E-cadherin and β-catenin. SP600125 abrogated DSS-induced Tyr-phosphorylation of junctional proteins. Recombinant JNK2 induced threonine phosphorylation and auto phosphorylation of c-Src. This study demonstrates that Ca2+-Ask1-MKK7-JNK2-cSrc signaling cascade mediates DSS-induced tight junction disruption and barrier dysfunction. PMID:25377781

  18. Quercetin ameliorates ischemia/reperfusion-induced cognitive deficits by inhibiting ASK1/JNK3/caspase-3 by enhancing the Akt signaling pathway.

    PubMed

    Pei, Bing; Yang, Miaomiao; Qi, Xiaoyan; Shen, Xin; Chen, Xing; Zhang, Fayong

    2016-09-09

    Cerebral ischemia/reperfusion (I/R) is a major cause of severe disability and death all worldwide. However, therapeutic options to minimize the detrimental effects of cerebral I/R injury are limited. Recent research has demonstrated that quercetin mediates neuroprotective effects associated with the activation of the Akt signaling pathway in the cerebral I/R brain. Therefore, the aim of this study was to further investigate the mechanisms of cognitive deficits induced by cerebral I/R injury and the effects of quercetin on these mechanisms. First, we assessed anxiety-like behavioral and cognitive impairment using the open field test and the Morris water maze test, respectively. Next, we examined the severity of apoptosis by staining hippocampal neurons by the Cresyl violet method. Third, we used western blot analysis to investigate the expression of total and phosphorylated Akt, ASK1, JNK3, c-Jun and caspase-3 after I/R injury. Our results revealed that mice subjected to bilateral common carotid occlusion exhibited severe anxiety-like behavior, learning and memory impairment, cell damage and apoptosis. These severe effects were attenuated by administration of quercetin. Further, western blot analysis revealed that quercetin increased p-Akt expression and decreased p-ASK1, p-JNK3 and cleaved caspase-3 expression after cerebral I/R injury and led to inhibition of neuronal apoptosis. Conversely, treatment with LY294002 (a selective inhibitor of Akt1) reversed the effects of quercetin. In conclusion, these findings highlight the important role of quercetin in protecting against cognitive deficits and inhibiting neuronal apoptosis via the Akt signaling pathway. We believe that quercetin might prove to be a useful therapeutic component in treating cerebral I/R diseases in the near future. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1 induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

    PubMed Central

    Yang, Lili; Wu, Defeng; Wang, Xiaodong; Cederbaum, Arthur I

    2011-01-01

    Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1 induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40–60% in E47 but not C34 cells with 80–90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity by either a glutathione synthesis inhibitor or TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 hrs of siRNA treatment. JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1 induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress. PMID:21557999

  20. Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    PubMed Central

    Chan, Vera S. F.; Chung, Nancy P. Y.; Wang, Shu-Rong; Li, Zhongye; Ma, Jing; Lin, Chia-Wei; Hsieh, Ya-Ju; Chang, Kao-Ping; Kung, Sui-Sum; Wu, Yi-Chia; Chu, Cheng-Wei; Tai, Hsiao-Ting; Gao, George F.; Zheng, Bojian; Yokoyama, Kazunari K.; Austyn, Jonathan M.; Lin, Chen-Lung S.

    2013-01-01

    During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to

  1. Drowning rescue, throw assist (image)

    MedlinePlus

    Drowning may occur wherever there is water, whether it is only a few inches in the bottom of the tub, or thousands of feet in the ocean. People should be aware of life-saving techniques from rescue to resuscitation.

  2. Drowning rescue, board assist (image)

    MedlinePlus

    Drowning may occur wherever there is water, whether it is only a few inches in the bottom of the tub or thousands of feet in the ocean. People should be aware of life-saving techniques from rescue to resuscitation.

  3. Drowning rescue, reaching assist (image)

    MedlinePlus

    Drowning may occur wherever there is water, whether it is only a few inches in the bottom of the tub or thousands of feet in the ocean. People should be aware of life-saving techniques from rescue to resuscitation.

  4. Community rescue in experimental metacommunities

    PubMed Central

    Low-Décarie, Etienne; Kolber, Marcus; Homme, Paige; Lofano, Andrea; Dumbrell, Alex; Gonzalez, Andrew; Bell, Graham

    2015-01-01

    The conditions that allow biodiversity to recover following severe environmental degradation are poorly understood. We studied community rescue, the recovery of a viable community through the evolutionary rescue of many populations within an evolving community, in metacommunities of soil microbes adapting to a herbicide. The metacommunities occupied a landscape of crossed spatial gradients of the herbicide (Dalapon) and a resource (glucose), whereas their constituent communities were either isolated or connected by dispersal. The spread of adapted communities across the landscape and the persistence of communities when that landscape was degraded were strongly promoted by dispersal, and the capacity to adapt to lethal stress was also related to community size and initial diversity. After abrupt and lethal stress, community rescue was most frequent in communities that had previously experienced sublethal levels of stress and had been connected by dispersal. Community rescue occurred through the evolutionary rescue of both initially common taxa, which remained common, and of initially rare taxa, which grew to dominate the evolved community. Community rescue may allow productivity and biodiversity to recover from severe environmental degradation. PMID:26578777

  5. 46 CFR 199.262 - Rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Rescue boats. 199.262 Section 199.262 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Cargo Vessels § 199.262 Rescue boats. (a) Each cargo vessel must carry at least one rescue boat. Each rescue boat must be approved under approval...

  6. 46 CFR 199.262 - Rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Rescue boats. 199.262 Section 199.262 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Cargo Vessels § 199.262 Rescue boats. (a) Each cargo vessel must carry at least one rescue boat. Each rescue boat must be approved under approval...

  7. 46 CFR 199.262 - Rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Rescue boats. 199.262 Section 199.262 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Cargo Vessels § 199.262 Rescue boats. (a) Each cargo vessel must carry at least one rescue boat. Each rescue boat must be approved under approval...

  8. 46 CFR 199.262 - Rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Rescue boats. 199.262 Section 199.262 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Cargo Vessels § 199.262 Rescue boats. (a) Each cargo vessel must carry at least one rescue boat. Each rescue boat must be approved under approval...

  9. 46 CFR 199.262 - Rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Rescue boats. 199.262 Section 199.262 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Cargo Vessels § 199.262 Rescue boats. (a) Each cargo vessel must carry at least one rescue boat. Each rescue boat must be approved under approval...

  10. 46 CFR 108.560 - Rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Rescue boats. 108.560 Section 108.560 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Lifesaving Equipment § 108.560 Rescue boats. Each unit must carry at least one rescue boat. Each rescue...

  11. Immature embryo rescue and culture.

    PubMed

    Shen, Xiuli; Gmitter, Fred G; Grosser, Jude W

    2011-01-01

    Embryo culture techniques have many significant applications in plant breeding, as well as basic studies in physiology and biochemistry. Immature embryo rescue and culture is a particularly attractive technique for recovering plants from sexual crosses where the majority of embryos cannot survive in vivo or become dormant for long periods of time. Overcoming embryo inviability is the most common reason for the application of embryo rescue techniques. Recently, fruit breeding programs have greatly increased the interest in exploiting interploid hybridization to combine desirable genetic traits of complementary parents at the triploid level for the purpose of developing improved seedless fruits. However, the success of this approach has only been reported in limited number of species due to various crossing barriers and embryo abortion at very early stages. Thus, immature embryo rescue provides an alternative means to recover triploid hybrids, which usually fail to completely develop in vivo. This chapter will provide a brief discussion of the utilization of interploid crosses between a monoembryonic diploid female with an allotetraploid male in a citrus cultivar improvement program, featuring a clear and comprehensive illustration of successful protocols for immature embryo rescue and culture. The protocols will cover the complete process from embryo excision to recovered plant in the greenhouse and can easily be adapted to other plant commodities. Factors affecting the success and failure of immature embryo rescue to recover triploid progeny from interploid crosses will be discussed.

  12. Overexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancer.

    PubMed

    Wang, Zhao-Xia; Xue, Dong; Liu, Zhi-Li; Lu, Bin-Bin; Bian, Hai-Bo; Pan, Xuan; Yin, Yong-Mei

    2012-01-01

    Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P=0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326-3.557; P=0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G(2)/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback

  13. Fenretinide-dependent upregulation of death receptors through ASK1 and p38α enhances death receptor ligand-induced cell death in Ewing's sarcoma family of tumours.

    PubMed

    White, D E; Burchill, S A

    2010-10-26

    Sustained p38(MAPK) phosphorylation upregulates p75 neurotrophin (p75(NTR)) and induces apoptosis in Ewing's sarcoma family of tumours (ESFT). As fenretinide induces ESFT death through sustained p38(MAPK) phosphorylation, we hypothesised that this may be effected through upregulation of death receptors (DRs) and that treatment of fenretinide plus DR ligands may enhance apoptosis. DR expression was determined by flow cytometry. Trypan blue exclusion assays, caspase-8 flow cytometry and immunoblotting for Bid were used to measure cell death. Fenretinide upregulated cell surface expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors, FAS and p75(NTR), in an ASK1- and p38α-dependent manner. Cotreatment with fenretinide and DR ligands resulted in synergistic death compared with either agent alone; caspase-8 and Bid were cleaved in a time-dependent manner. Fenretinide did not increase DR expression in non-malignant cells. Furthermore, fenretinide, TRAIL or a combination of both agents was non-cytotoxic to non-malignant cells. Etoposide and actinomycin D increased expression of all DRs examined, whereas vincristine increased FAS alone. Only actinomycin D and TRAIL, and etoposide with TRAIL or FasL, enhanced death compared with either agent alone. The synergistic death observed with fenretinide and DR ligands suggests that this combination may be an attractive strategy for the treatment of ESFT.

  14. Fenretinide-dependent upregulation of death receptors through ASK1 and p38α enhances death receptor ligand-induced cell death in Ewing's sarcoma family of tumours

    PubMed Central

    White, D E; Burchill, S A

    2010-01-01

    Background: Sustained p38MAPK phosphorylation upregulates p75 neurotrophin (p75NTR) and induces apoptosis in Ewing's sarcoma family of tumours (ESFT). As fenretinide induces ESFT death through sustained p38MAPK phosphorylation, we hypothesised that this may be effected through upregulation of death receptors (DRs) and that treatment of fenretinide plus DR ligands may enhance apoptosis. Methods: DR expression was determined by flow cytometry. Trypan blue exclusion assays, caspase-8 flow cytometry and immunoblotting for Bid were used to measure cell death. Results: Fenretinide upregulated cell surface expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors, FAS and p75NTR, in an ASK1- and p38α-dependent manner. Cotreatment with fenretinide and DR ligands resulted in synergistic death compared with either agent alone; caspase-8 and Bid were cleaved in a time-dependent manner. Fenretinide did not increase DR expression in non-malignant cells. Furthermore, fenretinide, TRAIL or a combination of both agents was non-cytotoxic to non-malignant cells. Etoposide and actinomycin D increased expression of all DRs examined, whereas vincristine increased FAS alone. Only actinomycin D and TRAIL, and etoposide with TRAIL or FasL, enhanced death compared with either agent alone. Conclusion: The synergistic death observed with fenretinide and DR ligands suggests that this combination may be an attractive strategy for the treatment of ESFT. PMID:20877355

  15. Joseph Conrad's tormented Rescue (fantasy).

    PubMed

    Freedman, William

    2014-02-01

    Joseph Conrad was a notoriously tormented writer for whom the creative act was often a punishment severe enough to drive him into paralyzing depressions that delayed the completion of his novels, sometimes for years. By far the most agonizing of these projects was The Rescue, a novel he began in 1898, abandoned a year later, tried unsuccessfully to continue several times over the next two decades, but was only able to resume in 1918 and to complete, after another tortured two-year struggle, in 1920. An explanation for this incapacity, that is powerfully suggested by the novel's evocative title and perhaps unintentionally ironic subtitle (A Romance of the Shallows) has not yet been explored. Using Freud's 1910 essay on the rescue fantasy, "Contributions to the Psychology of Love: A Special Type of Choice of Object Made by Men," and Emanuel Berman's instructive revision and expansion of the concept in his 2003 American Imago essay, "Ferenczi, Rescue, and Utopia," I argue that a substantial explanation for Conrad's tormented history with The Rescue is ascribable to its quite remarkably faithful treatment of a rescue fantasy with deep and disabling resonance for its author. More specifically, the difficulty was compounded by the novel's dramatization of the soul-crushing conflict between two such fantasies: one in the service of the masculine ideal of unflinching dedication to a heroic purpose, the other promising satisfaction to the equally potent demands of emotional and sexual desire. Features of Conrad's narrative fit so tightly and consistently with the theory as Freud (and Abraham) proposed and as Berman elaborated it that The Rescue offers itself as one of those rare and reinforcing instances wherein the literary text seems to validate the psychoanalytic theory at least as persuasively as the theory "understands" the text.

  16. Sex, mitochondria, and genetic rescue

    PubMed Central

    Havird, Justin C.; Fitzpatrick, Sarah W.; Kronenberger, John; Funk, W. Chris; Angeloni, Lisa M.; Sloan, Daniel B.

    2015-01-01

    Genetic rescue is a potentially effective management tool to offset the effects of reduced genetic diversity in imperiled populations. However, implementation requires complex choices. Here, we address the consequences of introducing males vs. females, highlighting the possibility that introduced females might lead to maladapted mitonuclear genomes and reduced offspring fitness. PMID:26712562

  17. Rotorcraft and Enabling Robotic Rescue

    NASA Technical Reports Server (NTRS)

    Young, Larry A.

    2010-01-01

    This paper examines some of the issues underlying potential robotic rescue devices (RRD) in the context where autonomous or manned rotorcraft deployment of such robotic systems is a crucial attribute for their success in supporting future disaster relief and emergency response (DRER) missions. As a part of this discussion, work related to proof-of-concept prototyping of two notional RRD systems is summarized.

  18. Role and mechanism of action of leucine-rich repeat kinase 1 in bone

    PubMed Central

    Xing, Weirong R; Goodluck, Helen; Zeng, Canjun; Mohan, Subburaman

    2017-01-01

    Leucine-rich repeat kinase 1 (LRRK1) plays a critical role in regulating cytoskeletal organization, osteoclast activity, and bone resorption with little effect on bone formation parameters. Deficiency of Lrrk1 in mice causes a severe osteopetrosis in the metaphysis of the long bones and vertebrae bones, which makes LRRK1 an attractive alternative drug target for the treatment of osteoporosis and other high-turnover bone diseases. This review summarizes recent advances on the functions of the Lrrk1-related family members, Lrrk1 deficiency-induced skeletal phenotypes, LRRK1 structure–function, potential biological substrates and interacting proteins, and the mechanisms of LRRK1 action in osteoclasts. PMID:28326224

  19. Death Associated Protein Kinase 1 (DAPK1): A Regulator of Apoptosis and Autophagy

    PubMed Central

    Singh, Pratibha; Ravanan, Palaniyandi; Talwar, Priti

    2016-01-01

    Death-Associated Protein Kinase 1 (DAPK1) belongs to a family of five serine/threonine (Ser/Thr) kinases that possess tumor suppressive function and also mediate a wide range of cellular processes, including apoptosis and autophagy. The loss and gain-of–function of DAPK1 is associated with various cancer and neurodegenerative diseases respectively. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms involved in DAPK1-mediated autophagy/apoptosis. In the present review, we have discussed the structural information and various cellular functions of DAPK1 in a comprehensive manner. PMID:27445685

  20. Reliability of Search and Rescue Action

    NASA Astrophysics Data System (ADS)

    Burciu, Zbigniew

    2012-06-01

    Determination of the reliability of Search and Rescue action system allows the SAR Mission Coordinator to increase the effectiveness of the action through the proper selection of operational characteristics of the system elements, in particular the selection of the rescue units and auxiliary units. The paper presents the example of the influence of water temperature and time of the action on the reliability of search and rescue action in the case of rescuing a survivor in the water.

  1. 46 CFR 133.135 - Rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Rescue boats. 133.135 Section 133.135 Shipping COAST... Requirements for All OSVs § 133.135 Rescue boats. (a) Each OSV must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156 and equipped as specified in table 133.175 of this...

  2. 46 CFR 133.135 - Rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Rescue boats. 133.135 Section 133.135 Shipping COAST... Requirements for All OSVs § 133.135 Rescue boats. (a) Each OSV must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156 and equipped as specified in table 133.175 of this...

  3. 46 CFR 133.135 - Rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Rescue boats. 133.135 Section 133.135 Shipping COAST... Requirements for All OSVs § 133.135 Rescue boats. (a) Each OSV must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156 and equipped as specified in table 133.175 of this...

  4. 46 CFR 133.135 - Rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Rescue boats. 133.135 Section 133.135 Shipping COAST... Requirements for All OSVs § 133.135 Rescue boats. (a) Each OSV must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.056 and equipped as specified in table 133.175 of this...

  5. 46 CFR 133.135 - Rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Rescue boats. 133.135 Section 133.135 Shipping COAST... Requirements for All OSVs § 133.135 Rescue boats. (a) Each OSV must carry at least one rescue boat. Each rescue boat must be approved under approval series 160.156 and equipped as specified in table 133.175 of this...

  6. Rescues conducted by surfers on Australian beaches.

    PubMed

    Attard, Anna; Brander, Robert W; Shaw, Wendy S

    2015-09-01

    This study describes the demographics, occurrence, location, primary hazards and outcomes involved in rescues performed by surfers on Australian beaches. Conservative estimates suggest that the number of rescues conducted by Australian surfers each year is on par with the number conducted by volunteer surf lifesavers. Surfers perform a considerable number of serious rescues in both lifesaver/lifeguard patrolled (45%) and unpatrolled (53%) beach locations. Rip currents represent the major physical hazard leading to rescue (75%) and the dominant emotional response of people rescued is one of panic (85%). Most surfer rescue events occur during conditions of moderate waves and sunny, fine weather with the highest proportion of rescues occurring on quiet beaches with few people around (26%). Swimming is the activity associated with most rescue events (63%), followed by board riding (25%). Males aged 18-29 represent the largest demographic of people rescued. Surfers with prior water-safety training are more likely to perform a higher number of rescues, however ability to perform rescues is not associated with formal training, but rather number of years' experience surfing. Seventy-eight percent of surfers were happy to help, while 28% expressed feelings of annoyance or inconvenience, generally towards unwary swimmers. Results of this research suggest that 63% of surfers feel they have saved a life. This value may be enhanced through improved training of surfers in basic water safety rescue techniques. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. 46 CFR 117.210 - Rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Rescue boats. 117.210 Section 117.210 Shipping COAST... Number and Type of Survival Craft § 117.210 Rescue boats. (a) Each vessel must carry at least one rescue boat unless the cognizant OCMI determines that: (1) The vessel is sufficiently maneuverable, arranged...

  8. 46 CFR 117.210 - Rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Rescue boats. 117.210 Section 117.210 Shipping COAST... Number and Type of Survival Craft § 117.210 Rescue boats. (a) Each vessel must carry at least one rescue boat unless the cognizant OCMI determines that: (1) The vessel is sufficiently maneuverable, arranged...

  9. 46 CFR 199.202 - Rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Rescue boats. 199.202 Section 199.202 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Passenger Vessels § 199.202 Rescue boats... least one rescue boat approved under approval series 160.156 that is equipped as specified in table 199...

  10. 46 CFR 199.202 - Rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Rescue boats. 199.202 Section 199.202 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Passenger Vessels § 199.202 Rescue boats... least one rescue boat approved under approval series 160.156 that is equipped as specified in table 199...

  11. 46 CFR 180.210 - Rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Rescue boats. 180.210 Section 180.210 Shipping COAST...) LIFESAVING EQUIPMENT AND ARRANGEMENTS Number and Type of Survival Craft § 180.210 Rescue boats. (a) A vessel of more than 19.8 meters (65 feet) in length must carry at least one rescue boat unless the cognizant...

  12. 46 CFR 169.517 - Rescue boat.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Rescue boat. 169.517 Section 169.517 Shipping COAST... and Firefighting Equipment Primary Lifesaving Equipment § 169.517 Rescue boat. All vessels certificated for exposed or partially protected waters service must have a suitable motor rescue boat, except...

  13. 46 CFR 169.517 - Rescue boat.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Rescue boat. 169.517 Section 169.517 Shipping COAST... and Firefighting Equipment Primary Lifesaving Equipment § 169.517 Rescue boat. All vessels certificated for exposed or partially protected waters service must have a suitable motor rescue boat, except...

  14. 46 CFR 180.210 - Rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Rescue boats. 180.210 Section 180.210 Shipping COAST...) LIFESAVING EQUIPMENT AND ARRANGEMENTS Number and Type of Survival Craft § 180.210 Rescue boats. (a) A vessel of more than 19.8 meters (65 feet) in length must carry at least one rescue boat unless the cognizant...

  15. 46 CFR 117.210 - Rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Rescue boats. 117.210 Section 117.210 Shipping COAST... Number and Type of Survival Craft § 117.210 Rescue boats. (a) Each vessel must carry at least one rescue boat unless the cognizant OCMI determines that: (1) The vessel is sufficiently maneuverable, arranged...

  16. 46 CFR 180.210 - Rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Rescue boats. 180.210 Section 180.210 Shipping COAST...) LIFESAVING EQUIPMENT AND ARRANGEMENTS Number and Type of Survival Craft § 180.210 Rescue boats. (a) A vessel of more than 19.8 meters (65 feet) in length must carry at least one rescue boat unless the cognizant...

  17. 46 CFR 169.517 - Rescue boat.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Rescue boat. 169.517 Section 169.517 Shipping COAST... and Firefighting Equipment Primary Lifesaving Equipment § 169.517 Rescue boat. All vessels certificated for exposed or partially protected waters service must have a suitable motor rescue boat, except...

  18. 46 CFR 117.210 - Rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Rescue boats. 117.210 Section 117.210 Shipping COAST... Number and Type of Survival Craft § 117.210 Rescue boats. (a) Each vessel must carry at least one rescue boat unless the cognizant OCMI determines that: (1) The vessel is sufficiently maneuverable, arranged...

  19. 46 CFR 199.202 - Rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Rescue boats. 199.202 Section 199.202 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Passenger Vessels § 199.202 Rescue boats... least one rescue boat approved under approval series 160.156 that is equipped as specified in table 199...

  20. 46 CFR 199.202 - Rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Rescue boats. 199.202 Section 199.202 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Passenger Vessels § 199.202 Rescue boats... least one rescue boat approved under approval series 160.156 that is equipped as specified in table 199...

  1. 46 CFR 180.210 - Rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Rescue boats. 180.210 Section 180.210 Shipping COAST...) LIFESAVING EQUIPMENT AND ARRANGEMENTS Number and Type of Survival Craft § 180.210 Rescue boats. (a) A vessel of more than 19.8 meters (65 feet) in length must carry at least one rescue boat unless the cognizant...

  2. 46 CFR 169.517 - Rescue boat.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Rescue boat. 169.517 Section 169.517 Shipping COAST... and Firefighting Equipment Primary Lifesaving Equipment § 169.517 Rescue boat. All vessels certificated for exposed or partially protected waters service must have a suitable motor rescue boat, except...

  3. 46 CFR 117.210 - Rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Rescue boats. 117.210 Section 117.210 Shipping COAST... Number and Type of Survival Craft § 117.210 Rescue boats. (a) Each vessel must carry at least one rescue boat unless the cognizant OCMI determines that: (1) The vessel is sufficiently maneuverable, arranged...

  4. 46 CFR 169.517 - Rescue boat.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Rescue boat. 169.517 Section 169.517 Shipping COAST... and Firefighting Equipment Primary Lifesaving Equipment § 169.517 Rescue boat. All vessels certificated for exposed or partially protected waters service must have a suitable motor rescue boat, except...

  5. 46 CFR 199.202 - Rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Rescue boats. 199.202 Section 199.202 Shipping COAST... SYSTEMS FOR CERTAIN INSPECTED VESSELS Additional Requirements for Passenger Vessels § 199.202 Rescue boats... least one rescue boat approved under approval series 160.156 that is equipped as specified in table 199...

  6. 46 CFR 180.210 - Rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Rescue boats. 180.210 Section 180.210 Shipping COAST...) LIFESAVING EQUIPMENT AND ARRANGEMENTS Number and Type of Survival Craft § 180.210 Rescue boats. (a) A vessel of more than 19.8 meters (65 feet) in length must carry at least one rescue boat unless the cognizant...

  7. Kinematic Simulation of a universal rescue vehicle

    NASA Astrophysics Data System (ADS)

    Vasiliev, I. A.; Smirnova, E. Y.; Krasheninnikov, M. S.; Koshurina, A. A.; Dorofeev, R. A.; E Gai, V.

    2017-01-01

    The rescue of people in disaster through autonomous means of evacuation in some cases is the only way to save their lives. Rescue Mission often takes place in remote locations. The paper studies modeling of a universal rescue vehicle with a rotary-screw propeller.

  8. Phosphorylation of Synaptic Vesicle Protein 2A at Thr84 by Casein Kinase 1 Family Kinases Controls the Specific Retrieval of Synaptotagmin-1

    PubMed Central

    Zhang, Ning; Gordon, Sarah L.; Fritsch, Maximilian J.; Esoof, Noor; Campbell, David G.; Gourlay, Robert; Velupillai, Srikannathasan; Macartney, Thomas; Peggie, Mark; van Aalten, Daan M.F.

    2015-01-01

    Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau–tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis. PMID:25673844

  9. Activation of serum/glucocorticoid-induced kinase 1 (SGK1) underlies increased glycogen levels, mTOR activation, and autophagy defects in Lafora disease.

    PubMed

    Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

    2013-12-01

    Lafora disease (LD), a fatal genetic form of myoclonic epilepsy, is characterized by abnormally high levels of cellular glycogen and its accumulation as Lafora bodies in affected tissues. Therefore the two defective proteins in LD-laforin phosphatase and malin ubiquitin ligase-are believed to be involved in glycogen metabolism. We earlier demonstrated that laforin and malin negatively regulate cellular glucose uptake by preventing plasma membrane targeting of glucose transporters. We show here that loss of laforin results in activation of serum/glucocorticoid-induced kinase 1 (SGK1) in cellular and animals models and that inhibition of SGK1 in laforin-deficient cells reduces the level of plasma membrane-bound glucose transporter, glucose uptake, and the consequent glycogen accumulation. We also provide evidence to suggest that mammalian target of rapamycin (mTOR) activates SGK1 kinase in laforin-deficient cells. The mTOR activation appears to be a glucose-dependent event, and overexpression of dominant-negative SGK1 suppresses mTOR activation, suggesting the existence of a feedforward loop between SGK1 and mTOR. Our findings indicate that inhibition of SGK1 activity could be an effective therapeutic approach to suppress glycogen accumulation, inhibit mTOR activity, and rescue autophagy defects in LD.

  10. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia

    PubMed Central

    Maynard, Sharon E.; Min, Jiang-Yong; Merchan, Jaime; Lim, Kee-Hak; Li, Jianyi; Mondal, Susanta; Libermann, Towia A.; Morgan, James P.; Sellke, Frank W.; Stillman, Isaac E.; Epstein, Franklin H.; Sukhatme, Vikas P.; Karumanchi, S. Ananth

    2003-01-01

    Preeclampsia, a syndrome affecting 5% of pregnancies, causes substantial maternal and fetal morbidity and mortality. The pathophysiology of preeclampsia remains largely unknown. It has been hypothesized that placental ischemia is an early event, leading to placental production of a soluble factor or factors that cause maternal endothelial dysfunction, resulting in the clinical findings of hypertension, proteinuria, and edema. Here, we confirm that placental soluble fms-like tyrosine kinase 1 (sFlt1), an antagonist of VEGF and placental growth factor (PlGF), is upregulated in preeclampsia, leading to increased systemic levels of sFlt1 that fall after delivery. We demonstrate that increased circulating sFlt1 in patients with preeclampsia is associated with decreased circulating levels of free VEGF and PlGF, resulting in endothelial dysfunction in vitro that can be rescued by exogenous VEGF and PlGF. Additionally, VEGF and PlGF cause microvascular relaxation of rat renal arterioles in vitro that is blocked by sFlt1. Finally, administration of sFlt1 to pregnant rats induces hypertension, proteinuria, and glomerular endotheliosis, the classic lesion of preeclampsia. These observations suggest that excess circulating sFlt1 contributes to the pathogenesis of preeclampsia. PMID:12618519

  11. Phosphorylation of synaptic vesicle protein 2A at Thr84 by casein kinase 1 family kinases controls the specific retrieval of synaptotagmin-1.

    PubMed

    Zhang, Ning; Gordon, Sarah L; Fritsch, Maximilian J; Esoof, Noor; Campbell, David G; Gourlay, Robert; Velupillai, Srikannathasan; Macartney, Thomas; Peggie, Mark; van Aalten, Daan M F; Cousin, Michael A; Alessi, Dario R

    2015-02-11

    Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau-tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis. Copyright © 2015 Zhang et al.

  12. Tripartite Motif 8 Contributes to Pathological Cardiac Hypertrophy Through Enhancing Transforming Growth Factor β-Activated Kinase 1-Dependent Signaling Pathways.

    PubMed

    Chen, Lijuan; Huang, Jia; Ji, Yan-Xiao; Mei, Fanghua; Wang, Pi-Xiao; Deng, Ke-Qiong; Jiang, Xi; Ma, Genshan; Li, Hongliang

    2017-02-01

    Tripartite motif (TRIM) 8 functions as an E3 ubiquitin ligase, interacting with and ubiquitinating diverse substrates, and is implicated in various pathological processes. However, the function of TRIM8 in the heart remains largely uncharacterized. This study aims to explore the role of TRIM8 in the development of pathological cardiac hypertrophy. Mice and isolated neonatal rat cardiomyocytes overexpressing or lacking TRIM8 were examined in several experiments. The effect of aortic banding-induced cardiac hypertrophy was analyzed by echocardiographic, pathological and molecular analyses. Our results indicated that the TRIM8 overexpression in hearts exacerbated the cardiac hypertrophy triggered by aortic banding. In contrast, the development of pathological cardiac hypertrophy was profoundly blocked in TRIM8-deficient hearts. Mechanistically, our study suggests that TRIM8 may elicit cardiodetrimental effects by promoting the activation of transforming growth factor β-activated kinase 1 (TAK1)-p38/JNK signaling pathways. Similar results were observed in cultured neonatal rat cardiomyocytes treated with angiotensin II. The rescue experiments using the TAK1-specific inhibitor 5z-7-ox confirmed the requirement of TAK1 activation in TRIM8-mediated pathological cardiac hypertrophy. Furthermore, TRIM8 contributed to TAK1 activation by binding to and promoting TAK1 ubiquitination. In conclusion, our study demonstrates that TRIM8 plays a deleterious role in pressure overload-induced cardiac hypertrophy by accelerating the activation of TAK1-dependent signaling pathways.

  13. Loss of p21Cip1/CDKN1A renders cancer cells susceptible to Polo-like kinase 1 inhibition.

    PubMed

    Kreis, Nina-Naomi; Louwen, Frank; Zimmer, Brigitte; Yuan, Juping

    2015-03-30

    The deregulation of Polo-like kinase 1 is inversely linked to the prognosis of patients with diverse human tumors. Targeting Polo-like kinase 1 has been widely considered as one of the most promising strategies for molecular anticancer therapy. While the preclinical results are encouraging, the clinical outcomes are rather less inspiring by showing limited anticancer activity. It is thus of importance to identify molecules and mechanisms responsible for the sensitivity of Polo-like kinase 1 inhibition. We have recently shown that p21Cip1/CDKN1A is involved in the regulation of mitosis and its loss prolongs the mitotic duration accompanied by defects in chromosome segregation and cytokinesis in various tumor cells. In the present study, we demonstrate that p21 affects the efficacy of Polo-like kinase 1 inhibitors, especially Poloxin, a specific inhibitor of the unique Polo-box domain. Intriguingly, upon treatment with Polo-like kinase 1 inhibitors, p21 is increased in the cytoplasm, associated with anti-apoptosis, DNA repair and cell survival. By contrast, deficiency of p21 renders tumor cells more susceptible to Polo-like kinase 1 inhibition by showing a pronounced mitotic arrest, DNA damage and apoptosis. Furthermore, long-term treatment with Plk1 inhibitors induced fiercely the senescent state of tumor cells with functional p21. We suggest that the p21 status may be a useful biomarker for predicting the efficacy of Plk1 inhibition.

  14. Casein kinase 1δ/ε inhibitor PF-5006739 attenuates opioid drug-seeking behavior.

    PubMed

    Wager, Travis T; Chandrasekaran, Ramalakshmi Y; Bradley, Jenifer; Rubitski, David; Berke, Helen; Mente, Scot; Butler, Todd; Doran, Angela; Chang, Cheng; Fisher, Katherine; Knafels, John; Liu, Shenping; Ohren, Jeff; Marconi, Michael; DeMarco, George; Sneed, Blossom; Walton, Kevin; Horton, David; Rosado, Amy; Mead, Andy

    2014-12-17

    Casein kinase 1 delta (CK1δ) and casein kinase 1 epsilon (CK1ε) inhibitors are potential therapeutic agents for a range of psychiatric disorders. The feasibility of developing a CNS kinase inhibitor has been limited by an inability to identify safe brain-penetrant compounds with high kinome selectivity. Guided by structure-based drug design, potent and selective CK1δ/ε inhibitors have now been identified that address this gap, through the design and synthesis of novel 4-[4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl]pyrimidin-2-amine derivatives. PF-5006739 (6) possesses a desirable profile, with low nanomolar in vitro potency for CK1δ/ε (IC50 = 3.9 and 17.0 nM, respectively) and high kinome selectivity. In vivo, 6 demonstrated robust centrally mediated circadian rhythm phase-delaying effects in both nocturnal and diurnal animal models. Further, 6 dose-dependently attenuated opioid drug-seeking behavior in a rodent operant reinstatement model in animals trained to self-administer fentanyl. Collectively, our data supports further development of 6 as a promising candidate to test the hypothesis of CK1δ/ε inhibition in treating multiple indications in the clinic.

  15. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    PubMed

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  16. Mountain rescue stretchers: usability trial.

    PubMed

    Hignett, Sue; Willmott, Joseph Wayne; Clemes, Stacy

    2009-01-01

    In the UK mountain rescues are carried out by highly trained volunteers in all weather conditions and at any time of the day/night. They interface with other services when they hand over the casualty to either land or air ambulances. The design of the stretcher is important to the safety of both the volunteers and casualties. This paper reports a usability trial to evaluate the features of mountain rescue stretchers and identify characteristics for future design. Two mountain rescue teams in the English Lake District participated in a five week field experiment. Data were collected using postural analysis with Rapid Entire Body Analysis, Body Part Discomfort Surveys, Rated Perceived Exertion and focus groups to compare the performance of four stretchers: Split Thomas, Ferno Titan, MacInnes mark 6 and MacInnes mark 7. None of the stretchers had an overall advantage, with benefits for some features counterbalanced by disadvantages resulting from others. All the stretchers produced shoulder discomfort with the Split Thomas and MacInnes 6 lowering the postural risks through the use of skids/wheel in the carrying phase. The key design features for future MR stretchers include: reduced unloaded weight (e.g. light weight materials and mesh platforms); undercarriage system to reduce the carrying load; adjustable handles at the front and back positions; flexible carrying system with an optional harness attachment; ease of assembly in adverse environmental conditions; large carrying capacity. It is suggested that military emergency evacuation should be considered in addition to mountain rescue tasks to identify a larger commercial market for development.

  17. Toward a behavioral ecology of rescue behavior.

    PubMed

    Hollis, Karen L; Nowbahari, Elise

    2013-07-18

    Although the study of helping behavior has revolutionized the field of behavioral ecology, scientific examination of rescue behavior remains extremely rare, except perhaps in ants, having been described as early as 1874. Nonetheless, recent work in our laboratories has revealed several new patterns of rescue behavior that appear to be much more complex than previously studied forms. This precisely-directed rescue behavior bears a remarkable resemblance to what has been labeled empathy in rats, and thus raises numerous philosophical and theoretical questions: How should rescue behavior (or empathy) be defined? What distinguishes rescue from other forms of altruism? In what ways is rescue behavior in ants different from, and similar to, rescue in other non-human animals? What selection pressures dictate its appearance? In this paper, we review our own experimental studies of rescue in both laboratory and field, which, taken together, begin to reveal some of the behavioral ecological conditions that likely have given rise to rescue behavior in ants. Against this background, we also address important theoretical questions involving rescue, including those outlined above. In this way, we hope not only to encourage further experimental analysis of rescue behavior, but also to highlight important similarities and differences in very distant taxa.

  18. Soft Selective Sweeps in Evolutionary Rescue

    PubMed Central

    Wilson, Benjamin A.; Pennings, Pleuni S.; Petrov, Dmitri A.

    2017-01-01

    Evolutionary rescue occurs when a population that is declining in size because of an environmental change is rescued from extinction by genetic adaptation. Evolutionary rescue is an important phenomenon at the intersection of ecology and population genetics, and the study of evolutionary rescue is critical to understanding processes ranging from species conservation to the evolution of drug and pesticide resistance. While most population-genetic models of evolutionary rescue focus on estimating the probability of rescue, we focus on whether one or more adaptive lineages contribute to evolutionary rescue. We find that when evolutionary rescue is likely, it is often driven by soft selective sweeps where multiple adaptive mutations spread through the population simultaneously. We give full analytic results for the probability of evolutionary rescue and the probability that evolutionary rescue occurs via soft selective sweeps. We expect that these results will find utility in understanding the genetic signatures associated with various evolutionary rescue scenarios in large populations, such as the evolution of drug resistance in viral, bacterial, or eukaryotic pathogens. PMID:28213477

  19. Adaptive mobility for rescue robots

    NASA Astrophysics Data System (ADS)

    Blitch, John G.

    2003-09-01

    It has often been observed that the most daunting aspect of any crisis response is managing the "unknown unknowns" that inevitably plague incident commanders and emergency personnel at all levels responsible for life and death decisions on a minute by minute basis. In structural collapse situations, for example, rescue crews rarely have even a coarse picture of the number or disposition of people or material scattered amongst the twisted beams and piles of concrete that typically entomb would-be survivors. How can the incident commander decide which beam to lift or even which section of the building to search first in the absence of information of what lies beneath. Even the slightest tug on a concrete slab can collapse potential life harboring void spaces below killing potential survivors in the process. In deploying mobile robots to assist in rescue operations we combined the traditional advantages of machine immunity to fatigue, hazardous materials and environmental controls, with the mechanical design freedom that allowed small platforms to penetrate deep into rubble to expand both situational awareness and operational influence of rescue services at the World Trade Center and mountainous snow-bound caves in Afghanistan. We learned a great deal from these experiences with regard to robot emloyment and design. This paper endeavors to share a few of our more prominent lessons learned regarding portable robot mobility as a means to manage user expectations and stimulate more innovative and adaptive design.

  20. Ligand-protein interactions of selective casein kinase 1δ inhibitors.

    PubMed

    Mente, Scot; Arnold, Eric; Butler, Todd; Chakrapani, Subramanyam; Chandrasekaran, Ramalakshmi; Cherry, Kevin; DiRico, Ken; Doran, Angela; Fisher, Katherine; Galatsis, Paul; Green, Michael; Hayward, Matthew; Humphrey, John; Knafels, John; Li, Jianke; Liu, Shenping; Marconi, Michael; McDonald, Scott; Ohren, Jeff; Paradis, Vanessa; Sneed, Blossom; Walton, Kevin; Wager, Travis

    2013-09-12

    Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.

  1. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis.

    PubMed

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-12-10

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1-CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis.

  2. The doublecortin and doublecortin-like kinase 1 genes cooperate in murine hippocampal development.

    PubMed

    Tanaka, Teruyuki; Koizumi, Hiroyuki; Gleeson, Joseph G

    2006-07-01

    The doublecortin (Dcx) and doublecortin-like kinase 1 (Dclk) genes are developmentally expressed neuronal microtubule-associated proteins. Humans with DCX mutations show a severe defect in hippocampal development, but targeted deletion in mouse shows only a defect in pyramidal neuron lamination. There is significant sequence overlap between Dcx and Dclk, suggesting functional redundancy. Here we show that the two genes display overlapping expression patterns in developing mouse hippocampus. Targeted deletion of Dclk shows no appreciable developmental defect in the hippocampus, but removal of both genes shows severe hippocampal lamination defects involving the entire cornu ammonis and dentate gyrus fields that mimic the human phenotype. These results suggest these genes are partially functionally redundant in the formation of the murine hippocampus.

  3. Downregulation of Death-Associated Protein Kinase 1 (DAPK1) in Chronic Lymphocytic Leukemia

    PubMed Central

    Raval, Aparna; Tanner, Stephan M.; Byrd, John C.; Angerman, Elizabeth B.; Perko, James D.; Chen, Shih-Shih; Hackanson, Björn; Grever, Michael R.; Lucas, David M.; Matkovic, Jennifer J.; Lin, Thomas S.; Kipps, Thomas J.; Murray, Fiona; Weisenburger, Dennis; Sanger, Warren; Lynch, Jane; Watson, Patrice; Jansen, Mary; Yoshinaga, Yuko; Rosenquist, Richard; de Jong, Pieter J.; Coggill, Penny; Beck, Stephan; Lynch, Henry; de la Chapelle, Albert; Plass, Christoph

    2007-01-01

    Summary The heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype. PMID:17540169

  4. Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

    SciTech Connect

    Hammond, Marlys; Washburn, David G.; Hoang, Tram H.; Manns, Sharada; Frazee, James S.; Nakamura, Hiroko; Patterson, Jaclyn R.; Trizna, Walter; Wu, Charlene; Azzarano, Leonard M.; Nagilla, Rakesh; Nord, Melanie; Trejo, Rebecca; Head, Martha S.; Zhao, Baoguang; Smallwood, Angela M.; Hightower, Kendra; Laping, Nicholas J.; Schnackenberg, Christine G.; Thompson, Scott K.

    2010-09-27

    The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {l_brace}4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl{r_brace}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.

  5. Reduced formation of advanced glycation endproducts via interactions between glutathione peroxidase 3 and dihydroxyacetone kinase 1.

    PubMed

    Lee, Hana; Chi, Seung Wook; Lee, Phil Young; Kang, Sunghyun; Cho, Sayeon; Lee, Chong-Kil; Bae, Kwang-Hee; Park, Byoung Chul; Park, Sung Goo

    2009-11-06

    Dihydroxyacetone (DHA) induces the formation of advanced glycation endproducts (AGEs), which are involved in several diseases. Earlier, we identified dihydroxyacetone kinase 1 (Dak1) as a candidate glutathione peroxidase 3 (Gpx3)-interacting protein in Saccharomyces cerevisiae. This finding is noteworthy, as no clear evidence on the involvement of oxidative stress systems in DHA-induced AGE formation has been found to date. Here, we demonstrate that Gpx3 interacts with Dak1, alleviates DHA-mediated stress by upregulating Dak activity, and consequently suppresses AGE formation. Based on these results, we propose that defense systems against oxidative stress and DHA-induced AGE formation are related via interactions between Gpx3 and Dak1.

  6. p21-Activated Kinase 1 Plays a Critical Role in Cellular Activation by Nef

    PubMed Central

    Fackler, Oliver T.; Lu, Xiaobin; Frost, Jeffrey A.; Geyer, Matthias; Jiang, Bing; Luo, Wen; Abo, Arie; Alberts, Arthur S.; Peterlin, B. Matija

    2000-01-01

    The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef. PMID:10713183

  7. Decoding Polo-like kinase 1 signaling along the kinetochore-centromere axis

    PubMed Central

    Lera, Robert F.; Potts, Gregory K.; Suzuki, Aussie; Johnson, James M.; Salmon, Edward D.; Coon, Joshua J.; Burkard, Mark E.

    2016-01-01

    Protein kinase signaling along the kinetochore-centromere axis is crucial to assure mitotic fidelity, yet its spatial coordination is obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore-centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection/resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1’s kinetochore functions. PMID:27043190

  8. The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 is involved in the perception of insect feeding

    PubMed Central

    2011-01-01

    The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 (LecRK1) has been recently identified as a component of the mechanism used by plants to suppress the Manduca sexta-triggered accumulation of salicylic acid (SA). The suppression of the SA burst by LecRK1 allows for the unfettered induction of jasmonic acid (JA)-mediated defense responses against M. sexta herbivory. LecRK1 contains a multi-domain extracellular region composed of a G-type Lectin domain and a PAN-AP domain separated by a variable sequence with low similarity to an EGF domain. The LecRK1 intracellular region is composed of a single domain structure with predicted Ser/Thr protein kinase activity. The multi-domain structure of the extracellular region of LecRK1 adds a level of complexity in terms of the potential ligands that this receptor protein could recognize. PMID:22105024

  9. [Peculiarities of phosphoglycerate kinase-1 pseudogene evolution in Schrenck salamander (Salamandrella schrenckii Strauch, 1870)].

    PubMed

    Malyarchuk, B A; Denisova, G A; Derenko, M V

    2013-07-01

    Processed copies of genes generally evolve in neutral mode as pseudogenes, however, some of them might be important sources of new functional genes. The psiPGK1 pseudogene has been discovered in Schrenck salamander (Salamandrella schrenckii, Amphibia, Caudata, Hynobiidae) via polymerase chain reaction used to amplify the phosphoglycerate kinase 1 gene (PGK1). This pseudogene is an intronless copy of PGK1 gene absent of exon 6. Analysis of psiPGK1 pseudogene polymorphism has demonstrated that it lacks mutations, which results in shifts in the stop codons and reading frames, as well as that the interspecies variation of this pseudogene was inconsistent with the neutral model of evolution. In addition, the pattern of phylogeographic differentiation of the psiPGK1 variants mainly coincides with that observed in mitochondrial DNA. These observations allow it to be suggested that the psiPGK1 pseudogene is a new functional gene in the Schrenck salamander.

  10. Greatwall and Polo-like Kinase 1 Coordinate to Promote Checkpoint Recovery*

    PubMed Central

    Peng, Aimin; Wang, Ling; Fisher, Laura A.

    2011-01-01

    Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G2/M DNA damage checkpoint and efficient checkpoint recovery. PMID:21708943

  11. Inhibition of Apoptosis-Regulated Signaling Kinase-1 and Prevention of Congestive Heart Failure by Estrogen

    PubMed Central

    Satoh, Minoru; Matter, Christian M.; Ogita, Hisakazu; Takeshita, Kyosuke; Wang, Chao-Yung; Dorn, Gerald W.; Liao, James K.

    2008-01-01

    Background Epidemiological studies have shown gender differences in the incidence of congestive heart failure (CHF); however, the role of estrogen in CHF is not known. We hypothesize that estrogen prevents cardiomyocyte apoptosis and the development of CHF. Methods and Results 17β-Estradiol (E2, 0.5 mg/60-day release) or placebo pellet was implanted subcutaneously into male Gαq transgenic (Gq) mice. After 8 weeks, E2 treatment decreased the extent of cardiac hypertrophy and dilation and improved contractility in Gq mice. E2 treatment also attenuated nicotinamide adenine dinucleotide phosphate oxidase activity and superoxide anion production via downregulation of Rac1. This correlated with reduced apoptosis in cardiomyocytes of Gq mice. The antioxidative properties of E2 were also associated with increased expression of thioredoxin (Trx), Trx reductases, and Trx reductase activity in the hearts of Gq mice. Furthermore, the activation of apoptosis signal-regulating kinase 1 and its downstream effectors, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, in the hearts of Gq mice was reduced by long-term E2 treatment. Indeed, E2 (10 nmol/L)-treated cardiomyocytes were much more resistant to angiotensin II–induced apoptosis. These antiapoptotic and cardioprotective effects of E2 were blocked by an estrogen receptor antagonist (ICI 182,780) and by a Trx reductase inhibitor (azelaic acid). Conclusions These findings indicate that long-term E2 treatment improves CHF by antioxidative mechanisms that involve the upregulation of Trx and inhibition of Rac1-mediated attenuated nicotinamide adenine dinucleotide phosphate oxidase activity and apoptosis signal-regulating kinase 1 /c-Jun N-terminal kinase/p38 mitogen-activated protein kinase–mediated apoptosis. These results suggest that estrogen may be a useful adjunctive therapy for patients with CHF. PMID:17562954

  12. Interactions between hematopoietic progenitor kinase 1 and its adaptor proteins (Review).

    PubMed

    Zhang, Qing; Ding, Shu; Zhang, Huilin

    2017-09-13

    Hematopoietic progenitor kinase 1 (HPK1), also known as mitogen‑activated protein kinase kinase kinase kinase 1 is a serine/threonine protein kinase. It is involved in various cellular events, including mitogen‑activated protein kinase signaling, nuclear factor‑κB signaling, cytokine signaling, cellular proliferation and apoptosis, T cell receptor/B cell receptor signaling and T/B/dendritic cell‑mediated immune responses. Therefore, HPK1 has variety of roles in immunity, and is associated with the pathogenesis of autoimmune diseases, cancer, and the inflammatory response. In these cellular and immune events, HPK1 interacts with several adaptor proteins, including caspase recruitment domain family, member 11, hematopoietic cell‑specific protein 1, HPK1‑interacting protein of 55 kDa, the growth factor receptor‑bound protein 2 family, linker for activated T‑cells, the SH2 domain‑containing leukocyte protein of 76 kDa family, the v‑crk avian sarcoma virus CT10 oncogene homolog family, B‑cell adaptor molecule of 32 kDa and non‑catalytic region of tyrosine kinase adaptor protein. These adaptor proteins can couple HPK1 with various effector molecules, leading to the transmission of upstream signals to downstream targets. They are crucial in regulating the relocation, phosphorylation, activation and functions of HPK1. HPK1 can also phosphorylate certain proteins, consequently modulating their functions. This review aims to describe the adaptor proteins, which interact with HPK1, particularly focusing on their modes of interaction with HPK1, and the effects that these interactions cause.

  13. Lunar mission safety and rescue: Escape/rescue analysis and plan

    NASA Technical Reports Server (NTRS)

    1971-01-01

    The results are presented of the technical analysis of escape/rescue/survival situations, crew survival techniques, alternate escape/rescue approaches and vehicles, and the advantages and disadvantages of each for advanced lunar exploration. Candidate escape/rescue guidelines are proposed and elements of a rescue plan developed. The areas of discussions include the following: lunar arrival/departure operations, lunar orbiter operations, lunar surface operations, lunar surface base escape/rescue analysis, lander tug location operations, portable airlock, emergency pressure suit, and the effects of no orbiting lunar station, no lunar surface base, and no foreign lunar orbit/surface operations on the escape/rescue plan.

  14. Search and rescue by satellite

    NASA Technical Reports Server (NTRS)

    Ehrlich, E.; Wilder, F. N.

    1978-01-01

    A system of satellites, ground stations and user equipments is proposed to provide an operational demonstration, using existing technology, for the timely detection and position location of general aviation aircraft and marine distress incidents so that rescue efforts can be started as soon as possible. The spaceborne equipment will consist of a transponder capable of receiving signals from existing and planned distress beacons at 121.5, 243.0 and 406.0 MHz, and transmitting at 1543 MHz. The program has generated international interest with Canada, France and the USSR presently planning to participate jointly with the U.S. in the development of the space and ground hardware.

  15. Battle of the eternal rivals: restoring functional p53 and inhibiting Polo-like kinase 1 as cancer therapy.

    PubMed

    Louwen, Frank; Yuan, Juping

    2013-07-01

    Polo-like kinase 1, a pivotal regulator of mitosis and cytokinesis, is highly expressed in a broad spectrum of tumors and its expression correlates often with poor prognosis, suggesting its potential as a therapeutic target. p53, the guardian of the genome, is the most important tumor suppressor. In this review, we address the intertwined relationship of these two key molecules by fighting each other as eternal rivals in many signaling pathways. p53 represses the promoter of Polo-like kinase 1, whereas Polo-like kinase 1 inhibits p53 and its family members p63 and p73 in cancer cells lacking functional p53. Plk1 inhibitors target all rapidly dividing cells irrespective of tumor cells or non-transformed normal but proliferating cells. Upon treatment with Plk1 inhibitors, p53 in tumor cells is activated and induces strong apoptosis, whereas tumor cells with inactive p53 arrest in mitosis with DNA damage. Thus, inactive p53 is not associated with a susceptible cytotoxicity of Polo-like kinase 1 inhibition and could rather foster the induction of polyploidy/aneuploidy in surviving cells. In addition, compared to the mono-treatment, combination of Polo-like kinase 1 inhibition with anti-mitotic or DNA damaging agents boosts more severe mitotic defects, effectually triggers apoptosis and strongly inhibits proliferation of cancer cells with functional p53. In this regard, restoration of p53 in tumor cells with loss or mutation of p53 will reinforce the cytotoxicity of combined Polo-like kinase 1 therapy and provide a proficient strategy for combating relapse and metastasis of cancer.

  16. Phaseolus immature embryo rescue technology.

    PubMed

    Geerts, Pascal; Toussaint, André; Mergeai, Guy; Baudoin, Jean-Pierre

    2011-01-01

    Predominant among the production constraints of the common bean Phaseolus vulgaris are infestation of Ascochyta blight, Bean Golden Mosaic virus (BGMV), and Bean Fly. Interbreeding with Phaseolus -coccineus L. and/or Phaseolus polyanthus Greenm has been shown to provide P. vulgaris with greater resistance to these diseases. For interspecific crosses to be successful, it is important to use P. coccineus and P. polyanthus as female parents; this prevents rapid reversal to the recurrent parent P. vulgaris. Although incompatibility barriers are post-zygotic, early hybrid embryo abortion limits the success of F1 crosses. While rescue techniques for globular and early heart-shaped embryos have improved in recent years, -success in hybridization remains very low. In this study, we describe six steps that allowed us to rescue 2-day-old P. vulgaris embryos using a pod culture technique. Our methods consisted of (i) pod culture, (ii) extraction and culture of immature embryos, (iii) dehydration of embryos, (iv) germination of embryos, (v) rooting of developed shoots, and (vi) hardening of plantlets.

  17. 46 CFR 133.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Stowage of rescue boats. 133.140 Section 133.140... SYSTEMS Requirements for All OSVs § 133.140 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each rescue boat...

  18. 46 CFR 133.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Stowage of rescue boats. 133.140 Section 133.140... SYSTEMS Requirements for All OSVs § 133.140 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each rescue boat...

  19. 46 CFR 133.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Stowage of rescue boats. 133.140 Section 133.140... SYSTEMS Requirements for All OSVs § 133.140 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each rescue boat...

  20. 46 CFR 133.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Stowage of rescue boats. 133.140 Section 133.140... SYSTEMS Requirements for All OSVs § 133.140 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each rescue boat...

  1. 46 CFR 133.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Stowage of rescue boats. 133.140 Section 133.140... SYSTEMS Requirements for All OSVs § 133.140 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each rescue boat...

  2. Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1.

    PubMed

    Pickar, Adrian; Zengel, James; Xu, Pei; Li, Zhuo; He, Biao

    2015-11-25

    The viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294. It has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1

    PubMed Central

    Pickar, Adrian; Zengel, James; Xu, Pei; Li, Zhuo

    2015-01-01

    ABSTRACT The viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294. IMPORTANCE It has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P. PMID:26608325

  4. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    PubMed

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  5. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    PubMed Central

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  6. Reduced activin receptor-like kinase 1 activity promotes cardiac fibrosis in heart failure.

    PubMed

    Morine, Kevin J; Qiao, Xiaoying; Paruchuri, Vikram; Aronovitz, Mark J; Mackey, Emily E; Buiten, Lyanne; Levine, Jonathan; Ughreja, Keshan; Nepali, Prerna; Blanton, Robert M; Oh, S Paul; Karas, Richard H; Kapur, Navin K

    2017-07-18

    Activin receptor-like kinase 1 (ALK1) mediates signaling via the transforming growth factor beta-1 (TGFβ1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. In patients with advanced heart failure referred for left ventricular (LV) assist device implantation, LV Alk1 mRNA and protein levels were lower than control LV obtained from patients without heart failure. To investigate the role of ALK1 in heart failure, Alk1 haploinsufficient (Alk1(+/-)) and wild-type (WT) mice were studied 2 weeks after severe transverse aortic constriction (TAC). LV and lung weights were higher in Alk1(+/-) mice after TAC. Cardiomyocyte area and LV mRNA levels of brain natriuretic peptide and β-myosin heavy chain were increased similarly in Alk1(+/-) and WT mice after TAC. Alk-1 mice exhibited reduced Smad 1 phosphorylation and signaling compared to WT mice after TAC. Compared to WT, LV fibrosis and Type 1 collagen mRNA and protein levels were higher in Alk1(+/-) mice. LV fractional shortening was lower in Alk1(+/-) mice after TAC. Reduced expression of ALK1 promotes cardiac fibrosis and impaired LV function in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication

    PubMed Central

    Pang, Jinke; Zhang, Geng; Lin, Yong; Xie, Zhanglian; Liu, Hongyan; Tang, Libo; Lu, Mengji; Yan, Ran; Guo, Haitao; Sun, Jian; Hou, Jinlin; Zhang, Xiaoyong

    2017-01-01

    Hepatitis B Virus (HBV) replication in hepatocytes is restricted by the host innate immune system and related intracellular signaling pathways. Transforming growth factor β-activated kinase 1 (TAK1) is a key mediator of toll-like receptors and pro-inflammatory cytokine signaling pathways. Here, we report that silencing or inhibition of endogenous TAK1 in hepatoma cell lines leads to an upregulation of HBV replication, transcription, and antigen expression. In contrast, overexpression of TAK1 significantly suppresses HBV replication, while an enzymatically inactive form of TAK1 exerts no effect. By screening TAK1-associated signaling pathways with inhibitors and siRNAs, we found that the MAPK-JNK pathway was involved in TAK1-mediated HBV suppression. Moreover, TAK1 knockdown or JNK pathway inhibition induced the expression of farnesoid X receptor α, a transcription factor that upregulates HBV transcription. Finally, ectopic expression of TAK1 in a HBV hydrodynamic injection mouse model resulted in lower levels of HBV DNA and antigens in both liver and serum. In conclusion, our data suggest that TAK1 inhibits HBV primarily at viral transcription level through activation of MAPK-JNK pathway, thus TAK1 represents an intrinsic host restriction factor for HBV replication in hepatocytes. PMID:28045080

  8. Adipocyte lipolysis-stimulated interleukin-6 production requires sphingosine kinase 1 activity.

    PubMed

    Zhang, Wenliang; Mottillo, Emilio P; Zhao, Jiawei; Gartung, Allison; VanHecke, Garrett C; Lee, Jen-Fu; Maddipati, Krishna R; Xu, Haiyan; Ahn, Young-Hoon; Proia, Richard L; Granneman, James G; Lee, Menq-Jer

    2014-11-14

    Adipocyte lipolysis can increase the production of inflammatory cytokines such as interleukin-6 (IL-6) that promote insulin resistance. However, the mechanisms that link lipolysis with inflammation remain elusive. Acute activation of β3-adrenergic receptors (ADRB3) triggers lipolysis and up-regulates production of IL-6 in adipocytes, and both of these effects are blocked by pharmacological inhibition of hormone-sensitive lipase. We report that stimulation of ADRB3 induces expression of sphingosine kinase 1 (SphK1) and increases sphingosine 1-phosphate production in adipocytes in a manner that also depends on hormone-sensitive lipase activity. Mechanistically, we found that adipose lipolysis-induced SphK1 up-regulation is mediated by the c-Jun N-terminal kinase (JNK)/activating protein-1 signaling pathway. Inhibition of SphK1 by sphingosine kinase inhibitor 2 diminished the ADRB3-induced IL-6 production both in vitro and in vivo. Induction of IL-6 by ADRB3 activation was suppressed by siRNA knockdown of Sphk1 in cultured adipocytes and was severely attenuated in Sphk1 null mice. Conversely, ectopic expression of SphK1 increased IL-6 expression in adipocytes. Collectively, these data demonstrate that SphK1 is a critical mediator in lipolysis-triggered inflammation in adipocytes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Casein kinase 1 is recruited to nuclear speckles by FAM83H and SON

    PubMed Central

    Kuga, Takahisa; Kume, Hideaki; Adachi, Jun; Kawasaki, Naoko; Shimizu, Maiko; Hoshino, Isamu; Matsubara, Hisahiro; Saito, Youhei; Nakayama, Yuji; Tomonaga, Takeshi

    2016-01-01

    In some fibroblasts, casein kinase 1α (CK1α) is localized to nuclear speckles, which are sub-nuclear compartments supplying splicing factors, whereas it is recruited on keratin filaments in colorectal cancer cells such as DLD1 cells. In order to obtain a deeper understanding of why CK1α is localized to these different subcellular sites, we herein elucidated the mechanisms underlying its localization to nuclear speckles. CK1α and FAM83H were localized to nuclear speckles in RKO and WiDr colorectal cancer cells, which do not express simple epithelial keratins, and in DLD1 cells transfected with siRNAs for type I keratins. The localization of FAM83H to nuclear speckles was also detected in colorectal cancer cells with a poorly organized keratin cytoskeleton in colorectal cancer tissues. Using an interactome analysis of FAM83H, we identified SON, a protein present in nuclear speckles, as a scaffold protein to which FAM83H recruits CK1α. This result was supported by the knockdown of FAM83H or SON delocalizing CK1α from nuclear speckles. We also found that CK1δ and ε are localized to nuclear speckles in a FAM83H-dependent manner. These results suggest that CK1 is recruited to nuclear speckles by FAM83H and SON in the absence of an intact keratin cytoskeleton. PMID:27681590

  10. Casein kinase 1 α phosphorylates the Wnt regulator Jade-1 and modulates its activity.

    PubMed

    Borgal, Lori; Rinschen, Markus M; Dafinger, Claudia; Hoff, Sylvia; Reinert, Matthäus J; Lamkemeyer, Tobias; Lienkamp, Soeren S; Benzing, Thomas; Schermer, Bernhard

    2014-09-19

    Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.

  11. Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Sumpio, B. E.

    1998-01-01

    We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain. Copyright 1998 Academic Press.

  12. Identification of Polo-like kinase 1 interaction inhibitors using a novel cell-based assay

    PubMed Central

    Normandin, Karine; Lavallée, Jean-François; Futter, Marie; Beautrait, Alexandre; Duchaine, Jean; Guiral, Sébastien; Marinier, Anne; Archambault, Vincent

    2016-01-01

    Polo-like kinase 1 (Plk1) plays several roles in cell division and it is a recognized cancer drug target. Plk1 levels are elevated in cancer and several types of cancer cells are hypersensitive to Plk1 inhibition. Small molecule inhibitors of the kinase domain (KD) of Plk1 have been developed. Their selectivity is limited, which likely contributes to their toxicity. Polo-like kinases are characterized by a Polo-Box Domain (PBD), which mediates interactions with phosphorylation substrates or regulators. Inhibition of the PBD could allow better selectivity or result in different effects than inhibition of the KD. In vitro screens have been used to identify PBD inhibitors with mixed results. We developed the first cell-based assay to screen for PBD inhibitors, using Bioluminescence Resonance Energy Transfer (BRET). We screened through 112 983 compounds and characterized hits in secondary biochemical and biological assays. Subsequent Structure-Activity Relationship (SAR) analysis on our most promising hit revealed that it requires an alkylating function for its activity. In addition, we show that the previously reported PBD inhibitors thymoquinone and Poloxin are also alkylating agents. Our cell-based assay is a promising tool for the identification of new PBD inhibitors with more drug-like profiles using larger and more diverse chemical libraries. PMID:27874094

  13. Protein receptor for activated C kinase 1 is involved in morphine reward in mice.

    PubMed

    Wan, L; Su, L; Xie, Y; Liu, Y; Wang, Y; Wang, Z

    2009-07-07

    Opiate addiction is associated with upregulation of cAMP signaling in the brain. cAMP-responsive element binding protein (CREB), a nuclear transcription factor, is a downstream component of the extracellular signal-regulated protein kinase (ERK) pathway, which has been shown to regulate different physiological and psychological responses of drug addiction. RACK1, the protein receptor for activated C kinase 1, is a multifunctional scaffolding protein known to be a key regulator of various signaling cascades in the CNS. RACK1 functions specifically in integrin mediated activation of ERK cascade and targets active ERK. We examined if RACK1 is involved in the mechanism of drug addiction by regulating CREB in mouse hippocampus and prefrontal cortex. Several expressions were observed. Chronic administration of morphine made the expression of RACK1 and CREB mRNA increase in hippocampus and prefrontal cortex. The expression of RACK1 and CREB protein was strongly positive in CA1, CA3 and dentate gyrus (DG) of the hippocampus of morphine-treated mice brain, especially the pyramidal neurons in the DG of the hippocampus. Using the small interfering RNA technology, we determined that the expression of CREB mRNA was decreased in hippocampus and prefrontal cortex of morphine-treated mice. The expression of RACK1 and CREB protein was negative in CA1, CA3 and DG of hippocampus. These findings suggest that morphine reward can influence the expression of RACK1 in mouse hippocampus and prefrontal cortex through regulating CREB transcription.

  14. Design of Targeted Inhibitors of Polo-like Kinase 1 (Plk1)

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    2011-03-01

    Computational design of small molecule inhibitors of Polo-like Kinase 1 (Plk1) is presented. Plk1, which regulates cell cycle, is often overexpressed in cancers. Its downregulation was shown to inhibit cancer progression. Most inhibitors of kinases' interact with the highly conserved ATP binding site. This makes the development of Plk1-specific inhibitors challenging, since different kinases have similar ATP sites. However, Plk1 also contains the polo-box domain (PBD), which is absent from other kinases. In this study, the PBD site was used as a target for designed Plk1 inhibitors. Common structural features of experimentally known Plk1 ligands were first identified. The information was used to design putative small molecules that specifically bonded Plk1. Druglikeness and possible toxicities of the designed molecules were determined. Molecules with no implied toxicities and optimal druglikeness were used for docking studies. The docking studies identified several molecules that made stable complexes with the Plk1 PBD site. Possible utilization of the designed molecules in drugs against cancers with overexpressed Plk1 is discussed.

  15. Putting a bit into the polo-box domain of polo-like kinase 1.

    PubMed

    Park, Jung-Eun; Kim, Tae-Sung; Meng, Lingjun; Bang, Jeong K; Kim, Bo Y; Lee, Kyung S

    Polo-like kinase 1 (Plk1) plays key roles in regulating various mitotic processes that are critical for cellular proliferation. A growing body of evidence suggests that Plk1 overexpression is tightly associated with the development of human cancers. Interestingly, various types of cancer cells are shown to be addicted to a high level of Plk1, and the reversal of Plk1 addiction appears to be an effective strategy for selectively killing cancer cells, but not normal cells. Therefore, Plk1 is considered an attractive anticancer drug target. Over the years, a large number of inhibitors that target the catalytic activity of Plk1 have been developed. However, these inhibitors exhibit significant levels of cross-reactivity with related kinases, including Plk2 and Plk3. Consequently, as an alternative approach for developing anti-Plk1 therapeutics, substantial effort is under way to develop inhibitors that target the C-terminal protein-protein interaction domain of Plk1, called the polo-box domain (PBD). In this communication, I will discuss the pros and cons of targeting the PBD in comparison to those of targeting the ATP-binding site within the kinase domain.

  16. Designed inhibitor for nuclear localization signal of polo-like kinase 1 induces mitotic arrest.

    PubMed

    Chen, Fangjin; Zhuo, Xiaolong; Qin, Tan; Guo, Xiao; Zhang, Chuanmao; Lai, Luhua

    2016-11-24

    Polo-like kinase 1 (Plk1), a member of polo-like kinase family, regulates multiple essential steps of the cell cycle progression. Plk1 is overexpressed in multiple cancer cell lines and considered to be a prime anticancer target. Plk1 accumulates in the nucleus during S and G2 phases by its bipartite nuclear localization signal (NLS) sequence, which is crucial for Plk1 regulation during normal cell cycle progression. Here, through combined computational and experimental studies, we identified compound D110, which inhibits Plk1 kinase activity with an IC50 of 85 nm and blocks the nuclear localization of Plk1 during S and G2 phases. D110-treated cancer cells were arrested at mitosis with monopolar spindle, indicating the inhibition of the Plk1 kinase activity in cell. As D110 interacts with both the ATP site and the NLS in Plk1, it demonstrates good selectivity toward Plk2 and Plk3. The strategy of simultaneously inhibiting kinase activity and its subcellular translocations offers a novel approach for selective kinase inhibitor design.

  17. Discovery of Non-ATP-Competitive Inhibitors of Polo-like Kinase 1.

    PubMed

    Yun, Taikangxiang; Qin, Tan; Liu, Ying; Lai, Luhua

    2016-04-05

    Polo-like kinase 1 (Plk1) is an evolutionarily conserved serine/threonine kinase, and its N-terminal kinase domain (KD) controls cell signaling through phosphorylation. Inhibitors of Plk1 are potential anticancer drugs. Most known Plk1 KD inhibitors are ATP-competitive compounds, which may suffer from low selectivity. In this study we discovered novel non-ATP-competitive Plk1 KD inhibitors by virtual screening and experimental studies. Potential binding sites in Plk1 KD were identified by using the protein binding site detection program Cavity. The identified site was subjected to molecular-docking-based virtual screening. The activities of top-ranking compounds were evaluated by in vitro enzyme assay with full-length Plk1 and direct binding assay with Plk1 KD. Several compounds showed inhibitory activity, and the most potent was found to be 3-((2-oxo-2-(thiophen-2-yl)ethyl)thio)-6-(pyridin-3-ylmethyl)-1,2,4-triazin-5(4H)-one (compound 4) with an IC50 value of 13.1 ± 1.7 μm. Our work provides new insight into the design of kinase inhibitors that target non-ATP binding sites.

  18. Doublecortin-like kinase 1-positive enterocyte - a new cell type in human intestine.

    PubMed

    Leppänen, Joni; Helminen, Olli; Huhta, Heikki; Kauppila, Joonas H; Miinalainen, Ilkka; Ronkainen, Veli-Pekka; Saarnio, Juha; Lehenkari, Petri P; Karttunen, Tuomo J

    2016-11-01

    Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease.

  19. Soluble fms-like tyrosine kinase 1 promotes angiotensin II sensitivity in preeclampsia

    PubMed Central

    Burke, Suzanne D.; Zsengellér, Zsuzsanna K.; Khankin, Eliyahu V.; Lo, Agnes S.; Rajakumar, Augustine; DuPont, Jennifer J.; McCurley, Amy; Moss, Mary E.; Zhang, Dongsheng; Clark, Christopher D.; Seely, Ellen W.; Kang, Peter M.; Stillman, Isaac E.; Jaffe, Iris Z.

    2016-01-01

    Preeclampsia is a hypertensive disorder of pregnancy in which patients develop profound sensitivity to vasopressors, such as angiotensin II, and is associated with substantial morbidity for the mother and fetus. Enhanced vasoconstrictor sensitivity and elevations in soluble fms-like tyrosine kinase 1 (sFLT1), a circulating antiangiogenic protein, precede clinical signs and symptoms of preeclampsia. Here, we report that overexpression of sFlt1 in pregnant mice induced angiotensin II sensitivity and hypertension by impairing endothelial nitric oxide synthase (eNOS) phosphorylation and promoting oxidative stress in the vasculature. Administration of the NOS inhibitor l-NAME to pregnant mice recapitulated the angiotensin sensitivity and oxidative stress observed with sFlt1 overexpression. Sildenafil, an FDA-approved phosphodiesterase 5 inhibitor that enhances NO signaling, reversed sFlt1-induced hypertension and angiotensin II sensitivity in the preeclampsia mouse model. Sildenafil treatment also improved uterine blood flow, decreased uterine vascular resistance, and improved fetal weights in comparison with untreated sFlt1-expressing mice. Finally, sFLT1 protein expression inversely correlated with reductions in eNOS phosphorylation in placental tissue of human preeclampsia patients. These data support the concept that endothelial dysfunction due to high circulating sFLT1 may be the primary event leading to enhanced vasoconstrictor sensitivity that is characteristic of preeclampsia and suggest that targeting sFLT1-induced pathways may be an avenue for treating preeclampsia and improving fetal outcomes. PMID:27270170

  20. Application of phosphoproteomics to find targets of casein kinase 1 in the flagellum of chlamydomonas.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum.

  1. Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. PMID:23316220

  2. Sphingosine kinase 1 (SK1) allosteric inhibitors that target the dimerization site.

    PubMed

    Bayraktar, Ozge; Ozkirimli, Elif; Ulgen, Kutlu

    2017-08-01

    The sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) signaling pathway is a crucial target for numerous human diseases from cancer to cardiovascular diseases. However, available SK1 inhibitors that target the active site suffer from poor potency, selectivity and pharmacokinetic properties. The selectivity issue of the kinases, which share a highly-conserved ATP-pocket, can be overcome by targeting the less-conserved allosteric sites. SK1 is known to function minimally as a dimer; however, the crystal structure of the SK1 dimer has not been determined. In this study, a template-based algorithm implemented in PRISM was used to predict the SK1 dimer structure and then the possible allosteric sites at the dimer interface were determined via SiteMap. These sites were used in a virtual screening campaign that includes an integrated workflow of structure-based pharmacophore modeling, virtual screening, molecular docking, re-screening of common scaffolds to propose a series of compounds with different scaffolds as potential allosteric SK1 inhibitors. Finally, the stability of the SK1-ligand complexes was analyzed by molecular dynamics simulations. As a final outcome, ligand 7 having a 4,9-dihydro-1H-purine scaffold and ligand 12 having a 2,3,4,9-tetrahydro-1H-β-carboline scaffold were found to be potential selective inhibitors for SK1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

    PubMed Central

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-01-01

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis. PMID:26656453

  4. Casein Kinase 1 Functions as both Penultimate and Ultimate Kinase in Regulating Cdc25A Destruction

    PubMed Central

    Honaker, Yuchi; Piwnica-Worms, Helen

    2010-01-01

    The Cdc25A protein phosphatase drives cell cycle transitions by activating cyclin-dependent protein kinases. Failure to regulate Cdc25A leads to deregulated cell cycle progression, bypass of cell cycle checkpoints and genome instability. Ubiquitin-mediated proteolysis plays an important role in balancing Cdc25A levels. Cdc25A contains a DS82G motif whose phosphorylation is targeted by β-TrCP E3 ligase during interphase. Targeting of β-TrCP to Cdc25A requires phosphorylation of serines 79 (S79) and 82 (S82). Here, we report that casein kinase 1 alpha (CK1α) phosphorylates Cdc25A on both S79 and S82 in a hierarchical manner requiring prior phosphorylation of serine 76 by Chk1 or GSK-3β. This facilitates β-TrCP binding and ubiquitin-mediated proteolysis of Cdc25A throughout interphase and following exposure to genotoxic stress. The priming of Cdc25A by at least three kinases (Chk1, GSK-3β, CK1α), some of which also require priming, ensures diverse extra- and intra-cellular signals interface with Cdc25A to precisely control cell division. PMID:20348946

  5. Inhibition of glycogen synthase (casein) kinase-1 by different divalent metal ions

    SciTech Connect

    Singh, T.J.

    1987-05-01

    The specificity of glycogen synthase (casein) kinase-1 (CK-1) for different divalent metal ions was explored in this study. Of the eight metal ions (Mg, Mn, Zn, Cu, Ca, Ni, Co, Fe) tested only Mg/sup 2 +/ supported significant kinase activity. Several of the other metals, however, inhibited the Mg/sup 2 +/-stimulated kinase activity. Half-maximal inhibition by Zn, Mn, Co, Fe and Ni were observed at 50, 55, 110, 125 and 300 ..mu..M, respectively. Kinetic analyses using Zn/sup 2 +/ indicate that at a constant (ATP) Zn/sup 2 +/ increases the K/sub m/ for casein, the V/sub max/ being unaffected +/- Zn/sup 2 +/. At a constant (casein), Zn/sup 2 +/ decreased the V/sub max/ for ATP, the K/sub m/ remaining unchanged +/- Zn/sup 2 +/. The inhibition of CK-1 by the different metals is reversed by EGTA or EDTA. These results suggest that divalent metal ions such as Zn/sup 2 +/ and Mn/sup 2 +/ that do not support CK-1 activity may serve as inhibitors of the kinase in vivo.

  6. Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Sumpio, B. E.

    1998-01-01

    We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain. Copyright 1998 Academic Press.

  7. Down-regulation of soluble fms-like tyrosine kinase 1 expression in invasive placentation.

    PubMed

    Shainker, Scott A; Dannheim, Katelyn; Gerson, Kristin D; Neo, Dayna; Zsengeller, Zsuzsanna K; Pernicone, Elizabeth; Karumanchi, S Ananth; Hacker, Michele R; Hecht, Jonathan L

    2017-08-01

    To confirm reduced expression of soluble fms-like tyrosine kinase 1 (sFlt-1) in accreta/increta. Formalin-fixed tissue sections from 11 peripartum hysterectomies with invasive placentation and 5 controls were stained for sFlt-1. Stain intensity was scored in selected 100× microscopic fields. We compared sFlt-1 expression in invasive areas among cases, non-invasive areas among cases and areas from control placentas. Chorionic villi displayed significantly decreased sFlt-1 expression in invasive areas of cases compared to control placentas (p = 0.003), as well as in non-invasive areas of cases compared to control placentas (p = 0.01). There was no difference in sFlt-1 expression between invasive and non-invasive areas among cases. Expression of sFlt-1 is diminished in villous trophoblasts from patients with placenta increta or percreta. Local depth of invasion was not associated with sFlt-1 expression, suggesting a more global abnormality across the implantation site rather than localized to areas of histologic invasion.

  8. Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1.

    PubMed

    Feldman, Richard I; Wu, James M; Polokoff, Mark A; Kochanny, Monica J; Dinter, Harald; Zhu, Daguang; Biroc, Sandra L; Alicke, Bruno; Bryant, Judi; Yuan, Shendong; Buckman, Brad O; Lentz, Dao; Ferrer, Mike; Whitlow, Marc; Adler, Marc; Finster, Silke; Chang, Zheng; Arnaiz, Damian O

    2005-05-20

    The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.

  9. SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

    PubMed

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-02-06

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.

  10. Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.

    PubMed

    Davidson, Gary; Wu, Wei; Shen, Jinlong; Bilic, Josipa; Fenger, Ursula; Stannek, Peter; Glinka, Andrei; Niehrs, Christof

    2005-12-08

    Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.

  11. Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2.

    PubMed

    McAlpine, Fiona; Williamson, Leon E; Tooze, Sharon A; Chan, Edmond Y W

    2013-03-01

    Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that occurs constitutively in cells, but can also be induced by stressors such as nutrient starvation or protein aggregation. Autophagy has been implicated in multiple disease mechanisms including neurodegeneration and cancer, with both tumor suppressive and oncogenic roles. Uncoordinated 51-like kinase 1 (ULK1) is a critical autophagy protein near the apex of the hierarchal regulatory pathway that receives signals from the master nutrient sensors MTOR and AMP-activated protein kinase (AMPK). In mammals, ULK1 has a close homolog, ULK2, although their functional distinctions have been unclear. Here, we show that ULK1 and ULK2 both function to support autophagy activation following nutrient starvation. Increased autophagy following amino acid or glucose starvation was disrupted only upon combined loss of ULK1 and ULK2 in mouse embryonic fibroblasts. Generation of PtdIns3P and recruitment of WIPI2 or ZFYVE1/DFCP1 to the phagophore following amino acid starvation was blocked by combined Ulk1/2 double knockout. Autophagy activation following glucose starvation did not involve recruitment of either WIPI1 or WIPI2 to forming autophagosomes. Consistent with a PtdIns3P-independent mechanism, glucose-dependent autophagy was resistant to wortmannin. Our findings support functional redundancy between ULK1 and ULK2 for nutrient-dependent activation of autophagy and furthermore highlight the differential pathways that respond to amino acid and glucose deprivation.

  12. Pathological Role of Serum- and Glucocorticoid-Regulated Kinase 1 in Adverse Ventricular Remodeling

    PubMed Central

    Das, Saumya; Aiba, Takeshi; Rosenberg, Michael; Hessler, Katherine; Xiao, Chunyang; Quintero, Pablo A.; Ottaviano, Filomena G.; Knight, Ashley C.; Graham, Evan L.; Boström, Pontus; Morissette, Michael R.; del Monte, Federica; Begley, Michael J.; Cantley, Lewis C.; Ellinor, Patrick T.; Tomaselli, Gordon F.; Rosenzweig, Anthony

    2012-01-01

    Background Heart failure is a growing cause of morbidity and mortality. Cardiac PI3-kinase signaling promotes cardiomyocyte survival and function but is paradoxically activated in heart failure, suggesting chronic activation of this pathway may become maladaptive. Here we investigated the downstream PI3-kinase effector, SGK1 (serum- and glucocorticoid-regulated kinase-1), in heart failure and its complications. Methods and Results We found that cardiac SGK1 is activated in human and murine heart failure. We investigated the role of SGK1 in the heart using cardiac-specific expression of constitutively-active or dominant-negative SGK1. Cardiac-specific activation of SGK1 in mice increased mortality, cardiac dysfunction, and ventricular arrhythmias. The pro-arrhythmic effects of SGK1 were linked to biochemical and functional changes in the cardiac sodium channel and could be reversed by treatment with ranolazine, a blocker of the late sodium current. Conversely, cardiac-specific inhibition of SGK1 protected mice after hemodynamic stress from fibrosis, heart failure, and sodium channel alterations. Conclusions SGK1 appears both necessary and sufficient for key features of adverse ventricular remodeling and may provide a novel therapeutic target in cardiac disease. PMID:23019294

  13. Casein Kinase 1 Coordinates Cohesin Cleavage, Gametogenesis, and Exit from M Phase in Meiosis II.

    PubMed

    Argüello-Miranda, Orlando; Zagoriy, Ievgeniia; Mengoli, Valentina; Rojas, Julie; Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Zachariae, Wolfgang

    2017-01-09

    Meiosis consists of DNA replication followed by two consecutive nuclear divisions and gametogenesis or spore formation. While meiosis I has been studied extensively, less is known about the regulation of meiosis II. Here we show that Hrr25, the conserved casein kinase 1δ of budding yeast, links three mutually independent key processes of meiosis II. First, Hrr25 induces nuclear division by priming centromeric cohesin for cleavage by separase. Hrr25 simultaneously phosphorylates Rec8, the cleavable subunit of cohesin, and removes from centromeres the cohesin protector composed of shugoshin and the phosphatase PP2A. Second, Hrr25 initiates the sporulation program by inducing the synthesis of membranes that engulf the emerging nuclei at anaphase II. Third, Hrr25 mediates exit from meiosis II by activating pathways that trigger the destruction of M-phase-promoting kinases. Thus, Hrr25 synchronizes formation of the single-copy genome with gamete differentiation and termination of meiosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Serum- and glucocorticoid-inducible kinase 1 in the regulation of renal and extrarenal potassium transport.

    PubMed

    Lang, Florian; Vallon, Volker

    2012-02-01

    Serum- and glucocorticoid inducible-kinase 1 (SGK1) is an early gene transcriptionally upregulated by cell stress such as cell shrinkage and hypoxia and several hormones including gluco- and mineralocorticoids. It is activated by insulin and growth factors. SGK1 is a powerful regulator of a wide variety of channels and transporters. The present review describes the role of SGK1 in the regulation of potassium (K(+)) channels, K(+) transporters and K(+) homeostasis. SGK1-regulated K(+) channels include renal outer medullary K+ channel, Kv1.3, Kv1.5, KCNE1/KCNQ1, KCNQ4 and, via regulation of calcium (Ca(2+)) entry, Ca(2+)-sensitive K(+) channels. SGK1-sensitive transporters include sodium-potassium-chloride cotransporter 2 and sodium/potassium-adenosine triphosphatase. SGK1-dependent regulation of K(+) channels and K(+) transport contributes to the stimulation of renal K(+) excretion following high K(+) intake, to insulin-induced cellular K(+) uptake and hypokalemia, to inhibition of insulin release by glucocorticoids, to stimulation of mast cell degranulation and gastric acid secretion, and to cardiac repolarization. Thus, SGK1 has a profound effect on K(+) homeostasis and on a multitude of K(+)-sensitive cellular functions.

  15. Comparative aspects of the proliferation marker thymidine kinase 1 in human and canine tumour diseases.

    PubMed

    von Euler, H; Eriksson, S

    2011-03-01

    As cell proliferation is one of the hallmarks of cancer, various types of proliferation markers are used as important tools in diagnosis, prognosis, treatment decision-making and follow-up in clinical oncology. The S phase-specific protein thymidine kinase 1 (TK1) can be used in immunohistochemistry for RNA/protein expression in tissue specimens and for activity or protein/peptide levels in serum from patients. TK1 has been used mainly in haematologic malignancies in humans, but also found beneficial in canine malignancies. As the protein sequence homology is high between humans and dogs, findings in canine models will have a high comparative value in further human research as well. In the present review, we will focus on the recent results concerning TK1's S phase-correlated expression, increased serum levels of TK1 in patients with malignancies and the relevance for veterinary and comparative oncology. Finally, the benefit of recently developed specific anti-TK1 antibodies suitable for immunologic assay is discussed.

  16. Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

    SciTech Connect

    Boel, Gaby-Fleur . E-mail: boel@mail.dife.de; Jurrmann, Nadine; Brigelius-Flohe, Regina

    2005-06-24

    Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4{sup IRAK-1-K239S}). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24 kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4{sup IRAK-1-K239S} cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate.

  17. Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity*

    PubMed Central

    Borgal, Lori; Rinschen, Markus M.; Dafinger, Claudia; Hoff, Sylvia; Reinert, Matthäus J.; Lamkemeyer, Tobias; Lienkamp, Soeren S.; Benzing, Thomas; Schermer, Bernhard

    2014-01-01

    Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner. PMID:25100726

  18. SUMOylation Regulates Polo-like Kinase 1-interacting Checkpoint Helicase (PICH) during Mitosis*

    PubMed Central

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-01-01

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres. PMID:25564610

  19. Death-associated protein kinase 1 promotes growth of p53-mutant cancers.

    PubMed

    Zhao, Jing; Zhao, Dekuang; Poage, Graham M; Mazumdar, Abhijit; Zhang, Yun; Hill, Jamal L; Hartman, Zachary C; Savage, Michelle I; Mills, Gordon B; Brown, Powel H

    2015-07-01

    Estrogen receptor-negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor-negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.

  20. An integrative study to identify novel scaffolds for sphingosine kinase 1 inhibitors.

    PubMed

    Vettorazzi, Marcela; Angelina, Emilio; Lima, Santiago; Gonec, Tomas; Otevrel, Jan; Marvanova, Pavlina; Padrtova, Tereza; Mokry, Petr; Bobal, Pavel; Acosta, Lina M; Palma, Alirio; Cobo, Justo; Bobalova, Janette; Csollei, Jozef; Malik, Ivan; Alvarez, Sergio; Spiegel, Sarah; Jampilek, Josef; Enriz, Ricardo D

    2017-10-20

    Sphingosine kinase 1 (SphK1), the enzyme that produces the bioactive sphingolipid metabolite, sphingosine-1-phosphate, is a promising new molecular target for therapeutic intervention in cancer and inflammatory diseases. In view of its importance, the main objective of this work was to find new and more potent inhibitors for this enzyme possessing different structural scaffolds than those of the known inhibitors. Our theoretical and experimental study has allowed us to identify two new structural scaffolds (three new compounds), which could be used as starting structures for the design and then the development of new inhibitors of SphK1. Our study was carried out in different steps: virtual screening, synthesis, bioassays and molecular modelling. From our results, we propose a new dihydrobenzo[b]pyrimido[5,4-f]azepine and two alkyl{3-/4-[1-hydroxy-2-(4-arylpiperazin-1-yl)ethyl]phenyl}carbamates as initial structures for the development of new inhibitors. In addition, our molecular modelling study using QTAIM calculations, allowed us to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analyses indicate that the cationic head of the different compounds must be refined in order to obtain an increase in the binding affinity of these ligands. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. Polo-like kinase-1 regulates kinetochore–microtubule dynamics and spindle checkpoint silencing

    PubMed Central

    Liu, Dan; Davydenko, Olga

    2012-01-01

    Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore–microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore–microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore–microtubule attachments and subsequently silencing the spindle checkpoint. PMID:22908307

  2. The activity regulation of the mitotic centromere-associated kinesin by Polo-like kinase 1.

    PubMed

    Ritter, Andreas; Sanhaji, Mourad; Steinhäuser, Kerstin; Roth, Susanne; Louwen, Frank; Yuan, Juping

    2015-03-30

    The mitotic centromere-associated kinesin (MCAK), a potent microtubule depolymerase, is involved in regulating microtubule dynamics. The activity and subcellular localization of MCAK are tightly regulated by key mitotic kinases, such as Polo-like kinase 1 (Plk1) by phosphorylating multiple residues in MCAK. Since Plk1 phosphorylates very often different residues of substrates at different stages, we have dissected individual phosphorylation of MCAK by Plk1 and characterized its function in more depth. We have recently shown that S621 in MCAK is the major phosphorylation site of Plk1, which is responsible for regulating MCAK's degradation by promoting the association of MCAK with APC/CCdc20. In the present study, we have addressed another two residues phosphorylated by Plk1, namely S632/S633 in the C-terminus of MCAK. Our data suggest that Plk1 phosphorylates S632/S633 and regulates its catalytic activity in mitosis. This phosphorylation is required for proper spindle assembly during early phases of mitosis. The subsequent dephosphorylation of S632/S633 might be necessary to timely align the chromosomes onto the metaphase plate. Therefore, our studies suggest new mechanisms by which Plk1 regulates MCAK: the degradation of MCAK is controlled by Plk1 phosphorylation on S621, whereas its activity is modulated by Plk1 phosphorylation on S632/S633 in mitosis.

  3. Targeted knockdown of polo-like kinase 1 alters metabolic regulation in melanoma.

    PubMed

    Gutteridge, Rosie Elizabeth Ann; Singh, Chandra K; Ndiaye, Mary Ann; Ahmad, Nihal

    2017-05-28

    A limited number of studies have indicated an association of the mitotic kinase polo-like kinase 1 (PLK1) and cellular metabolism. Here, employing an inducible RNA interference approach in A375 melanoma cells coupled with a PCR array and multiple validation approaches, we demonstrated that PLK1 alters a number of genes associated with cellular metabolism. PLK1 knockdown resulted in a significant downregulation of IDH1, PDP2 and PCK1 and upregulation of FBP1. Ingenuity Pathway Analysis (IPA) identified that 1) glycolysis and the pentose phosphate pathway are major canonical pathways associated with PLK1, and 2) PLK1 inhibition-modulated genes were largely associated with cellular proliferation, with FBP1 being the key modulator. Further, BI 6727-mediated inhibition of PLK1 caused a decrease in PCK1 and increase in FBP1 in A375 melanoma cell implanted xenografts in vivo. Furthermore, an inverse correlation between PLK1 and FBP1 was found in melanoma cells, with FBP1 expression significantly downregulated in a panel of melanoma cells. In addition, BI 6727 treatment resulted in an upregulation in FBP1 in A375, Hs294T and G361 melanoma cells. Overall, our study suggests that PLK1 may be an important regulator of metabolism maintenance in melanoma cells. Published by Elsevier B.V.

  4. Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants

    PubMed Central

    Hsieh, Louise Tzung-Harn; Nastase, Madalina-Viviana; Roedig, Heiko; Zeng-Brouwers, Jinyang; Poluzzi, Chiara; Schwalm, Stephanie; Fork, Christian; Tredup, Claudia; Brandes, Ralf P.; Wygrecka, Malgorzata; Huwiler, Andrea; Pfeilschifter, Josef; Schaefer, Liliana

    2017-01-01

    In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions. PMID:28282921

  5. The Expression of Sphingosine Kinase-1 in Head and Neck Carcinoma

    PubMed Central

    Facchinetti, María M.; Gandini, Norberto A.; Fermento, María E.; Sterin-Speziale, Norma B.; Ji, Youngmi; Patel, Vyomesh; Gutkind, J. Silvio; Rivadulla, Maria G.; Curino, Alejandro C.

    2010-01-01

    Sphingosine kinase-1 (SPHK1) modulates the proliferation, apoptosis and differentiation of keratinocytes through the regulation of ceramide and sphingosine-1-phosphate levels. However, studies on the expression of SPHK1 in human head and neck squamous cell carcinoma (HNSCC) specimens are lacking. Therefore, the aim of the present work was to evaluate SPHK1 expression in human primary HNSCCs and to correlate the results with clinical and anatomopathological parameters. We investigated the expression of this protein by immunohistochemistry performed in tissue microarrays of HNSCC and in an independent cohort of 37 paraffin-embedded specimens. SPHK1 expression was further validated by real-time PCR performed on laser capture-microdissected tissue samples. The positive rate of SPHK1 protein in the cancerous tissues was significantly higher (74%) than that in the nontumor oral tissues (23%), and malignant tissues showed stronger immunoreactivity for SPHK1 than normal matching samples. These results were confirmed by real-time PCR quantification of SPHK1 mRNA. Interestingly, the positive expression of SPHK1 was associated with shorter patient survival time (Kaplan-Meier survival curves) and with the loss of p21 expression. Taken together, these results demonstrate that SPHK1 is upregulated in HNSCC and provide clues of the role SPHK1 might play in tumor progression. PMID:20606403

  6. Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

    PubMed Central

    Park, Choon-Ho; Choi, Bo-Hwa; Jeong, Min-Woo; Kim, Sangjune; Kim, Wanil; Song, Yun Seon; Kim, Kyong-Tai

    2011-01-01

    Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage–induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner. PMID:21346188

  7. Fire Service Training. Rescue Practices. (Revised).

    ERIC Educational Resources Information Center

    North Carolina State Dept. of Community Colleges, Raleigh.

    One of a set of fourteen instructional outlines for use in a course to train novice firemen, this guide covers the topic of rescue operations. Two types of rescue functions are recognized: the primary one consists of locating and saving trapped victims, and the secondary one of recovering bodies and making the area safe for other workers and…

  8. Rescue coronary stenting in acute myocardial infarction

    NASA Astrophysics Data System (ADS)

    Barbieri, Enrico; Meneghetti, Paolo; Molinari, Gionata; Zardini, Piero

    1996-01-01

    Failed rescue coronary angioplasty is a high risk situation because of high mortality. Coronary stent has given us the chance of improving and maintaining the patency of the artery. We report our preliminary experience of rescue stenting after unsuccessful coronary angioplasty.

  9. Teaching Holocaust Rescue: A Problematic Pedagogy

    ERIC Educational Resources Information Center

    Lindquist, David H.

    2008-01-01

    Determining how to teach about rescue during the Holocaust presents many dilemmas to teachers as they plan Holocaust curricula. Rescue is often overemphasized, and faulty perspectives about rescuers and their actions may cause students to develop distorted views about this aspect of Holocaust history. This article explores several factors that…

  10. Satellite search and rescue analysis

    NASA Astrophysics Data System (ADS)

    Bailey, J. T.

    The success of rescue operations in the case of the survivors of aircraft crashes depends crucially on the rapid detection of the aircraft location. Similar considerations apply in the case of marine distress. For this reason, the U.S. is currently participating in a program called Cospas/Sarsat, an international cooperative humanitarian effort designed to assist in saving the lives of aviators and mariners in distress. The other original participants in the program include France, Canada, and the Soviet Union. The program began as an experiment with the launch of the first spacecraft, Cospas I, in June 1982. The Cospas/Sarsat partners are engaged in work concerning a second experiment, involving a new generation distress beacon operating on a frequency of 406 MHz. Details regarding the Cospas/Sarsat constellation are discussed, and attention is given to the immediate and the long-term outlook.

  11. 46 CFR 160.156-13 - Approval inspections and tests for prototype rescue boats and fast rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... boats and fast rescue boats. 160.156-13 Section 160.156-13 Shipping COAST GUARD, DEPARTMENT OF HOMELAND... EQUIPMENT Rescue Boats and Fast Rescue Boats (SOLAS) § 160.156-13 Approval inspections and tests for prototype rescue boats and fast rescue boats. (a) After the Commandant notifies the manufacturer that the...

  12. Lunar mission safety and rescue: Technical summary

    NASA Technical Reports Server (NTRS)

    1971-01-01

    A technical summary is presented of the escape/rescue and the hazards analyses for manned missions and operations in the 1980 time frame. Hazards are interpreted as hazards to man, not to equipment, program schedule, or program objectives. Hazards in 39 individual areas are analyzed, and corrective measures are recommended. Over 200 safety guidelines are proposed, based on significant hazards. Escape and rescue situtations and requirements are identified and analyzed, and escape/survival/rescue concepts are defined to cope with each escape/rescue situation. Areas in which research or technical development efforts could improve mission safety are identified. It is concluded that the primary emphasis should be on survival and escape provisions, with rescue required only where self-help cannot bring the endangered crewmen to a safe haven.

  13. Hubble Space Telescope Crew Rescue Analysis

    NASA Technical Reports Server (NTRS)

    Hamlin, Teri L.; Canga, Michael; Boyer, Roger; Thigpen, Eric

    2009-01-01

    In the aftermath of the 2003 Columbia accident NASA removed the Hubble Space Telescope (HST) Servicing Mission 4 (SM4) from the Space Shuttle manifest. Reasons cited included concerns that the risk of flying the mission would be too high. There was at the time no viable technique to repair the orbiter s thermal protection system if it were to be damaged by debris during ascent. Furthermore in the event of damage, since the mission was not to the International Space Station, there was no safe haven for the crew to wait for an extended period of time for a rescue. The HST servicing mission was reconsidered because of improvements in the ascent debris environment, the development of techniques for the astronauts to perform on orbit repairs to damage thermal protection, and the development of a strategy to provide a crew rescue capability. However, leading up to the launch of servicing mission, the HST crew rescue capability was a recurring topic. For HST there was a limited amount of time available to perform a crew rescue because of the limited consumables available on the Orbiter. The success of crew rescue depends upon several factors including when a problem is identified, when and to what extent power down procedures are begun, and where the rescue vehicle is in its ground processing cycle. Severe power downs maximize crew rescue success but would eliminate the option for the orbiter servicing the HST to attempt a landing. Therefore, crew rescue success needed to be weighed against preserving the ability of the orbiter to have landing option in case there was a problem with the rescue vehicle. This paper focuses on quantification of the HST mission loss of crew rescue capability using Shuttle historical data and various power down capabilities. That work supported NASA s decision to proceed with the HST service mission, which was successfully completed on May 24th 2009.

  14. Search and rescue in Alaska's national parks.

    PubMed

    Heggie, Travis W

    2008-11-01

    Recreational travel to wilderness destinations such as national parks is increasing. The inherent risks present in such destinations can result in injury and illness, have a significant impact on local medical resources, and end with expensive search and rescue operations. In order to increase our understanding of the activities and situations which lead to wilderness search and rescue incidents, this study examines search and rescue operations from National Park Service units in Alaska. A retrospective review of all search and rescue incident reports filed by National Park Service units in Alaska during 2002. During 2002 there were 25 reported search and rescue incidents involving 38 individuals. The majority of incidents (19 of 25) occurred at Denali National Park and Preserve. Thirteen fatalities were reported in six incidents, nine incidents involved traumatic injuries, eight involved illnesses, and two involved both injuries and an illness. Mountain climbing (20) and hiking (8) were the most common subject activities at the time search and rescue assistance was required. Climbing solo (4), uneven and wet terrain (4), falls into crevasses (3), and a lack of experience or ability (3) were the factors most commonly contributing to search and rescue incidents. Nineteen helicopters were utilized in 15 operations and fixed-wing aircraft were utilized in seven operations. Males accounted for 33 of the 38 individuals involved in all search and rescue incidents and United States citizens accounted for 74% of the individuals involved. The mountain environment higher than 4500m was the most common search and rescue environment (11). The average cost was USD $6253. Search and rescue operations in Alaska can be expensive and end with severe health consequences. Preventive education efforts at park visitor centers and at the lower and upper base camps on Mt. McKinley should be continued. In addition, pre-departure travel education efforts via the internet should be expanded

  15. IRE1α-TRAF2-ASK1 pathway is involved in CSTMP-induced apoptosis and ER stress in human non-small cell lung cancer A549 cells.

    PubMed

    Zhang, Jiexia; Liang, Ying; Lin, Yongbin; Liu, Yuanbin; YouYou; Yin, Weiqiang

    2016-08-01

    CSTMP, a Tetramethylpyrazine (TMP) analogue, is designed and synthesized based on the pharmacophores of TMP and resveratrol. Recent studies showed that CSTMP had strong protective effects in endothelial cells apoptosis by its anti-oxidant activity. However, the pharmacological function of CSTMP in cancer have not been elucidated to date. The objective of this study was to investigate the anti-cancer effect of CSTMP against human non-small cell lung cancer (NSCLC) A549 cells and the underlying mechanisms. The cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Caspases activity was determined spectrophotometricaly at 405nm using a microtiter plate reader. Western blot and real-time PCR was used to assess the protein and mRNA expression. Immunoprecipitation was used to examine the protein-protein interactions. CSTMP inhibited the proliferation and induced cell cycle arrest and apoptosis of A549 cells. Caspase3, 8, 9 and PARP-1 activation, and Bax/Bcl-2 ratio analyses demonstrated that the anti-cancer effect of CSTMP in A549 cells was mediated by promoting caspase- and mitochondria-dependent apoptosis. Furthermore, CSTMP induced ER stress in A549 cells as evidenced by elevated levels of GRP78, GRP94, CHOP, IRE1α, TRAF2, p-ASK1 and p-JNK, activation of caspase12 and 4, and enhanced formation of an IRE1α-TRAF2-ASK1 complex. Knockdown of IRE1α by siRNA suppressed activation of IRE1α, TRAF2, p-ASK1 and p-JNK in CSTMP treated A549 cells. In addition, the effects of CSTMP on the formation of an IRE1α-TRAF2-ASK1 complex, caspase- and mitochondria-dependent apoptosis were also reversed by IRE1α siRNA in A549 cells. Collectively, we showed that CSTMP induced apoptosis of A549 cells were through IRE1α-TRAF2-ASK1 complex-mediated ER stress, JNK activation, and mitochondrial dysfunction. These insights on this novel compound CSTMP may provide a novel anti-cancer candidate for the treatment of NSCLC. Copyright © 2016 Elsevier Masson SAS. All

  16. Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy.

    PubMed

    Ojo, Kayode K; Dangoudoubiyam, Sriveny; Verma, Shiv K; Scheele, Suzanne; DeRocher, Amy E; Yeargan, Michelle; Choi, Ryan; Smith, Tess R; Rivas, Kasey L; Hulverson, Matthew A; Barrett, Lynn K; Fan, Erkang; Maly, Dustin J; Parsons, Marilyn; Dubey, Jitender P; Howe, Daniel K; Van Voorhis, Wesley C

    2016-12-01

    Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5μM range but interfere with intracellular division at 2.5μM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis.

  17. Involvement of Activated Cdc42 Kinase1 in Colitis and Colorectal Neoplasms.

    PubMed

    Lv, Chaolan; Zhao, Xinmei; Gu, Hongxiang; Huang, Liyun; Zhou, Sanxi; Zhi, Fachao

    2016-12-07

    BACKGROUND Activated Cdc42 kinase1 (ACK1) is a non-receptor tyrosine kinase which is critical for cell survival, proliferation, and migration. Genomic amplification of ACK1 has been reported in multiple human cancers. We aimed to investigate ACK1 protein expression in colorectal mucosa with inflammation and neoplasm, and to evaluate its correlation with disease activity and severity. MATERIAL AND METHODS A total of 250 individuals who underwent total colonoscopy were collected randomly from January 2007 to May 2013 in Nanfang Hospital, Guangzhou, China. Colorectal mucosal biopsy specimens were obtained by endoscopy from 78 patients with ulcerative colitis (UC), 22 with Crohn's disease (CD), 20 with infectious colitis, 26 with non-IBD and noninfectious colitis, 16 with sporadic adenomas, 4 with dysplasia-associated lesions or masses, 10 with sporadic colorectal cancer (CRC), 4 with UC-related CRC, 10 with hyperplastic polyps, and 60 without colonic abnormalities. ACK1 protein levels were determined immunohistochemically. The correlations of ACK1 expression with disease activity and severity were also evaluated. RESULTS Significantly increased ACK1 expression was observed in epithelial cells of colorectal mucosa with inflammation and dysplasia compared to controls (P<0.05). ACK1 expression correlated with clinical activity in IBD (χ²=4.57, P=0.033 for UC; χ²=5.68, P=0.017 for CD), as well as grade of dysplasia in preneoplastic lesions (P<0.05). No significant differences in ACK1 expression were found between UC and CD, or between IBD and non-IBD conditions (P>0.05). CONCLUSIONS ACK1 protein is increased extensively in colitis and colorectal dysplasia. ACK1 overexpression may play a role in colorectal inflammation and neoplasms.

  18. Mitogen- and stress-activated protein kinase 1 modulates photic entrainment of the suprachiasmatic circadian clock.

    PubMed

    Cao, Ruifeng; Butcher, Greg Q; Karelina, Kate; Arthur, J Simon; Obrietan, Karl

    2013-01-01

    The master circadian clock in mammals, the suprachiasmatic nucleus (SCN), is under the entraining influence of the external light cycle. At a mechanistic level, intracellular signaling via the p42/44 mitogen-activated protein kinase pathway appears to play a central role in light-evoked clock entrainment; however, the precise downstream mechanisms by which this pathway influences clock timing are not known. Within this context, we have previously reported that light stimulates activation of the mitogen-activated protein kinase effector mitogen-stress-activated kinase 1 (MSK1) in the SCN. In this study, we utilised MSK1(-/-) mice to further investigate the potential role of MSK1 in circadian clock timing and entrainment. Locomotor activity analysis revealed that MSK1 null mice entrained to a 12 h light/dark cycle and exhibited circadian free-running rhythms in constant darkness. Interestingly, the free-running period in MSK1 null mice was significantly longer than in wild-type control animals, and MSK1 null mice exhibited a significantly greater variance in activity onset. Further, MSK1 null mice exhibited a significant reduction in the phase-delaying response to an early night light pulse (100 lux, 15 min), and, using an 8 h phase-advancing 'jet-lag' experimental paradigm, MSK1 knockout animals exhibited a significantly delayed rate of re-entrainment. At the molecular level, early night light-evoked cAMP response element-binding protein (CREB) phosphorylation, histone phosphorylation and Period1 gene expression were markedly attenuated in MSK1(-/-) animals relative to wild-type mice. Together, these data provide key new insights into the molecular mechanisms by which MSK1 affects the SCN clock.

  19. PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy

    PubMed Central

    Ruf, Stefanie; Heberle, Alexander Martin; Langelaar-Makkinje, Miriam; Gelino, Sara; Wilkinson, Deepti; Gerbeth, Carolin; Schwarz, Jennifer Jasmin; Holzwarth, Birgit; Warscheid, Bettina; Meisinger, Chris; van Vugt, Marcel A. T. M.; Baumeister, Ralf; Hansen, Malene; Thedieck, Kathrin

    2017-01-01

    ABSTRACT Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1′s and PLK1′s functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy. PMID:28102733

  20. Polo-like kinase 1 is a therapeutic target in high-risk neuroblastoma.

    PubMed

    Ackermann, Sandra; Goeser, Felix; Schulte, Johannes H; Schramm, Alexander; Ehemann, Volker; Hero, Barbara; Eggert, Angelika; Berthold, Frank; Fischer, Matthias

    2011-02-15

    High-risk neuroblastoma remains a therapeutic challenge for pediatric oncologists. The Polo-like kinase 1 (PLK1) is highly expressed in many human cancers and is a target of the novel small-molecule inhibitor BI 2536, which has shown promising anticancer activity in adult malignancies. Here, we investigated the effect of BI 2536 on neuroblastoma cells in vitro and in vivo to explore PLK1 as a potential target in high-risk neuroblastoma therapy. PLK1 transcript levels were analyzed by microarrays in 476 primary neuroblastoma specimens, and correlation with prognostic markers and patient outcome was examined. To explore the effect of PLK1 inhibition on neuroblastoma cells, 7 cell lines were treated with BI 2536 and changes in growth properties were determined. Furthermore, nude mice with IMR-32 and SK-N-AS xenografts were treated with BI 2536. PLK1 is highly expressed in unfavorable neuroblastoma and in neuroblastoma cell lines. Expression of PLK1 is associated with unfavorable prognostic markers such as stage 4, age >18 months, MYCN amplification, unfavorable gene expression-based classification, and adverse patient outcome (P < 0.001 each). On treatment with nanomolar doses of BI 2536, all neuroblastoma cell lines analyzed showed significantly reduced proliferation, cell cycle arrest, and cell death. Moreover, BI 2536 abrogated growth of neuroblastoma xenografts in nude mice. Elevated PLK1 expression is significantly associated with high-risk neuroblastoma and unfavorable patient outcome. Inhibition of PLK1 using BI 2536 exhibits strong antitumor activity on human neuroblastoma cells in vitro and in vivo, opening encouraging new perspectives for the treatment of high-risk neuroblastoma. ©2010 AACR.

  1. The phosphoinositide-dependent protein kinase 1 inhibitor, UCN-01, induces fragmentation: possible role of metalloproteinases.

    PubMed

    Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; García-Sáinz, J Adolfo

    2014-10-05

    Phosphoinositide-dependent protein kinase 1 (PDK1) is a key enzyme, master regulator of cellular proliferation and metabolism; it is considered a key target for pharmacological intervention. Using membranes obtained from DDT1 MF-2 cells, phospho-PDK1 was identified by Western blotting, as two major protein bands of Mr 58-68 kDa. Cell incubation with the PDK1 inhibitor, UCN-01, induced a time- and concentration-dependent decrease in the amount of phospho-PDK1 with a concomitant appearance of a ≈42 kDa phosphorylated fragment. Knocking down PDK1 diminished the amount of phospho-PDK1 detected in membranes, accompanied by similarly decreased fragment generation. UCN-01-induced fragment generation was also observed in membranes from cells stably expressing a myc-tagged PDK1 construct. Other PDK1 inhibitors were also tested: OSU-03012 induced a clear decrease in phospho-PDK1 and increased the presence of the phosphorylated fragment in membrane preparations; in contrast, GSK2334470 and staurosporine induced only marginal increases in the amount of PDK1 fragment. Galardin and batimastat, two metalloproteinase inhibitors, markedly attenuated inhibitor-induced PDK1 fragment generation. Metalloproteinases 2, 3, and 9 co-immunoprecipitated with myc-PDK1 under baseline conditions and this interaction was stimulated by UCN-01; batimastat also markedly diminished this effect of the PDK1 inhibitor. Our results indicate that a series of protein kinase inhibitors, namely UCN-01 and OSU-03012 and to a lesser extent GSK2334470 and staurosporine induce PDK1 fragmentation and suggest that metalloproteinases could participate in this effect. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Elevation of serum thymidine kinase 1 in a bacterial infection: canine pyometra.

    PubMed

    Sharif, H; Hagman, R; Wang, L; Eriksson, S

    2013-01-01

    Pyometra is a bacterial infection of the uterus that is common in dogs and is potentially life-threatening if delayed in diagnosis and/or treatment. Thymidine kinase 1 (TK1) is a cytosolic enzyme involved in DNA precursor synthesis, and it is also present in serum from patients with malignant diseases. TK1 has been used as a cell proliferation biomarker for many years in human medicine and recently in dogs. However, little is known regarding serum TK1 levels in individuals with bacterial infection. The objective of this study was to determine the activity of serum TK1 in dogs with pyometra and compare it with hematologic and biochemical parameters, e.g., acute phase proteins and inflammatory mediators such as C-reactive protein and Prostaglandin F(2α). Serum and plasma TK1 activity of 40 healthy female dogs and 54 dogs with pyometra were analyzed using an optimized [(3)H]-thymidine phosphorylation assay. TK1 activities in serum or plasma were significantly higher in dogs with pyometra as compared with healthy female dogs (mean ± SD: 4.0 ± 7.3 pmol/min/mL in the pyometra group and 1.07 ± 0.34 pmol/min/mL in healthy control group). However, there was no difference in TK1 activity between systemic inflammatory response syndrome (SIRS) positive (n = 38) and SIRS negative (n = 16) pyometra cases. Furthermore, the plasma TK1 activity decreased in six and increased in one pyometra patients (n = 10), 24 h after ovariohysterectomy. No significant correlations (P > 0.05) were found between TK1 activity and hematological or other biochemical parameters. In conclusion, the TK1 activity was significantly elevated in dogs with pyometra. Further studies are needed to evaluate the mechanism and role of serum TK1 activity in bacterial infections and its possible diagnostic or prognostic value.

  3. Luteolin Suppresses Cancer Cell Proliferation by Targeting Vaccinia-Related Kinase 1

    PubMed Central

    Shin, Joon; Harikishore, Amaravadhi; Lim, Jong-Kwan; Jung, Youngseob; Lyu, Ha-Na; Baek, Nam-In; Choi, Kwan Yong; Yoon, Ho Sup; Kim, Kyong-Tai

    2014-01-01

    Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy. PMID:25310002

  4. Involvement of Activated Cdc42 Kinase1 in Colitis and Colorectal Neoplasms

    PubMed Central

    Lv, Chaolan; Gu, Hongxiang; Zhao, Xinmei; Huang, Liyun; Zhou, Sanxi; Zhi, Fachao

    2016-01-01

    Background Activated Cdc42 kinase1 (ACK1) is a non-receptor tyrosine kinase which is critical for cell survival, proliferation, and migration. Genomic amplification of ACK1 has been reported in multiple human cancers. We aimed to investigate ACK1 protein expression in colorectal mucosa with inflammation and neoplasm, and to evaluate its correlation with disease activity and severity. Material/Methods A total of 250 individuals who underwent total colonoscopy were collected randomly from January 2007 to May 2013 in Nanfang Hospital, Guangzhou, China. Colorectal mucosal biopsy specimens were obtained by endoscopy from 78 patients with ulcerative colitis (UC), 22 with Crohn’s disease (CD), 20 with infectious colitis, 26 with non-IBD and noninfectious colitis, 16 with sporadic adenomas, 4 with dysplasia-associated lesions or masses, 10 with sporadic colorectal cancer (CRC), 4 with UC-related CRC, 10 with hyperplastic polyps, and 60 without colonic abnormalities. ACK1 protein levels were determined immunohistochemically. The correlations of ACK1 expression with disease activity and severity were also evaluated. Results Significantly increased ACK1 expression was observed in epithelial cells of colorectal mucosa with inflammation and dysplasia compared to controls (P<0.05). ACK1 expression correlated with clinical activity in IBD (χ2=4.57, P=0.033 for UC; χ2=5.68, P=0.017 for CD), as well as grade of dysplasia in preneoplastic lesions (P<0.05). No significant differences in ACK1 expression were found between UC and CD, or between IBD and non-IBD conditions (P>0.05). Conclusions ACK1 protein is increased extensively in colitis and colorectal dysplasia. ACK1 overexpression may play a role in colorectal inflammation and neoplasms. PMID:27926694

  5. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  6. Sphingosine Kinase 1 Deficiency Confers Protection against Hyperoxia-Induced Bronchopulmonary Dysplasia in a Murine Model

    PubMed Central

    Harijith, Anantha; Pendyala, Srikanth; Reddy, Narsa M.; Bai, Tao; Usatyuk, Peter V.; Berdyshev, Evgeny; Gorshkova, Irina; Huang, Long Shuang; Mohan, Vijay; Garzon, Steve; Kanteti, Prasad; Reddy, Sekhar P.; Raj, J. Usha; Natarajan, Viswanathan

    2014-01-01

    Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1−/− (Sphk1−/−), sphingosine kinase 2−/− (Sphk2−/−), and S1P lyase+/− (Sgpl1+/−) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1−/−, but not Sphk2−/− or Sgpl1+/−, mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling–regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia. PMID:23933064

  7. Impairment of Angiogenic Sphingosine Kinase-1/Sphingosine-1-Phosphate Receptors Pathway in Preeclampsia

    PubMed Central

    Dobierzewska, Aneta; Palominos, Macarena; Sanchez, Marianela; Dyhr, Michael; Helgert, Katja; Venegas-Araneda, Pia; Tong, Stephen; Illanes, Sebastian E.

    2016-01-01

    Preeclampsia (PE), is a serious pregnancy disorder characterized in the early gestation by shallow trophoblast invasion, impaired placental neo-angiogenesis, placental hypoxia and ischemia, which leads to maternal and fetal morbidity and mortality. Here we hypothesized that angiogenic sphingosine kinase-1 (SPHK1)/sphingosine-1-phosphate (S1P) receptors pathway is impaired in PE. We found that SPHK1 mRNA and protein expression are down-regulated in term placentae and term chorionic villous explants from patients with PE or severe PE (PES), compared with controls. Moreover, mRNA expression of angiogenic S1PR1 and S1PR3 receptors were decreased in placental samples of PE and PES patients, whereas anti-angiogenic S1PR2 was up-regulated in chorionic villous tissue of PES subjects, pointing to its potential atherogenic and inflammatory properties. Furthermore, in in vitro (JAR cells) and ex vivo (chorionic villous explants) models of placental hypoxia, SPHK1 mRNA and protein were strongly up-regulated under low oxygen tension (1% 02). In contrast, there was no change in SPHK1 expression under the conditions of placental physiological hypoxia (8% 02). In both models, nuclear protein levels of HIF1A were increased at 1% 02 during the time course, but there was no up-regulation at 8% 02, suggesting that SPHK1 and HIF1A might be the part of the same canonical pathway during hypoxia and that both contribute to placental neovascularization during early gestation. Taken together, this study suggest the SPHK1 pathway may play a role in the human early placentation process and may be involved in the pathogenesis of PE. PMID:27284992

  8. Role of casein kinase 1 in the glucose sensor-mediated signaling pathway in yeast

    PubMed Central

    2010-01-01

    Background In yeast, glucose-dependent degradation of the Mth1 protein, a corepressor of the glucose transporter gene (HXT) repressor Rgt1, is a crucial event enabling expression of several HXT. This event occurs through a signaling pathway that involves the Rgt2 and Snf3 glucose sensors and yeast casein kinase 1 and 2 (Yck1/2). In this study, we examined whether the glucose sensors directly couple with Yck1/2 to convert glucose binding into an intracellular signal that leads to the degradation of Mth1. Results High levels of glucose induce degradation of Mth1 through the Rgt2/Snf3 glucose signaling pathway. Fluorescence microscopy analysis indicates that, under glucose-limited conditions, GFP-Mth1 is localized in the nucleus and does not shuttle between the nucleus and cytoplasm. If glucose-induced degradation is prevented due to disruption of the Rgt2/Snf3 pathway, GFP-Mth1 accumulates in the nucleus. When engineered to be localized to the cytoplasm, GFP-Mth1 is degraded regardless of the presence of glucose or the glucose sensors. In addition, removal of Grr1 from the nucleus prevents degradation of GFP-Mth1. These results suggest that glucose-induced, glucose sensor-dependent Mth1 degradation occurs in the nucleus. We also show that, like Yck2, Yck1 is localized to the plasma membrane via C-terminal palmitoylation mediated by the palmitoyl transferase Akr1. However, glucose-dependent degradation of Mth1 is not impaired in the absence of Akr1, suggesting that a direct interaction between the glucose sensors and Yck1/2 is not required for Mth1 degradation. Conclusion Glucose-induced, glucose sensor-regulated degradation of Mth1 occurs in the nucleus and does not require direct interaction of the glucose sensors with Yck1/2. PMID:20205947

  9. Polo-like kinase 1 is essential for the first mitotic division in the mouse embryo.

    PubMed

    Baran, V; Solc, P; Kovarikova, V; Rehak, P; Sutovsky, P

    2013-07-01

    Polo-like kinase 1 (PLK1), a member of the serine/threonine protein kinases family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little is known about PLK1 activities in the mammalian preimplantation embryo. We examined the role of PLK1 in the one-cell mouse embryo. Western blotting showed that the PLK1 protein content increased significantly during the S-phase of the one-cell stage and declined during the first mitotic division. Activation of PLK1 preceded nuclear envelope breakdown (NEBD) in both pronuclei at the entry to first embryo mitosis. Immunofluorescence revealed the presence of phosphorylated, active PLK1 (pThr(210) -PLK1) in both male and female pronuclei, and in the microtubule-organizing centers (MTOCs) shortly before NEBD. During the first mitotic metaphase, pThr(210) -PLK1 accumulated at the spindle poles and was also associated with condensed chromosomes. Inhibition of PLK1 activity with a specific PLK1 inhibitor, BI 2536, at the one-cell stage induced the formation of a bipolar spindle that displayed disordered microtubular arrangements and dislocated, condensed chromosomes. Although such embryos entered mitosis, they did not complete mitosis and arrested at metaphase. Time-lapse recording revealed progressive misalignment of condensed chromosomes during first mitotic metaphase. These data indicate that PLK1 activity is not essential for entry into first mitosis, but is required for the events leading up to metaphase-anaphase transition in the one-cell mouse embryo. Copyright © 2013 Wiley Periodicals, Inc.

  10. Enhanced alpha-kinase 1 accelerates multiple early nephropathies in streptozotocin-induced hyperglycemic mice.

    PubMed

    Kuo, Tzer-Min; Hsu, Hui-Ting; Chung, Chia-Min; Yeh, Kun-Tu; Wu, Cheng-Tien; Lee, Chi-Pin; Chiang, Shang-Lun; Huang, Chung-Ming; Ko, Ying-Chin

    2016-11-01

    Alpha-kinase 1 (ALPK1) is associated with chronic kidney disease (CKD), type 2 diabetes mellitus and gout. Elevated ALPK1 levels have been observed in the kidneys of patients with diabetes and the white blood cells of patients with gout. As renal injury is a common outcome of CKD, diabetes and gout, the aim of this study was to investigate the effect of ALPK1 in the development of renal injury in a hyperglycemic condition. Hyperglycemia was induced in wild-type and ALPK1 transgenic mice by an intraperitoneal injection of streptozotocin (STZ). Functional and histological examinations were performed after 3weeks. STZ-treated ALPK1 transgenic mice exclusively showed arteriolar sclerosis and fibrous thickening of the Bowman's capsule in the kidney. This was accompanied by body weight loss, severe hyperglycemia, and low serum insulin levels. Renal renin and serum renin protein levels were higher in STZ-treated ALPK1 transgenic mice, whereas cGKII protein level was decreased by ALPK1 in human embryonic kidney 293 (HEK293) cells. ALPK1 up-regulated TGF-beta1 levels and transcription of fibrosis-related genes, including MMP-9, FIBRONECTIN, and TIMP1. MSU crystals increased ALPK1 transcription in cultured kidney cells. Finally, ALPK1 enhanced production of MSU crystals-induced IL-1beta in mice. Stimulation of soluble sodium urate induced IL-1beta and Alpk1 mRNA production in mice kidney. Taken together, these data show that an increase in ALPK1 results in accelerated fibrotic nephropathies, primarily through the enhancement of renin, TGF-beta1, and IL-1beta. Renal or blood ALPK1 levels are involved in the induction of fibrotic renal injury in an experimental model of hyperglycemia.

  11. Polo-like kinase1 is required for recruitment of dynein to kinetochores during mitosis.

    PubMed

    Bader, Jason R; Kasuboski, James M; Winding, Michael; Vaughan, Patricia S; Hinchcliffe, Edward H; Vaughan, Kevin T

    2011-06-10

    Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J., Bader, J. R., Tauhata, S. B., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E. H., Vaughan, P. S., and Vaughan, K. T. (2008) J. Cell Biol. 183, 819-834), and phosphorylated and dephosphorylated forms of dynein coexist at prometaphase kinetochores. In this study, we measured the impact of inhibiting polo-like kinase 1 (Plk1) on both dynein populations. Phosphorylated dynein was ablated at kinetochores after inhibiting Plk1 with a small molecule inhibitor (5-Cyano-7-nitro-2-(benzothiazolo-N-oxide)-carboxamide) or chemical genetic approaches. The total complement of kinetochore dynein was also reduced but not eliminated, reflecting the presence of some dephosphorylated dynein after Plk1 inhibition. Although Plk1 inhibition had a profound effect on dynein, kinetochore populations of dynactin, spindly, and zw10 were not reduced. Plk1-independent dynein was reduced after p150(Glued) depletion, consistent with the binding of dephosphorylated dynein to dynactin. Plk1 phosphorylated dynein intermediate chains at Thr-89 in vitro and generated the phospho-Thr-89 phospho-epitope on recombinant dynein intermediate chains. Finally, inhibition of Plk1 induced defects in microtubule capture and persistent microtubule attachment, suggesting a role for phosphorylated dynein in these functions during prometaphase. These findings suggest that Plk1 is a dynein kinase required for recruitment of phosphorylated dynein to kinetochores.

  12. TANK-binding kinase-1 broadly affects oyster immune response to bacteria and viruses.

    PubMed

    Tang, Xueying; Huang, Baoyu; Zhang, Linlin; Li, Li; Zhang, Guofan

    2016-09-01

    As a benthic filter feeder of estuaries, the immune system of oysters provides one of the best models for studying the genetic and molecular basis of the innate immune pathway in marine invertebrates and examining the influence of environmental factors on the immune system. Here, the molecular function of molluscan TANK-binding kinase-1 (TBK1) (which we named CgTBK1) was studied in the Pacific oyster, Crassostrea gigas. Compared with known TBK1 proteins in other model organisms, CgTBK1 contains a conserved S-TKc domain and a coiled coil domain at the N- and C-terminals but lacks an important ubiquitin domain. Quantitative real-time PCR analysis revealed that the expression level of CgTBK1 was ubiquitous in all selected tissues, with highest expression in the gills. CgTBK1 expression was significantly upregulated in response to infections with Vibrio alginolyticus, ostreid herpesvirus 1 (OsHV-1 reference strain and μvar), and polyinosinic:polycytidylic acid sodium salt, suggesting its broad function in immune response. Subcellular localization showed the presence of CgTBK1 in the cytoplasm of HeLa cells, suggesting its potential function as the signal transducer between the receptor and transcription factor. We further demonstrated that CgTBK1 interacted with CgSTING in HEK293T cells, providing evidence that CgTBK1 could be activated by direct binding to CgSTING. In summary, we characterized the TBK1 gene in C. gigas and demonstrated its role in the innate immune response to pathogen infections.

  13. A Role for Casein Kinase 1 Epsilon in the Locomotor Stimulant Response to Methamphetamine

    PubMed Central

    Bryant, Camron D.; Graham, Melissa E.; Distler, Margaret G.; Munoz, Michaelanne B.; Li, Dongdong; Vezina, Paul; Sokoloff, Greta; Palmer, Abraham A.

    2009-01-01

    Rationale We previously co-localized a quantitative trait locus (QTL) for sensitivity to the locomotor stimulant effect of methamphetamine (MA) with a QTL for expression of casein kinase 1 epsilon (Csnk1-ε) in the nucleus accumbens (NAc). Subsequently, we identified a single nucleotide polymorphism in CSNK1E (rs135745) that was associated with increased sensitivity to the subjective effects of d-amphetamine in healthy human subjects. Based on these results, we hypothesized that differential expression of Csnk1-ε causes differential sensitivity to MA-induced locomotor activity in mice. Objective In the present study, we used PF-670462 (PF), which is a selective inhibitor of Csnk1-ε, to directly evaluate the role of Csnk1-ε in the locomotor response to MA in male C57BL/6J mice. Methods We administered vehicle, PF, MA or MA+PF, either via intraperitoneal injections or bilateral intra-NAc microinjections. We also examined Darpp-32 phosphorylation in mice receiving intraperitoneal injections. Results Intraperitoneal PF (20-40 mg/kg) attenuated the locomotor response to MA (2 mg/kg) without affecting baseline activity. The high dose of PF also significantly inhibited MA-induced phosphorylation of Darpp-32, providing a potential mechanism by which Csnk1-ε contributes to MA-induced locomotor activity. Furthermore, microinjection of PF (5 μg/side) into the NAc completely blocked the locomotor response to MA (2.5 μg/side) without affecting baseline activity. Conclusions These results provide direct evidence that Csnk1-ε is crucial for the locomotor stimulant response to a moderate dose of MA and are consistent with the hypothesis that genetic polymorphisms in Csnk1-ε influence sensitivity to amphetamines in both mice and humans. PMID:19050854

  14. Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

    PubMed

    Petrone, Adam; Adamo, Mark E; Cheng, Chao; Kettenbach, Arminja N

    2016-07-01

    Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Transient knock down of checkpoint kinase 1 in hematopoietic progenitors is linked to bone marrow toxicity.

    PubMed

    Hu, Wenyue; Zong, Qing; John-Baptiste, Annette; Jessen, Bart

    2011-07-28

    Checkpoint kinase 1 (Chk1) is required for both intra-S phase and G2/M checkpoints in cell cycle, and plays critical roles in maintaining genomic stability and transducing DNA damage response. Chk1 deficiency has been shown to inhibit T-cell differentiation and resulted in severe anemia in a Chk1 heterozygous mouse model. To date, there has been a good correlation between Chk1 inhibition and in vitro bone marrow toxicity among small molecule inhibitors. To better understand the role of Chk1 in hematopoiesis, we conducted transient Chk1 gene silencing in human bone marrow progenitor cells using siRNA and electroporation. At 48h post electroporation, approximately 70% inhibition of Chk1 was confirmed using real-time RT-PCR and immunoblotting, which resulted in more than 60% reduction in cell count when compared to the non-specific siRNA control on day 6 post-electroporation. This result was confirmed using a colony forming unit assay, where reduced number in both erythroid and granulocyte colonies was observed with Chk1 siRNA treatment. The Chk1 gene inhibition in bone marrow progenitor cells resulted in significant induction of apoptosis, but not cell cycle arrest, as assessed using flow cytometry. In this study an effective method to knock down a gene of interest was established in hard-to-transfect hematopoietic stem cells. Furthermore, our results support a direct role of Chk1 in maintaining normal hematopoiesis in the bone marrow. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Casein kinase 1-epsilon deletion increases mu opioid receptor-dependent behaviors and binge eating1.

    PubMed

    Goldberg, L R; Kirkpatrick, S L; Yazdani, N; Luttik, K P; Lacki, O A; Keith Babbs, R; Jenkins, D F; Evan Johnson, W; Bryant, C D

    2017-09-01

    Genetic and pharmacological studies indicate that casein kinase 1 epsilon (Csnk1e) contributes to psychostimulant, opioid, and ethanol motivated behaviors. We previously used pharmacological inhibition to demonstrate that Csnk1e negatively regulates the locomotor stimulant properties of opioids and psychostimulants. Here, we tested the hypothesis that Csnk1e negatively regulates opioid and psychostimulant reward using genetic inhibition and the conditioned place preference assay in Csnk1e knockout mice. Similar to pharmacological inhibition, Csnk1e knockout mice showed enhanced opioid-induced locomotor activity with the mu opioid receptor agonist fentanyl (0.2 mg/kg i.p.) as well as enhanced sensitivity to low-dose fentanyl reward (0.05 mg/kg). Interestingly, female knockout mice also showed a markedly greater escalation in consumption of sweetened palatable food - a behavioral pattern consistent with binge eating that also depends on mu opioid receptor activation. No difference was observed in fentanyl analgesia in the 52.5°C hot plate assay (0-0.4 mg/kg), naloxone conditioned place aversion (4 mg/kg), or methamphetamine conditioned place preference (0-4 mg/kg). To identify molecular adaptations associated with increased drug and food behaviors in knockout mice, we completed transcriptome analysis via mRNA sequencing of the striatum. Enrichment analysis identified terms associated with myelination and axon guidance and pathway analysis identified a differentially expressed gene set predicted to be regulated by the Wnt signaling transcription factor, Tcf7l2. To summarize, Csnk1e deletion increased mu opioid receptor-dependent behaviors, supporting previous studies indicating an endogenous negative regulatory role of Csnk1e in opioid behavior. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  17. Isoflurane protects against human endothelial cell apoptosis by inducing sphingosine kinase-1 via ERK MAPK.

    PubMed

    Bakar, Adnan M; Park, Sang Won; Kim, Mihwa; Lee, H Thomas

    2012-01-01

    Endothelial dysfunction is a major clinical problem affecting virtually every patient requiring critical care. Volatile anesthetics are frequently used during the perioperative period and protect the heart and kidney against ischemia and reperfusion injury. We aimed to determine whether isoflurane, the most commonly used volatile anesthetic in the USA, protects against endothelial apoptosis and necrosis and the mechanisms involved in this protection. Human endothelial EA.hy926 cells were pretreated with isoflurane or carrier gas (95% room air + 5% CO(2)) then subjected to apoptosis with tumor necrosis factor-α or to necrosis with hydrogen peroxide. DNA laddering and in situ Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick-End Labeling (TUNEL) staining determined EA.hy926 cell apoptosis and percent LDH released determined necrosis. We also determined whether isoflurane modulates the expression and activity of sphingosine kinase-1 (SK1) and induces the phosphorylation of extracellular signal regulated kinase (ERK MAPK) as both enzymes are known to protect against cell death. Isoflurane pretreatment significantly decreased apoptosis in EA.hy926 cells as evidenced by reduced TUNEL staining and DNA laddering without affecting necrosis. Mechanistically, isoflurane induces the phosphorylation of ERK MAPK and increased SK1 expression and activity in EA.hy926 cells. Finally, selective blockade of SK1 (with SKI-II) or S1P(1) receptor (with W146) abolished the anti-apoptotic effects of isoflurane. Taken together, we demonstrate that isoflurane, in addition to its potent analgesic and anesthetic properties, protects against endothelial apoptosis most likely via SK1 and ERK MAPK activation. Our findings have significant clinical implication for protection of endothelial cells during the perioperative period and patients requiring critical care.

  18. Neospora caninum Calcium-Dependent Protein Kinase 1 Is an Effective Drug Target for Neosporosis Therapy

    PubMed Central

    Ojo, Kayode K.; Reid, Molly C.; Kallur Siddaramaiah, Latha; Müller, Joachim; Winzer, Pablo; Zhang, Zhongsheng; Keyloun, Katelyn R.; Vidadala, Rama Subba Rao; Merritt, Ethan A.; Hol, Wim G. J.; Maly, Dustin J.; Fan, Erkang; Van Voorhis, Wesley C.; Hemphill, Andrew

    2014-01-01

    Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine

  19. Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543

    SciTech Connect

    Ju, TongFa; Gao, DaQuan; Fang, Zheng-yu

    2016-02-12

    In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrin A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. - Highlights: • PF-543 is anti-proliferative and cytotoxic to established and primary CRC cells. • PF-543 induces programmed necrosis, but not apoptosis, in CRC cells. • Modulation of mitochondrial protein cyclophilin-D alters PF-543's sensitivity. • PF-543 inhibits HCT-116 xenograft growth in SCID mice, improving mice survival. • Co-administration of cyclophilin-D inhibitor CsA inhibits PF-543's activity in vivo.

  20. Polo-like Kinase 1 Regulates Vimentin Phosphorylation at Ser-56 and Contraction in Smooth Muscle.

    PubMed

    Li, Jia; Wang, Ruping; Gannon, Olivia J; Rezey, Alyssa C; Jiang, Sixin; Gerlach, Brennan D; Liao, Guoning; Tang, Dale D

    2016-11-04

    Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Structure of the catalytic domain of human polo-like kinase 1.

    PubMed

    Kothe, Michael; Kohls, Darcy; Low, Simon; Coli, Rocco; Cheng, Alan C; Jacques, Suzanne L; Johnson, Theresa L; Lewis, Cristina; Loh, Christine; Nonomiya, Jim; Sheils, Alissa L; Verdries, Kimberly A; Wynn, Thomas A; Kuhn, Cyrille; Ding, Yuan-Hua

    2007-05-22

    Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.

  2. Regulation of microtubule-based microtubule nucleation by mammalian polo-like kinase 1.

    PubMed

    Johmura, Yoshikazu; Soung, Nak-Kyun; Park, Jung-Eun; Yu, Li-Rong; Zhou, Ming; Bang, Jeong K; Kim, Bo-Yeon; Veenstra, Timothy D; Erikson, Raymond L; Lee, Kyung S

    2011-07-12

    Bipolar spindle formation is pivotal for accurate segregation of mitotic chromosomes during cell division. A growing body of evidence suggests that, in addition to centrosome- and chromatin-based microtubule (MT) nucleation, MT-based MT nucleation plays an important role for proper bipolar spindle formation in various eukaryotic organisms. Although a recently discovered Augmin complex appears to play a central role in this event, how Augmin is regulated remains unknown. Here we provide evidence that a mammalian polo-like kinase 1 (Plk1) localizes to mitotic spindles and promotes MT-based MT nucleation by directly regulating Augmin. Mechanistically, we demonstrated that Cdc2-dependent phosphorylation on a γ-tubulin ring complex (γ-TuRC) recruitment protein, Nedd1/GCP-WD, at the previously uncharacterized S460 residue induces the Nedd1-Plk1 interaction. This step appeared to be critical to allow Plk1 to phosphorylate the Hice1 subunit of the Augmin complex to promote the Augmin-MT interaction and MT-based MT nucleation from within the spindle. Loss of either the Nedd1 S460 function or the Plk1-dependent Hice1 phosphorylation impaired both the Augmin-MT interaction and γ-tubulin recruitment to the spindles, thus resulting in improper bipolar spindle formation that ultimately leads to mitotic arrest and apoptotic cell death. Thus, via the formation of the Nedd1-Plk1 complex and subsequent Augmin phosphorylation, Plk1 regulates spindle MT-based MT nucleation to accomplish normal bipolar spindle formation and mitotic progression.

  3. Regulation of microtubule-based microtubule nucleation by mammalian polo-like kinase 1

    PubMed Central

    Johmura, Yoshikazu; Soung, Nak-Kyun; Park, Jung-Eun; Yu, Li-Rong; Zhou, Ming; Bang, Jeong K.; Kim, Bo-Yeon; Veenstra, Timothy D.; Erikson, Raymond L.; Lee, Kyung S.

    2011-01-01

    Bipolar spindle formation is pivotal for accurate segregation of mitotic chromosomes during cell division. A growing body of evidence suggests that, in addition to centrosome- and chromatin-based microtubule (MT) nucleation, MT-based MT nucleation plays an important role for proper bipolar spindle formation in various eukaryotic organisms. Although a recently discovered Augmin complex appears to play a central role in this event, how Augmin is regulated remains unknown. Here we provide evidence that a mammalian polo-like kinase 1 (Plk1) localizes to mitotic spindles and promotes MT-based MT nucleation by directly regulating Augmin. Mechanistically, we demonstrated that Cdc2-dependent phosphorylation on a γ-tubulin ring complex (γ-TuRC) recruitment protein, Nedd1/GCP-WD, at the previously uncharacterized S460 residue induces the Nedd1–Plk1 interaction. This step appeared to be critical to allow Plk1 to phosphorylate the Hice1 subunit of the Augmin complex to promote the Augmin–MT interaction and MT-based MT nucleation from within the spindle. Loss of either the Nedd1 S460 function or the Plk1-dependent Hice1 phosphorylation impaired both the Augmin–MT interaction and γ-tubulin recruitment to the spindles, thus resulting in improper bipolar spindle formation that ultimately leads to mitotic arrest and apoptotic cell death. Thus, via the formation of the Nedd1–Plk1 complex and subsequent Augmin phosphorylation, Plk1 regulates spindle MT-based MT nucleation to accomplish normal bipolar spindle formation and mitotic progression. PMID:21690413

  4. Metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate.

    PubMed

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-09-18

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling, while LRRK2 is implicated in the pathogenesis of Parkinson's disease. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with (32)P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing (32)P-orthophosphate. The (32)P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography ((32)P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

  5. Involvement of LIM kinase 1 in actin polarization in human CD4 T cells

    PubMed Central

    Xu, Xuehua; Guo, Jia; Vorster, Paul; Wu, Yuntao

    2012-01-01

    Chemokine binding to cognate receptors induces actin dynamics that are a major driving force for T cell migration and chemotactic motility. HIV-1 binding to the chemokine coreceptor CXCR4 initiates chemotactic signaling, mimicking chemokine-induced actin dynamics to facilitate infection processes such as entry, early DNA synthesis, and nuclear migration. Recently, we identified that HIV-triggered early actin polymerization is mediated through the Rac1-PAK1/2-LIMK1-cofilin pathway. Inhibition of LIMK1 (LIM domain kinase 1), a kinase phosphorylating cofilin, through shRNA knockdown decreases actin polymerization and T cell chemotaxis toward SDF-1. The LIMK1 knockdown T cells also supported lower viral entry, DNA synthesis and nuclear migration, suggesting a critical role of LIMK1-mediated actin dynamics in the initiation of HIV-1 infection. Surprisingly, LIMK1 knockdown in CEM-SS T cells did not lead to an overall change in the ratio of phospho-cofilin to total cofilin although there was a measurable decrease in the amount of actin filaments in cells. The decrease in filamentous actin in LIMK1 knockdown cells was found to mainly occur in polarized cap region rich in F-actin. These results suggest that LIMK1 may be involved in spontaneous actin polarization in transformed T cells. The inhibition of T cell chemotaxis by LIMK1 knockdown likely result from inhibition of localized LIMK1 activation and cofilin phosphorylation that are required for polarized actin polymerization for directional cell migration. The inhibition of HIV-1 infection by LIMK1 knockdown may also result from the decrease of actin-rich membrane protrusions that may be preferred viral entry sites in T cells. PMID:23060964

  6. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  7. Truncation and Activation of Dual Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I

    PubMed Central

    Jin, Nana; Yin, Xiaomin; Gu, Jianlan; Zhang, Xinhua; Shi, Jianhua; Qian, Wei; Ji, Yuhua; Cao, Maohong; Gu, Xiaosong; Ding, Fei; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei

    2015-01-01

    Hyperphosphorylation and dysregulation of exon 10 splicing of Tau are pivotally involved in pathogenesis of Alzheimer disease (AD) and/or other tauopathies. Alternative splicing of Tau exon 10, which encodes the second microtubule-binding repeat, generates Tau isoforms containing three and four microtubule-binding repeats, termed 3R-Taus and 4R-Taus, respectively. Dual specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A) lies at the Down syndrome critical region of chromosome 21. Overexpression of this kinase may contribute to the early Tau pathology in Down syndrome via phosphorylation of Tau and dysregulation of Tau exon 10. Here, we report that Dyrk1A was truncated at the C terminus and was associated with overactivation of calpain I in AD brain. Calpain I proteolyzed Dyrk1A in vitro first at the C terminus and further at the N terminus and enhanced its kinase activity toward Tau via increased Vmax but not Km. C-terminal truncation of Dyrk1A resulted in stronger activity than its full-length protein in promotion of exon 10 exclusion and phosphorylation of Tau. Dyrk1A was truncated in kainic acid-induced excitotoxic mouse brains and coincided with an increase in 3R-Tau expression and phosphorylation of Tau via calpain activation. Moreover, truncation of Dyrk1A was correlated with an increase in the ratio of 3R-Tau/4R-Tau and Tau hyperphosphorylation in AD brain. Collectively, these findings suggest that truncation/activation of Dyrk1A by Ca2+/calpain I might contribute to Tau pathology via promotion of exon 10 exclusion and hyperphosphorylation of Tau in AD brain. PMID:25918155

  8. BOTRYTIS-INDUCED KINASE1 Modulates Arabidopsis Resistance to Green Peach Aphids via PHYTOALEXIN DEFICIENT4.

    PubMed

    Lei, Jiaxin; A Finlayson, Scott; Salzman, Ron A; Shan, Libo; Zhu-Salzman, Keyan

    2014-08-01

    BOTRYTIS-INDUCED KINASE1 (BIK1) plays important roles in induced defense against fungal and bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Its tomato (Solanum lycopersicum) homolog is required for host plant resistance to a chewing insect herbivore. However, it remains unknown whether BIK1 functions in plant defense against aphids, a group of insects with a specialized phloem sap-feeding style. In this study, the potential role of BIK1 was investigated in Arabidopsis infested with the green peach aphid (Myzus persicae). In contrast to the previously reported positive role of intact BIK1 in defense response, loss of BIK1 function adversely impacted aphid settling, feeding, and reproduction. Relative to wild-type plants, bik1 displayed higher aphid-induced hydrogen peroxide accumulation and more severe lesions, resembling a hypersensitive response (HR) against pathogens. These symptoms were limited to the infested leaves. The bik1 mutant showed elevated basal as well as induced salicylic acid and ethylene accumulation. Intriguingly, elevated salicylic acid levels did not contribute to the HR-like symptoms or to the heightened aphid resistance associated with the bik1 mutant. Elevated ethylene levels in bik1 accounted for an initial, short-term repellence. Introducing a loss-of-function mutation in the aphid resistance and senescence-promoting gene PHYTOALEXIN DEFICIENT4 (PAD4) into the bik1 background blocked both aphid resistance and HR-like symptoms, indicating bik1-mediated resistance to aphids is PAD4 dependent. Taken together, Arabidopsis BIK1 confers susceptibility to aphid infestation through its suppression of PAD4 expression. Furthermore, the results underscore the role of reactive oxygen species and cell death in plant defense against phloem sap-feeding insects. © 2014 American Society of Plant Biologists. All Rights Reserved.

  9. The changes of vaccinia related kinase 1 in grafted heart after rat heart transplantation

    PubMed Central

    Qian, Shiguo; Yang, Xuechao; Wu, Kunpeng; Lv, Qiangsheng; Zhang, Yuanyuan; Dai, Jiahong; Chen, Cheng

    2014-01-01

    Objective To assess the expression and significance of vaccinia-related kinase 1 (VRK1) after rat heart transplantation. Materials and methods Lewis and Wistar rats weighing 250 to 300 g were used as donors and recipients. Allografts were from Wistar transplanted into Lewis, and isografts were transplanted from Lewis into Lewis. Grafts were harvested at 1, 3, 5, and 7 days after transplantation. We performed Western Blot of heart tissues after cardiac transplantation. To analyze VRK1 express between the isografts and allografts for immunohistochemical staining. At 5th day after heart transplantation use related cytokines VRK1 for immunohistochemical. We used double immunofluorescent staining on transverse cryosections of graft tissues by co-labeling with different markers, including those for VRK1, activate caspase-3, α-actinin, VCAM-1, CD4. Results Compared with rare expression in syngeneic Lewis rat hearts, VRK1 protein level in allogeneic hearts were detected at various survival times after heterotopic heart transplantation, which observably expressed on day 5 postoperative. In addition, we examined the expression of activate caspase-3 in allogeneic hearts, which has a similar expression with VRK1. Immunohistochemical and immunofluorescent method displayed that VRK1 was widely expressed in cytoplasm of cardiac tissue and activate caspase-3 was also expressed in cardiomyocytes. However, the VRK1 wasn’t express in inflammation. Conclusions The VRK1 expression has increased after heart transplantation in allograft and isograft, and VRK1 may play a significant role in myocardial apoptosis after heterotopic heart transplantation in rats. PMID:25589968

  10. Inhibition of the casein-kinase-1-ε/δ/ prevents relapse-like alcohol drinking.

    PubMed

    Perreau-Lenz, Stéphanie; Vengeliene, Valentina; Noori, Hamid R; Merlo-Pich, Emilio V; Corsi, Mauro A; Corti, Corrado; Spanagel, Rainer

    2012-08-01

    During the past decade, it has been shown that circadian clock genes have more than a simple circadian time-keeping role. Clock genes also modulate motivational processes and have been implicated in the development of psychiatric disorders such as drug addiction. Recent studies indicate that casein-kinase 1ε/δ (CK1ε/δ)--one of the components of the circadian molecular clockwork-might be involved in the etiology of addictive behavior. The present study was initiated to study the specific role of CK1ε/δ in alcohol relapse-like drinking using the 'Alcohol Deprivation Effect' model. The effect of CK1ε/δ inhibition was tested on alcohol consumption in long-term alcohol-drinking rats upon re-exposure to alcohol after deprivation using a four-bottle free-choice paradigm with water, 5%, 10%, and 20% ethanol solutions, as well as on saccharin preference in alcohol-naive rats. The inhibition of CK1ε/δ with systemic PF-670462 (0, 10, and 30 mg/kg) injections dose-dependently decreased, and at a higher dosage prevented the alcohol deprivation effect, as compared with vehicle-treated rats. The impact of the treatment was further characterized using nonlinear regression analyses on the daily profiles of drinking and locomotor activity. We reveal that CK1ε/δ inhibition blunted the high daytime alcohol intake typically observed upon alcohol re-exposure, and induced a phase shift of locomotor activity toward daytime. Only the highest dose of PF-670462 shifted the saccharin intake daily rhythm toward daytime during treatment, and decreased saccharin preference after treatment. Our data suggest that CK1 inhibitors may be candidates for drug treatment development for alcoholism.

  11. 33 CFR 127.1505 - Emergency response and rescue.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... (CONTINUED) WATERFRONT FACILITIES WATERFRONT FACILITIES HANDLING LIQUEFIED NATURAL GAS AND LIQUEFIED... and rescue pending the arrival of resources for firefighting or pollution control. Response and rescue...

  12. 33 CFR 127.1505 - Emergency response and rescue.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... (CONTINUED) WATERFRONT FACILITIES WATERFRONT FACILITIES HANDLING LIQUEFIED NATURAL GAS AND LIQUEFIED... and rescue pending the arrival of resources for firefighting or pollution control. Response and rescue...

  13. A new approach to road accident rescue.

    PubMed

    Morales, Alejandro; González-Aguilera, Diego; López, Alfonso I; Gutiérrez, Miguel A

    2016-01-01

    This article develops and validates a new methodology and tool for rescue assistance in traffic accidents, with the aim of improving its efficiency and safety in the evacuation of people, reducing the number of victims in road accidents. Different tests supported by professionals and experts have been designed under different circumstances and with different categories of damaged vehicles coming from real accidents and simulated trapped victims in order to calibrate and refine the proposed methodology and tool. To validate this new approach, a tool called App_Rescue has been developed. This tool is based on the use of a computer system that allows an efficient access to the technical information of the vehicle and sanitary information of the common passengers. The time spent during rescue using the standard protocol and the proposed method was compared. This rescue assistance system allows us to make vital information accessible in posttrauma care services, improving the effectiveness of interventions by the emergency services, reducing the rescue time and therefore minimizing the consequences involved and the number of victims. This could often mean saving lives. In the different simulated rescue operations, the rescue time has been reduced an average of 14%.

  14. Air Cushion Crash Rescue Vehicle (ACCRV)

    DTIC Science & Technology

    1987-10-01

    x 13.3 x 5.7 Battery Incl. Monitors 1 DC Defibril- 11.90 3.8 x 13.3 x 9.2 Battery Inc\\. lator 106 -) 0) ho cd o +-> w c cd 3...reverse if necessary and identify by block number) Current USAF crash rescue vehicles have been designed to operate on the roads, ramps, taxiways...Cushion Crash Rescue Vehicle (ACCRV) has been designed by integrating a retractable air cushion system with a crash rescue vehicle. This report

  15. Space safety and rescue 1984-1985

    NASA Astrophysics Data System (ADS)

    Heath, G. W.

    The present conference on spacecraft crew safety and rescue technologies and operations considers safety aspects of Space Shuttle ground processing, the Inmarsat and COSPAS/SARSAT emergency location satellite systems, emergency location and rescue communications using Geosat, the use of the Manned Maneuvering Unit for on-orbit rescue operations, NASA Space Station safety design and operational considerations, and the medico-legal implications of space station operation. Also discussed are the operational and environmental aspects of EPIRBS, mobile satellites for safety and disaster response, Inmarsat's role in the Future Global Maritime Distress and Safety System, and test results of the L-band satellite's EPIRB system.

  16. Conditional knockout of activin like kinase-1 (ALK-1) leads to heart failure without maladaptive remodeling.

    PubMed

    Morine, Kevin J; Qiao, Xiaoying; Paruchuri, Vikram; Aronovitz, Mark J; Mackey, Emily E; Buiten, Lyanne; Levine, Jonathan; Ughreja, Keshan; Nepali, Prerna; Blanton, Robert M; Karas, Richard H; Oh, S Paul; Kapur, Navin K

    2017-05-01

    Activin like kinase-1 (AlK-1) mediates signaling via the transforming growth factor beta (TGFβ) family of ligands. AlK-1 activity promotes endothelial proliferation and migration. Reduced AlK-1 activity is associated with arteriovenous malformations. No studies have examined the effect of global AlK-1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of AlK-1 promote maladaptive cardiac remodeling. To test this hypothesis, we employed AlK-1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele, whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre recombinase-mediated excision of floxed AlK-1 alleles. Tamoxifen treated wild-type (WT-TAM; n = 5) and vehicle treated AlK-1-cKO mice (cKO-CON; n = 5) served as controls for tamoxifen treated AlK-1-cKO mice (cKO-TAM; n = 15). AlK-1 cKO-TAM mice demonstrated reduced 14-day survival compared to cKO-CON controls (13 vs 100%, respectively, p < 0.01). Seven days after treatment, cKO-TAM mice exhibited reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14 days total body mass was reduced, but LV and lung mass increased in cKO-TAM not cKO-CON mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume were increased in cKO-TAM, not cKO-CON mice. LV AlK-1 mRNA levels were reduced in cKO-TAM, not cKO-CON mice. LV levels of other TGFβ-family ligands and receptors (AlK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFβ1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-TAM mice, but LV levels of β-MHC and SERCA were unchanged. No increase in markers of cardiac fibrosis, Type I collagen, CTGF, or PAI-1, were observed between groups. No differences were observed for any variable studied between cKO-CON and WT-TAM mice. Global deletion of AlK-1 is associated with the

  17. Phase I Study of LY2606368, a Checkpoint Kinase 1 Inhibitor, in Patients With Advanced Cancer

    PubMed Central

    Infante, Jeffrey; Janku, Filip; Jones, Suzanne; Nguyen, Ly M.; Burris, Howard; Naing, Aung; Bauer, Todd M.; Piha-Paul, Sarina; Johnson, Faye M.; Kurzrock, Razelle; Golden, Lisa; Hynes, Scott; Lin, Ji; Lin, Aimee Bence; Bendell, Johanna

    2016-01-01

    Purpose The primary objective was to determine safety, toxicity, and a recommended phase II dose regimen of LY2606368, an inhibitor of checkpoint kinase 1, as monotherapy. Patients and Methods This phase I, nonrandomized, open-label, dose-escalation trial used a 3 + 3 dose-escalation scheme and included patients with advanced solid tumors. Intravenous LY2606368 was dose escalated from 10 to 50 mg/m2 on schedule 1 (days 1 to 3 every 14 days) or from 40 to 130 mg/m2 on schedule 2 (day 1 every 14 days). Safety measures and pharmacokinetics were assessed, and pharmacodynamics were measured in blood, hair follicles, and circulating tumor cells. Results Forty-five patients were treated; seven experienced dose-limiting toxicities (all hematologic). The maximum-tolerated doses (MTDs) were 40 mg/m2 (schedule 1) and 105 mg/m2 (schedule 2). The most common related grade 3 or 4 treatment-emergent adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (typically < 5 days). Febrile neutropenia incidence was low (7%). The LY2606368 exposure over the first 72 hours (area under the curve from 0 to 72 hours) at the MTD for each schedule coincided with the exposure in mouse xenografts that resulted in maximal tumor responses. Minor intra- and intercycle accumulation of LY2606368 was observed at the MTDs for both schedules. Two patients (4.4%) had a partial response; one had squamous cell carcinoma (SCC) of the anus and one had SCC of the head and neck. Fifteen patients (33.3%) had a best overall response of stable disease (range, 1.2 to 6.7 months), six of whom had SCC. Conclusion An LY2606368 dose of 105 mg/m2 once every 14 days is being evaluated as the recommended phase II dose in dose-expansion cohorts for patients with SCC. PMID:27044938

  18. S6 kinase 1 knockout inhibits uninephrectomy- or diabetes-induced renal hypertrophy.

    PubMed

    Chen, Jian-Kang; Chen, Jianchun; Thomas, George; Kozma, Sara C; Harris, Raymond C

    2009-09-01

    Removal of one kidney stimulates synthesis of RNA and protein, with minimal DNA replication, in all nephron segments of the remaining kidney, resulting in cell growth (increase in cell size) with minimal cell proliferation (increase in cell number). In addition to the compensatory renal hypertrophy caused by nephron loss, pathophysiological renal hypertrophy can occur as a consequence of early uncontrolled diabetes. However, the molecular mechanism underlying renal hypertrophy in these conditions remains unclear. In the present study, we report that deletion of S6 kinase 1 (S6K1) inhibited renal hypertrophy seen following either contralateral nephrectomy or induction of diabetes. In wild-type mice, hypertrophic stimuli increased phosphorylation of 40S ribosomal protein S6 (rpS6), a known target of S6K1. Immunoblotting analysis revealed that S6K1(-/-) mice exhibited moderately elevated basal levels of rpS6, which did not increase further in response to the hypertrophic stimuli. Northern blotting indicated a moderate upregulation of S6K2 expression in the kidneys of S6K1(-/-) mice. Phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1, another downstream target of the mammalian target of rapamycin (mTOR), was stimulated to equivalent levels in S6K1(-/-) and S6K1(+/+) littermates during renal hypertrophy, indicating that mTOR was still activated in the S6K1(-/-) mice. The highly selective mTOR inhibitor, rapamycin, inhibited increased phosphorylation of rpS6 and blocked 60-70% of the hypertrophy seen in wild-type mice but failed to prevent the approximately 10% hypertrophy seen in S6K1(-/-) mice in response to uninephrectomy (UNX) although it did inhibit the basal rpS6 phosphorylation. Thus the present study provides the first genetic evidence that S6K1 plays a major role in the development of compensatory renal hypertrophy as well as diabetic renal hypertrophy and indicates that UNX- and diabetes-mediated mTOR activation can selectively

  19. Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells

    SciTech Connect

    Xu, Lin; Zhang, Yanan; Gao, Meng; Wang, Guangping; Fu, Yunfeng

    2016-04-15

    Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AML progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings. - Highlights: • A-674563 is cytotoxic and anti-proliferative in U937 and AML progenitor cells. • A-674563 activates caspase-3/9 and apoptosis in U937 and AML progenitor cells. • Whiling blocking Akt, A-674563 manipulates other signalings in AML cells. • A-674563 inhibits SphK1 activity in AML cells, independent of Akt blockage. • A-674563 injection inhibits U937 xenograft in vivo growth, and improves mice survival.

  20. Different approaches of teaching in chemistry. Membrane targeting mechanism of human sphingosine kinase 1

    NASA Astrophysics Data System (ADS)

    Hwang, Jeong-Hye

    Part 1. Biochemistry research involves elucidating the mechanism of membrane targeting of human sphingosine kinase 1 (hSK1). Sphingosine kinase (SK) is an enzyme that catalyzes phosphorylation of sphingosine to sphingosine-1-phosphate (S-1-P). hSK1 can be activated by its agonists resulting in rapid and transient increased production of S-1-P, resulting in enhancement of apoptosis. Upon activation by PMA, SK translocates to the plasma membrane. In vitro measurement demonstrated hSK1 selectively bound phosphatidylserine over anionic lipids and showed strong preference for the plasma membrane-mimetics. Mutational analysis of conserved Thr54 and Asn89 on putative membrane-binding surface from the model structure showed both in vivo and in vitro that these two residues are important for the membrane selectivity of hSK1. Part 2. Chemical education research focuses on three different ways of scientific learning and teaching. First, inquiry teaching that involves a writing method called the Science Writing Heuristic (SWH) is analyzed using grounded theory. This is done in a general education course for pre-service teachers. As a result, (1) students experience understanding of concept through mastery of their own methods and experience different aspects of inquiry processes; (2) SWH method allows instructors to detect misconceptions generated by students' incorrect, but logical interpretations of their data, and helps instructors to make changes to guide students; (3) as students experience meta-cognition, students gain understanding of concepts by relating mathematical progression to different parts of the experiments and also by applying what they learn into other situations. Second, statistical analysis on the long term effects of a combined math/chemistry program is analyzed through multiple linear regression and discriminant function analysis. The results demonstrate the program was beneficial to the underrepresented students when the college success was measured

  1. Ethanol Regulation of Serum Glucocorticoid Kinase 1 Expression in DBA2/J Mouse Prefrontal Cortex

    PubMed Central

    Costin, Blair N.; Dever, Seth M.; Miles, Michael F.

    2013-01-01

    Background We previously identified a group of glucocorticoid-responsive genes, including Serum Glucocorticoid kinase 1 (Sgk1), regulated by acute ethanol in prefrontal cortex of DBA2/J mice. Acute ethanol activates the hypothalamic pituitary adrenal axis (HPA) causing release of glucocorticoids. Chronic ethanol dysregulates the HPA response in both humans and rodents, possibly contributing to important interactions between stress and alcoholism. Because Sgk1 regulates ion channels and learning and memory, we hypothesized that Sgk1 contributes to HPA-dependent acute and adaptive neuronal responses to ethanol. These studies characterized acute and chronic ethanol regulation of Sgk1 mRNA and protein and their relationship with ethanol actions on the HPA axis. Results Acute ethanol increased Sgk1 mRNA expression in a dose and time dependent manner. Three separate results suggested that ethanol regulated Sgk1 via circulating glucocorticoids: acute ethanol increased glucocorticoid receptor binding to the Sgk1 promoter; adrenalectomy blocked ethanol induction of Sgk1 mRNA; and chronic ethanol exposure during locomotor sensitization down-regulated HPA axis activation and Sgk1 induction by acute ethanol. SGK1 protein had complex temporal responses to acute ethanol with rapid and transient increases in Ser422 phosphorylation at 15 min. following ethanol administration. This activating phosphorylation had functional consequences, as suggested by increased phosphorylation of the known SGK1 target, N-myc downstream-regulated gene 1 (NDRG1). After repeated ethanol administration during locomotor sensitization, basal SGK1 protein phosphorylation increased despite blunting of Sgk1 mRNA induction by ethanol. Conclusions These results suggest that HPA axis and glucocorticoid receptor signaling mediate acute ethanol induction of Sgk1 transcription in mouse prefrontal cortex. However, acute ethanol also causes complex changes in SGK1 protein expression and activity. Chronic ethanol

  2. Sphingosine kinase 1 and cancer: a systematic review and meta-analysis.

    PubMed

    Zhang, Yun; Wang, Yan; Wan, Zhi; Liu, Shiping; Cao, Yu; Zeng, Zhi

    2014-01-01

    Sphingosine kinase 1 (SK1) is a key regulator of the dynamic ceramide/sphingosine 1-phosphate rheostat balance and important in the pathological cancer genesis, progression, and metastasis processes. Many studies have demonstrated SK1 overexpressed in various cancers, but no meta-analysis has evaluated the relationship between SK1 and various cancers. We retrieved relevant articles from the PubMed, EBSCO, ISI, and OVID databases. A pooled odds ratio (OR) was used to assess the associations between SK1 expression and cancer; hazard ratios (HR) were used for 5-year and overall survival. Review Manager 5.0 was used for the meta-analysis, and publication bias was evaluated with STATA 12.0 (Egger's test). Thirty-four eligible studies (n=4,673 patients) were identified. SK1 positivity and high expression were significantly different between cancer, non-cancer, and benign tissues. SK1 mRNA and protein expression levels were elevated in the cancer tissues, compared with the normal tissues. SK1 positivity rates differed between various cancer types (lowest [27.3%] in estrogen receptor-positive breast cancer and highest [82.2%] in tongue squamous cell carcinoma). SK1 positivity and high expression were associated with 5-year survival; the HR was 1.86 (95% confidence interval [CI], 1.18-2.94) for breast cancer, 1.58 (1.08-2.31) for gastric cancer, and 2.68 (2.10-3.44) for other cancers; the total cancer HR was 2.21 (95% CI, 1.83-2.67; P < 0.00001). The overall survival HRs were 2.09 (95% CI, 1.35-3.22), 1.56 (1.08-2.25), and 2.62 (2.05-3.35) in breast, gastric, and other cancers, respectively. The total effect HR was 2.21 (95% CI, 1.83-2.66; P < 0.00001). SK1 positivity and high expression were significantly associated with cancer and a shorter 5-year and overall survival. SK1 positivity rates vary tremendously among the cancer types. It is necessary to further explore whether SK1 might be a predictive biomarker of outcomes in cancer patients.

  3. Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression.

    PubMed

    Watari, Akihiro; Hasegawa, Maki; Yagi, Kiyohito; Kondoh, Masuo

    2016-01-01

    Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.

  4. Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression

    PubMed Central

    Watari, Akihiro; Hasegawa, Maki; Yagi, Kiyohito; Kondoh, Masuo

    2016-01-01

    Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine. PMID:26727128

  5. Crizotinib inhibits NF2-associated schwannoma through inhibition of focal adhesion kinase 1

    PubMed Central

    Troutman, Scott; Moleirinho, Susana; Kota, Smitha; Nettles, Kendall; Fallahi, Mohammad; Johnson, Gary L.; Kissil, Joseph L.

    2016-01-01

    Neurofibromatosis type 2 (NF2) is a dominantly inherited autosomal disease characterized by schwannomas of the 8th cranial nerve. The NF2 tumor suppressor gene encodes for Merlin, a protein implicated as a suppressor of multiple cellular signaling pathways. To identify potential drug targets in NF2-associated malignancies we assessed the consequences of inhibiting the tyrosine kinase receptor MET. We identified crizotinib, a MET and ALK inhibitor, as a potent inhibitor of NF2-null Schwann cell proliferation in vitro and tumor growth in vivo. To identify the target/s of crizotnib we employed activity-based protein profiling (ABPP), leading to identification of FAK1 (PTK2) as the relevant target of crizotinib inhibition in NF2-null schwannoma cells. Subsequent studies confirm that inhibition of FAK1 is sufficient to suppress tumorigenesis in animal models of NF2 and that crizotinib-resistant forms of FAK1 can rescue the effects of treatment. These studies identify a FDA approved drug as a potential treatment for NF2 and delineate the mechanism of action in NF2-null Schwann cells. PMID:27363027

  6. The structure of human tau-tubulin kinase 1 both in the apo form and in complex with an inhibitor

    PubMed Central

    Kiefer, Susan E.; Chang, ChiehYing J.; Kimura, S. Roy; Gao, Mian; Xie, Dianlin; Zhang, Yaqun; Zhang, Guifen; Gill, Martin B.; Mastalerz, Harold; Thompson, Lorin A.; Cacace, Angela M.; Sheriff, Steven

    2014-01-01

    Tau-tubulin kinase 1 (TTBK1) is a dual-specificity (serine/threonine and tyrosine) kinase belonging to the casein kinase 1 superfamily. TTBK1 is a neuron-specific kinase that regulates tau phosphorylation. Hyperphosphorylation of tau is implicated in the pathogenesis of Alzheimer’s disease. Two kinase-domain constructs of TTBK1 were expressed in a baculovirus-infected insect-cell system and purified. The purified TTBK1 kinase-domain proteins were crystallized using the hanging-drop vapor-diffusion method. X-ray diffraction data were collected and the structure of TTBK1 was determined by molecular replacement both as an apo structure and in complex with a kinase inhibitor. PMID:24637750

  7. Fire and Rescue Technology. Resources in Technology.

    ERIC Educational Resources Information Center

    Valesey, Brigitte G.

    1997-01-01

    Provides occupational information about fire and rescue operations personnel, such as fire science, fire protection engineering, emergency medical technicians, and firefighters. Provides information about organizations in these fields. (JOW)

  8. Physics Involved in Air Search and Rescue.

    ERIC Educational Resources Information Center

    Egler, Robert A.

    1994-01-01

    Describes how a simple radio homing device can be used to allow students to participate in a search and rescue mission similar to that which the Civil Air Patrol engages in when locating planes that have crashed. (ZWH)

  9. NASA's Search-and-Rescue Technology

    NASA Image and Video Library

    This animation depicts the next-generation search and rescue system, the DASS. Under this system, instruments used to relay emergency beacon signals will be installed on GPS satellites. When one em...

  10. Physics Involved in Air Search and Rescue.

    ERIC Educational Resources Information Center

    Egler, Robert A.

    1994-01-01

    Describes how a simple radio homing device can be used to allow students to participate in a search and rescue mission similar to that which the Civil Air Patrol engages in when locating planes that have crashed. (ZWH)

  11. Drowning rescue on ice, board assist (image)

    MedlinePlus

    Drowning may occur wherever there is water, whether it is only a few inches in the bottom of the tub or thousands of feet in the ocean. People should be aware of life-saving techniques from rescue to resuscitation.

  12. Self-repair promotes microtubule rescue

    PubMed Central

    Gaillard, Jérémie; John, Karin; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Summary The dynamic instability of microtubules is characterised by slow growth phases stochastically interrupted by rapid depolymerisations called catastrophes. Rescue events can arrest the depolymerisation and restore microtubule elongation. However the origin of these rescue events remain unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints. PMID:27617929

  13. Fire and Rescue Technology. Resources in Technology.

    ERIC Educational Resources Information Center

    Valesey, Brigitte G.

    1997-01-01

    Provides occupational information about fire and rescue operations personnel, such as fire science, fire protection engineering, emergency medical technicians, and firefighters. Provides information about organizations in these fields. (JOW)

  14. An introduction to mountain search and rescue.

    PubMed

    Johnson, Lanny

    2004-05-01

    Alpine search and rescue teams must perform each incident response safely. To do so requires experience, organizational skills, technical training, and ability. In addition, teams should interface with emergency medical control advisors who are familiar with local terrain, mountain rescue operations, and the evacuation techniques employed. To facilitate safety and organization, each mission can be divided into four linked stages: location, reach, stabilize, and evacuate.

  15. Protective function of pyridoxamine on retinal photoreceptor cells via activation of the p‑Erk1/2/Nrf2/Trx/ASK1 signalling pathway in diabetic mice.

    PubMed

    Ren, Xiang; Sun, Hong; Zhang, Chenghong; Li, Chen; Wang, Jinlei; Shen, Jie; Yu, Dong; Kong, Li

    2016-07-01

    The present study aimed to investigate the mechanisms that mediate the protective effects of pyridoxamine (PM) on light‑damaged retinal photoreceptor cells in diabetic mice. A high‑fat diet and streptozotocin were used to induce a mouse model of type II diabetes. During the experiment, mice were divided the mice into three types of group, as follows: Control groups (negative control and light‑damaged groups); experimental groups (diabetic and diabetic light‑damaged groups); and treatment groups (25, 50 and 100 mg/kg PM‑treated groups). Using hematoxylin‑eosin staining, the number of nuclear layer cells were counted. Western blotting and immunohistochemistry were performed to measure the levels of thioredoxin (Trx), phospho‑extracellular signal‑regulated kinase 1/2 (p‑Erk1/2), nuclear factor erythroid 2‑related factor 2 (Nrf2) and apoptosis signal‑regulating kinase 1 (ASK1). The photoreceptor cell count in the outer nuclear layer of the light‑damaged, diabetic control and diabetic light‑damaged groups were significantly reduced compared with the negative control group (P<0.001). The cell counts in the PM‑treated groups were significantly increased compared with the diabetic group (P<0.001). Compared with the negative control group, the light‑damaged, diabetic and diabetic light‑damaged groups exhibited significantly decreased Trx, p‑Erk1/2 and Nrf2 expression levels (P<0.001), and significantly increased ASK1 expression levels (P<0.001). However, in the PM‑treated groups, Trx, p‑Erk1/2 and Nrf2 expression levels were significantly increased (P<0.001), and ASK1 expression was significantly decreased (P<0.001). The results of the present study demonstrate that PM protects retinal photoreceptor cells against light damage in diabetic mice, and that its mechanism may be associated with the upregulation of Trx, p‑Erk1/2 and Nrf2 expression, and the downregulation of ASK1 expression.

  16. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    PubMed Central

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  17. Hubble Space Telescope Crew Rescue Analysis

    NASA Technical Reports Server (NTRS)

    Hamlin, Teri L.; Canga, Michael A.; Cates, Grant R.

    2010-01-01

    In the aftermath of the 2003 Columbia accident, NASA removed the Hubble Space Telescope (HST) Servicing Mission 4 (SM4) from the Space Shuttle manifest. Reasons cited included concerns that the risk of flying the mission would be too high. The HST SM4 was subsequently reinstated and flown as Space Transportation System (STS)-125 because of improvements in the ascent debris environment, the development of techniques for astronauts to perform on orbit repairs to damaged thermal protection, and the development of a strategy to provide a viable crew rescue capability. However, leading up to the launch of STS-125, the viability of the HST crew rescue capability was a recurring topic. For STS-125, there was a limited amount of time available to perform a crew rescue due to limited consumables (power, oxygen, etc.) available on the Orbiter. The success of crew rescue depended upon several factors, including when a problem was identified; when and what actions, such as powering down, were begun to conserve consumables; and where the Launch on Need (LON) vehicle was in its ground processing cycle. Crew rescue success also needed to be weighed against preserving the Orbiter s ability to have a landing option in case there was a problem with the LON vehicle. This paper focuses on quantifying the HST mission loss of crew rescue capability using Shuttle historical data and various power down strategies. Results from this effort supported NASA s decision to proceed with STS-125, which was successfully completed on May 24th 2009.

  18. Factors influencing mine rescue team behaviors.

    PubMed

    Jansky, Jacqueline H; Kowalski-Trakofler, K M; Brnich, M J; Vaught, C

    2016-01-01

    A focus group study of the first moments in an underground mine emergency response was conducted by the National Institute for Occupational Safety and Health (NIOSH), Office for Mine Safety and Health Research. Participants in the study included mine rescue team members, team trainers, mine officials, state mining personnel, and individual mine managers. A subset of the data consists of responses from participants with mine rescue backgrounds. These responses were noticeably different from those given by on-site emergency personnel who were at the mine and involved with decisions made during the first moments of an event. As a result, mine rescue team behavior data were separated in the analysis and are reported in this article. By considering the responses from mine rescue team members and trainers, it was possible to sort the data and identify seven key areas of importance to them. On the basis of the responses from the focus group participants with a mine rescue background, the authors concluded that accurate and complete information and a unity of purpose among all command center personnel are two of the key conditions needed for an effective mine rescue operation.

  19. 30 CFR 49.18 - Training for mine rescue teams.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Training for mine rescue teams. 49.18 Section 49.18 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.18 Training for mine rescue...

  20. 30 CFR 49.18 - Training for mine rescue teams.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Training for mine rescue teams. 49.18 Section 49.18 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.18 Training for mine rescue...

  1. 30 CFR 49.15 - Mine rescue station.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Mine rescue station. 49.15 Section 49.15 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.15 Mine rescue station. (a) Every operator...

  2. 30 CFR 49.5 - Mine rescue station.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Mine rescue station. 49.5 Section 49.5 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Metal and Nonmetal Mines § 49.5 Mine rescue station. (a) Except...

  3. 30 CFR 49.15 - Mine rescue station.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Mine rescue station. 49.15 Section 49.15 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.15 Mine rescue station. (a) Every operator...

  4. 30 CFR 49.5 - Mine rescue station.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Mine rescue station. 49.5 Section 49.5 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Metal and Nonmetal Mines § 49.5 Mine rescue station. (a) Except...

  5. 30 CFR 49.15 - Mine rescue station.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Mine rescue station. 49.15 Section 49.15 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.15 Mine rescue station. (a) Every operator...

  6. 30 CFR 49.18 - Training for mine rescue teams.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Training for mine rescue teams. 49.18 Section 49.18 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.18 Training for mine rescue...

  7. 30 CFR 49.5 - Mine rescue station.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Mine rescue station. 49.5 Section 49.5 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Metal and Nonmetal Mines § 49.5 Mine rescue station. (a) Except...

  8. 30 CFR 49.18 - Training for mine rescue teams.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Training for mine rescue teams. 49.18 Section 49.18 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.18 Training for mine rescue...

  9. 30 CFR 49.15 - Mine rescue station.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Mine rescue station. 49.15 Section 49.15 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.15 Mine rescue station. (a) Every...

  10. 30 CFR 49.15 - Mine rescue station.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Mine rescue station. 49.15 Section 49.15 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.15 Mine rescue station. (a) Every...

  11. 46 CFR 108.565 - Stowage of rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Stowage of rescue boats. 108.565 Section 108.565... AND EQUIPMENT Lifesaving Equipment § 108.565 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each...

  12. 46 CFR 108.565 - Stowage of rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Stowage of rescue boats. 108.565 Section 108.565... AND EQUIPMENT Lifesaving Equipment § 108.565 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each...

  13. 46 CFR 108.565 - Stowage of rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Stowage of rescue boats. 108.565 Section 108.565... AND EQUIPMENT Lifesaving Equipment § 108.565 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each...

  14. 46 CFR 108.565 - Stowage of rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Stowage of rescue boats. 108.565 Section 108.565... AND EQUIPMENT Lifesaving Equipment § 108.565 Stowage of rescue boats. (a) Rescue boats must be stowed as follows: (1) Each rescue boat must be ready for launching in not more than 5 minutes. (2) Each...

  15. 30 CFR 49.2 - Availability of mine rescue teams.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... mine served by a mine rescue team shall be located more than two hours ground travel time from the mine... underground mine shall: (1) Establish at least two mine rescue teams which are available at all times when... least two mine rescue teams are available at all times when miners are underground. (b) Each mine rescue...

  16. 30 CFR 49.12 - Availability of mine rescue teams.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ground travel time from the mine rescue station with which the rescue team is associated. (g) As used in... all times when miners are underground; or (2) Enter into an arrangement for mine rescue services which assures that at least two mine rescue teams are available at all times when miners are underground. (b...

  17. 30 CFR 49.12 - Availability of mine rescue teams.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ground travel time from the mine rescue station with which the rescue team is associated. (g) As used in... all times when miners are underground; or (2) Enter into an arrangement for mine rescue services which assures that at least two mine rescue teams are available at all times when miners are underground. (b...

  18. 30 CFR 49.12 - Availability of mine rescue teams.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ground travel time from the mine rescue station with which the rescue team is associated. (g) As used in... all times when miners are underground; or (2) Enter into an arrangement for mine rescue services which assures that at least two mine rescue teams are available at all times when miners are underground. (b...

  19. 30 CFR 49.2 - Availability of mine rescue teams.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... than two hours ground travel time from the mine rescue station with which the rescue team is associated... teams which are available at all times when miners are underground; or (2) Enter into an arrangement for mine rescue services which assures that at least two mine rescue teams are available at all times when...

  20. 30 CFR 49.2 - Availability of mine rescue teams.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... mine served by a mine rescue team shall be located more than two hours ground travel time from the mine... underground mine shall: (1) Establish at least two mine rescue teams which are available at all times when... least two mine rescue teams are available at all times when miners are underground. (b) Each mine rescue...

  1. 30 CFR 49.12 - Availability of mine rescue teams.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ground travel time from the mine rescue station with which the rescue team is associated. (g) As used in... all times when miners are underground; or (2) Enter into an arrangement for mine rescue services which assures that at least two mine rescue teams are available at all times when miners are underground. (b...

  2. 30 CFR 49.2 - Availability of mine rescue teams.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... mine served by a mine rescue team shall be located more than two hours ground travel time from the mine... underground mine shall: (1) Establish at least two mine rescue teams which are available at all times when... least two mine rescue teams are available at all times when miners are underground. (b) Each mine rescue...

  3. 49 CFR 238.114 - Rescue access windows.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Rescue access windows. 238.114 Section 238.114... § 238.114 Rescue access windows. (a) Number and location. Except as provided in paragraph (a)(1)(ii) of... rescue access windows. At least one rescue access window shall be located in each side of the car...

  4. REGγ deficiency promotes premature aging via the casein kinase 1 pathway

    PubMed Central

    Li, Lei; Zhao, Dengpan; Wei, Haibin; Yao, Liangfang; Dang, Yongyan; Amjad, Ali; Xu, Jinjin; Liu, Jiang; Guo, Linjie; Li, Dongqing; Li, Zhen; Zuo, Di; Zhang, Yuanyuan; Liu, Jian; Huang, Shixia; Jia, Caifeng; Wang, Lu; Wang, Ying; Xie, Yifan; Luo, Jian; Zhang, Bianhong; Luo, Honglin; Donehower, Lawrence A.; Moses, Robb E.; Xiao, Jianru; O’Malley, Bert W.; Li, Xiaotao

    2013-01-01

    Our recent studies suggest a role for the proteasome activator REG (11S regulatory particles, 28-kDa proteasome activator)γ in the regulation of tumor protein 53 (p53). However, the molecular details and in vivo biological significance of REGγ-p53 interplay remain elusive. Here, we demonstrate that REGγ-deficient mice develop premature aging phenotypes that are associated with abnormal accumulation of casein kinase (CK) 1δ and p53. Antibody array analysis led us to identify CK1δ as a direct target of REGγ. Silencing CK1δ or inhibition of CK1δ activity prevented decay of murine double minute (Mdm)2. Interestingly, a massive increase of p53 in REGγ−/− tissues is associated with reduced Mdm2 protein levels despite that Mdm2 transcription is enhanced. Allelic p53 haplodeficiency in REGγ-deficient mice attenuated premature aging features. Furthermore, introducing exogenous Mdm2 to REGγ−/− MEFs significantly rescues the phenotype of cellular senescence, thereby establishing a REGγ-CK1-Mdm2-p53 regulatory pathway. Given the conflicting evidence regarding the “antiaging” and “proaging” effects of p53, our results indicate a key role for CK1δ-Mdm2-p53 regulation in the cellular aging process. These findings reveal a unique model that mimics acquired aging in mammals and indicates that modulating the activity of the REGγ-proteasome may be an approach for intervention in aging-associated disorders. PMID:23766372

  5. CYP2E1 Sensitizes the Liver to LPS- and TNF α-Induced Toxicity via Elevated Oxidative and Nitrosative Stress and Activation of ASK-1 and JNK Mitogen-Activated Kinases

    PubMed Central

    Cederbaum, Arthur I.; Yang, Lili; Wang, Xiaodong; Wu, Defeng

    2012-01-01

    The mechanisms by which alcohol causes cell injury are not clear. A major mechanism is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other; however, interactions between them, have not been extensively evaluated. Increased oxidative stress from induction of CYP2E1 sensitizes hepatocytes to LPS and TNFα toxicity and oxidants, activation of inducible nitric oxide synthase and p38 and JNK MAP kinases, and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNFα-potentiated hepatotoxicity. This paper will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved including activation of the mitogen-activated kinase kinase kinase ASK-1. Decreasing either cytosolic or mitochondrial thioredoxin in HepG2 cells expressing CYP2E1 causes loss of cell viability and elevated oxidative stress via an ASK-1/JNK-dependent mechanism. We hypothesize that similar interactions occur as a result of ethanol induction of CYP2E1 and TNFα. PMID:22028977

  6. 46 CFR 160.156-7 - Design, construction and performance of rescue boats and fast rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Design, construction and performance of rescue boats and... Boats and Fast Rescue Boats (SOLAS) § 160.156-7 Design, construction and performance of rescue boats and... immersion suits. (ii) Each rescue boat should be designed following standard human engineering practices...

  7. Kinetic Mechanism and Rate-Limiting Steps of Focal Adhesion Kinase-1

    SciTech Connect

    Schneck, Jessica L.; Briand, Jacques; Chen, Stephanie; Lehr, Ruth; McDevitt, Patrick; Zhao, Baoguang; Smallwood, Angela; Concha, Nestor; Oza, Khyati; Kirkpatrick, Robert; Yan, Kang; Villa, James P.; Meek, Thomas D.; Thrall, Sara H.

    2010-12-07

    Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH{sub 2}, FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k{sub cat} (0.052 {+-} 0.001 s{sup -1}), K{sub MgATP} (1.2 {+-} 0.1 {micro}M), K{sub iMgATP} (1.3 {+-} 0.2 {micro}M), K{sub FAK-tide} (5.6 {+-} 0.4 {micro}M), and K{sub iFAK-tide} (6.1 {+-} 1.1 {micro}M) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the {beta}{gamma}-bridge:{beta}-nonbridge positional oxygen exchange of [{gamma}-{sup 18}O{sub 4}]ATP in the presence of 1 mM [{gamma}-{sup 18}O{sub 4}]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k{sub x}/k{sub cat} = 0.14 {+-} 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k{sub cat}/K{sub FAK-tide}, while k{sub cat} and k{sub cat}/K{sub MgATP} were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k{sub cat}/K{sub m} for MgATP but not on k{sub cat}/K{sub m} for FAK-tide. From the positional isotope exchange, viscosity, and

  8. Organizations of food redistribution and rescue.

    PubMed

    Mousa, T Y; Freeland-Graves, J H

    2017-09-06

    Food insecurity affects 13.4% of the USA population, despite the fact that 30-40% of all food is deposited in a landfill. Food rescue nutrition is the process of redistribution of surplus food to the impoverished. The aim of this study is to document the extent of involvement of organizations in food rescue nutrition. In this cross-sectional study, a survey about organizations involved in food rescue nutrition was developed, validated, and then tested. Directors of 100 organizations involved in food rescue nutrition from eight Southwestern States in the USA participated in this research. These organizations provided an average of 2 million kg of food to more than 40,000 clients each month. Food assistance programs had an average of eight workers and 3081 volunteers. In addition to food, these organizations provided other services such as clothing, clinical, and childcare. The agencies encountered several challenges, including lack of resources that resulted in reducing food portions and turning away clients. The extent of involvement of community-based programs in food rescue nutrition was strong in eight Southwestern states in the USA. Organizations involved in food redistribution helped alleviate food insecurity in their clients. Sustainability of these charitable networks was dependent on availability of resources and sufficient volunteers. Health professionals should encourage these organizations by providing support through donations of time, money, and/or food. Copyright © 2017 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

  9. Reconstitution of CKMT1 expression fails to rescue cells from mitochondrial membrane potential dissipation: implications for controlling RNAi experiments.

    PubMed

    Datler, Christoph; Grimm, Stefan

    2013-12-01

    RNA interference (RNAi) is an essential method in molecular biology to reduce the expression of target genes and thereby determine their function. Since this tool is known to also have unspecific effects, control experiments are needed, chiefly among them the exclusion of off-target effects and the reconstitution of the genes' expression for the rescue of the cellular RNAi effects. We show here that the knock-down of the mitochondrial creatine kinase-1 (CKMT1) by RNA interference causes the dissipation of the mitochondrial membrane potential ΔΨm. This was accomplished with 11 different RNAi constructs designed to target 7 distinct exons as well as exon/intron junctions making unspecific off-target effects unlikely. However, all our attempts failed to rescue human cells from ΔΨm dissipation by the expression of CKMT1 alleles not targeted by RNAi. This included the transient and stable expression of the murine CKMT1 homologue, the expression of human codon usage-modified alleles, the transfection of a novel splice-isoform of CKMT1, and even the introduction of a human genomic clone for CKMT1 with codon usage changes. Our results indicate that while off-target effects of RNA interference can easily be addressed, the rescue of the knock-down phenotype is not necessarily achievable. © 2013 Elsevier B.V. All rights reserved.

  10. [Air rescue: current significance and practical issues].

    PubMed

    Schellhaaß, A; Popp, E

    2014-12-01

    Germany has a nationwide and powerful helicopter emergency medical services system (HEMS), which executes primary rescue missions and interhospital transfer of intensive care patients. In recent years the range of HEMS missions has become modified due to demographic changes and structural changes in the healthcare system. Furthermore, the number of HEMS missions is steadily increasing. If reasonably used air rescue contributes to desired reductions in overall preclinical time. Moreover, it facilitates prompt transport of patients to a hospital suitable for definitive medical care and treatment can be initiated earlier which is a particular advantage for severely injured and critically ill patients. Because of complex challenges during air rescue missions the qualifications of the HEMS personnel have to be considerably higher in comparison with ground based emergency medical services.

  11. Measurement of phosphorylated extracellular signal-regulated kinase 1 and 2 in an undergraduate teaching laboratory with ALPHAscreen technology.

    PubMed

    Hay, Debbie L

    2009-03-17

    Teaching the practical aspects of signal transduction to large undergraduate classes can be challenging when there is only a finite time frame in which to engage in laboratory activities. This teaching resource describes the use of bead-based ALPHAscreen technology for a class of 300 second-year biochemistry students, exposing the next generation of researchers to cutting-edge technology. Although in this case phosphorylated extracellular signal-regulated kinase 1 and 2 were measured, this technology is applicable to the measurement of many different signaling components. This resource provides a practical guide for instructors and exemplifies how such traditionally high-throughput research technologies can be used as teaching tools.

  12. Requirement for ribosomal protein S6 kinase 1 to mediate glycolysis and apoptosis resistance induced by Pten deficiency

    PubMed Central

    Tandon, Preeti; Gallo, Catherine A.; Khatri, Shikha; Barger, Jennifer F.; Yepiskoposyan, Hasmik; Plas, David R.

    2011-01-01

    Pten inactivation promotes cell survival in leukemia cells by activating glycolytic metabolism. We found that targeting ribosomal protein S6 kinase 1 (S6K1) in Pten-deficient cells suppressed glycolysis and induced apoptosis. S6K1 knockdown decreased expression of HIF-1α, and HIF-1α was sufficient to restore glycolysis and survival of cells lacking S6K1. In the Ptenfl/fl Mx1-Cre+ mouse model of leukemia, S6K1 deletion delayed the development of leukemia. Thus, S6K1 is a critical mediator of glycolytic metabolism, cell survival, and leukemogenesis in Pten-deficient cells. PMID:21262837

  13. RESCU: A real space electronic structure method

    NASA Astrophysics Data System (ADS)

    Michaud-Rioux, Vincent; Zhang, Lei; Guo, Hong

    2016-02-01

    In this work we present RESCU, a powerful MATLAB-based Kohn-Sham density functional theory (KS-DFT) solver. We demonstrate that RESCU can compute the electronic structure properties of systems comprising many thousands of atoms using modest computer resources, e.g. 16 to 256 cores. Its computational efficiency is achieved from exploiting four routes. First, we use numerical atomic orbital (NAO) techniques to efficiently generate a good quality initial subspace which is crucially required by Chebyshev filtering methods. Second, we exploit the fact that only a subspace spanning the occupied Kohn-Sham states is required, and solving accurately the KS equation using eigensolvers can generally be avoided. Third, by judiciously analyzing and optimizing various parts of the procedure in RESCU, we delay the O (N3) scaling to large N, and our tests show that RESCU scales consistently as O (N2.3) from a few hundred atoms to more than 5000 atoms when using a real space grid discretization. The scaling is better or comparable in a NAO basis up to the 14,000 atoms level. Fourth, we exploit various numerical algorithms and, in particular, we introduce a partial Rayleigh-Ritz algorithm to achieve efficiency gains for systems comprising more than 10,000 electrons. We demonstrate the power of RESCU in solving KS-DFT problems using many examples running on 16, 64 and/or 256 cores: a 5832 Si atoms supercell; a 8788 Al atoms supercell; a 5324 Cu atoms supercell and a small DNA molecule submerged in 1713 water molecules for a total 5399 atoms. The KS-DFT is entirely converged in a few hours in all cases. Our results suggest that the RESCU method has reached a milestone of solving thousands of atoms by KS-DFT on a modest computer cluster.

  14. Involvement of Polo-like Kinase 1 (Plk1) in Mitotic Arrest by Inhibition of Mitogen-activated Protein Kinase-Extracellular Signal-regulated Kinase-Ribosomal S6 Kinase 1 (MEK-ERK-RSK1) Cascade*

    PubMed Central

    Li, Ran; Chen, Dian-Fu; Zhou, Rong; Jia, Sheng-Nan; Yang, Jin-Shu; Clegg, James S.; Yang, Wei-Jun

    2012-01-01

    Cell division is controlled through cooperation of different kinases. Of these, polo-like kinase 1 (Plk1) and p90 ribosomal S6 kinase 1 (RSK1) play key roles. Plk1 acts as a G2/M trigger, and RSK1 promotes G1 progression. Although previous reports show that Plk1 is suppressed by RSK1 during meiosis in Xenopus oocytes, it is still not clear whether this is the case during mitosis or whether Plk1 counteracts the effects of RSK1. Few animal models are available for the study of controlled and transient cell cycle arrest. Here we show that encysted embryos (cysts) of the primitive crustacean Artemia are ideal for such research because they undergo complete cell cycle arrest when they enter diapause (a state of obligate dormancy). We found that Plk1 suppressed the activity of RSK1 during embryonic mitosis and that Plk1 was inhibited during embryonic diapause and mitotic arrest. In addition, studies on HeLa cells using Plk1 siRNA interference and overexpression showed that phosphorylation of RSK1 increased upon interference and decreased after overexpression, suggesting that Plk1 inhibits RSK1. Taken together, these findings provide insights into the regulation of Plk1 during cell division and Artemia diapause cyst formation and the correlation between the activity of Plk1 and RSK1. PMID:22427657

  15. The Duty to Rescue and Investigators' Obligations.

    PubMed

    MacKay, Douglas; Rulli, Tina

    We examine current applications of the moral duty to rescue to justify clinical investigators' duties of ancillary care and standard of care to subjects in resource-poor settings. These applications fail to explain why investigators possess obligations to research participants, in particular, and not to people in need, in general. Further, these applications fail to recognize the normative significance of the institutional role of the investigators. We offer a positive account of the duty to rescue for investigators as institutional agents, with duties to populations rather than merely individuals.

  16. Appropriate suction device in rescue medicine.

    PubMed

    Dahlgren, B E; Nilsson, H; Bjorn, P; Skedevik, C

    1987-12-01

    In rescue medicine, a suction apparatus must function in a variety of environmental conditions. To find an appropriate device for the Swedish Air Force air rescue service the Laerdal suction device 790,000 was selected for further testing according to international standards for aviation safety. Tests showed that vibrations had deleterious effects on the internal construction of the suction device. In addition, an electromagnetic field was generated affecting the navigation, autopilot, and communication systems. We conclude that the suction apparatus and probably other devices as well must be tested for their functioning in adverse environments and their ability to meet international aviation safety regulations.

  17. Medical Rescue of China International Search & Rescue Team (CISAR) in Nepal Earthquake.

    PubMed

    Yang, Jiong; Yang, Zhen; Lv, Qi; Liu, Hai-Feng; Ding, Hui; Yu, Meng-Yang; Zeng, Xi-Huan; Wang, Xin; Fan, Hao-Jun

    2016-05-18

    On April 25, 2015, a massive 8.1-magnitude earthquake struck Nepal at 2:11 pm (Beijing time). The 68-member-strong China International Search & Rescue Team (CISAR) left for Nepal at 6 am, April 26, to help with relief work. The CISAR was the first foreign team to rescue a survivor who was trapped beneath the rubble in the Gongabu area after the earthquake. On May 8, the team fulfilled the search-and-rescue mission and returned to Beijing. During the 2 weeks of rescue work, the team treated more than 3700 victims and cleared approximately 430 buildings. In this rescue mission, 10 experienced medical officers (including nine doctors and a nurse) from the General Hospital of Chinese People's Armed Police Force (PAP) comprised the medical team of CISAR. In this report, we focus on the medical rescues by CISAR and discuss the characteristics of the medical rescue in Nepal. (Disaster Med Public Health Preparedness. 2016;page 1 of 3).

  18. Effect of the sphingosine kinase 1 selective inhibitor, PF-543 on arterial and cardiac remodelling in a hypoxic model of pulmonary arterial hypertension.

    PubMed

    MacRitchie, Neil; Volpert, Giora; Al Washih, Mohammed; Watson, David G; Futerman, Anthony H; Kennedy, Simon; Pyne, Susan; Pyne, Nigel J

    2016-08-01

    Recent studies have demonstrated that the expression of sphingosine kinase 1, the enzyme that catalyses formation of the bioactive lipid, sphingosine 1-phosphate, is increased in lungs from patients with pulmonary arterial hypertension. In addition, Sk1(-/-) mice are protected from hypoxic-induced pulmonary arterial hypertension. Therefore, we assessed the effect of the sphingosine kinase 1 selective inhibitor, PF-543 and a sphingosine kinase 1/ceramide synthase inhibitor, RB-005 on pulmonary and cardiac remodelling in a mouse hypoxic model of pulmonary arterial hypertension. Administration of the potent sphingosine kinase 1 inhibitor, PF-543 in a mouse hypoxic model of pulmonary hypertension had no effect on vascular remodelling but reduced right ventricular hypertrophy. The latter was associated with a significant reduction in cardiomyocyte death. The protection involves a reduction in the expression of p53 (that promotes cardiomyocyte death) and an increase in the expression of anti-oxidant nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). In contrast, RB-005 lacked effects on right ventricular hypertrophy, suggesting that sphingosine kinase 1 inhibition might be nullified by concurrent inhibition of ceramide synthase. Therefore, our findings with PF-543 suggest an important role for sphingosine kinase 1 in the development of hypertrophy in pulmonary arterial hypertension.

  19. Casein kinase 1 controls the activation threshold of an α-arrestin by multisite phosphorylation of the interdomain hinge

    PubMed Central

    Herrador, Antonio; Livas, Daniela; Soletto, Lucía; Becuwe, Michel; Léon, Sébastien; Vincent, Olivier

    2015-01-01

    α-Arrestins play a key role as trafficking adaptors in both yeast and mammals. The yeast Rim8/Art9 α-arrestin mediates the recruitment of endosomal sorting complex required for transport (ESCRT) to the seven-transmembrane protein Rim21 in the ambient pH signaling RIM pathway. ESCRT is believed to function as a signaling platform that enables the proteolytic activation of the Rim101 transcription factor upon external alkalization. Here we provide evidence that the pH signal promotes the stable association of Rim8 with Rim21 at the plasma membrane. We show that Rim8 is phosphorylated in a pH-independent but Rim21-dependent manner by the plasma membrane–associated casein kinase 1 (CK1). We further show that this process involves a cascade of phosphorylation events within the hinge region connecting the arrestin domains. Strikingly, loss of casein kinase 1 activity causes constitutive activation of the RIM pathway, and, accordingly, pH signaling is activated in a phosphodeficient Rim8 mutant and impaired in the corresponding phosphomimetic mutant. Our results indicate that Rim8 phosphorylation prevents its accumulation at the plasma membrane at acidic pH and thereby inhibits RIM signaling. These findings support a model in which CK1-mediated phosphorylation of Rim8 contributes to setting a signaling threshold required to inhibit the RIM pathway at acidic pH. PMID:25851600

  20. Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity.

    PubMed

    Sun, Kaiqi; Zhang, Yujin; Bogdanov, Mikhail V; Wu, Hongyu; Song, Anren; Li, Jessica; Dowhan, William; Idowu, Modupe; Juneja, Harinder S; Molina, Jose G; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

    2015-03-05

    Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

  1. Mitochondria-translocated phosphoglycerate kinase 1 functions as a protein kinase to coordinate glycolysis and TCA cycle in tumorigenesis

    PubMed Central

    Li, Xinjian; Jiang, Yuhui; Meisenhelder, Jill; Yang, Weiwei; Hawke, David H.; Zheng, Yanhua; Xia, Yan; Aldape, Kenneth; He, Jie; Hunter, Tony; Wang, Liwei; Lu, Zhimin

    2016-01-01

    SUMMARY It is unclear how the Warburg effect that exemplifies enhanced glycolysis in the cytosol is coordinated with suppressed mitochondrial pyruvate metabolism. We demonstrate here that hypoxia, EGFR activation, and expression of K-Ras G12V and B-Raf V600E induce mitochondrial translocation of phosphoglycerate kinase 1 (PGK1); this is mediated by ERK-dependent PGK1 S203 phosphorylation and subsequent PIN1-mediated cis–trans isomerization. Mitochondrial PGK1 acts as a protein kinase to phosphorylate pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 to phosphorylate and inhibit the pyruvate dehydrogenase (PDH) complex. This reduces mitochondrial pyruvate utilization, suppresses reactive oxygen species production, increases lactate production, and promotes brain tumorigenesis. Furthermore, PGK1 S203 and PDHK1 T338 phosphorylation levels correlate with PDH S293 inactivating phosphorylation levels and poor prognosis in glioblastoma patients. This work highlights that PGK1 act as a protein kinase in coordinating glycolysis and the TCA cycle, which is instrumental in cancer metabolism and tumorigenesis. PMID:26942675

  2. EMERGENCY VICTIM CARE AND RESCUE, INSTRUCTOR'S MANUAL.

    ERIC Educational Resources Information Center

    MORANDO, ROCCO V.; STOVER, WILBUR F.

    DEVELOPED AT THE STATE LEVEL BY SQUADMEN AND TRADE AND INDUSTRIAL PERSONNEL, THIS MANUAL IS FOR USE BY A QUALIFIED SQUADMAN IN TEACHING FULL-TIME AND VOLUNTEER EMERGENCY AND RESCUE WORKERS IN AN EMERGENCY SQUAD STATION OR TRAINING CENTER. TEACHING GUIDES ARE PROVIDED FOR A 30-HOUR COURSE ON EMERGENCY VICTIM CARE AND A 20-HOUR COURSE ON VICTIM…

  3. Rescuing Dogs in the Frederick Community | Poster

    Cancer.gov

    Many Frederick National Lab employees have a favorite cause to which they volunteer a significant amount of time. For Dianna Kelly, IT program manager/scientific program analyst, Office of Scientific Operations, and Courtney Kennedy, associate technical project manager, Business Enterprise Systems, that cause is dog rescue.

  4. REM sleep rescues learning from interference.

    PubMed

    McDevitt, Elizabeth A; Duggan, Katherine A; Mednick, Sara C

    2015-07-01

    Classical human memory studies investigating the acquisition of temporally-linked events have found that the memories for two events will interfere with each other and cause forgetting (i.e., interference; Wixted, 2004). Importantly, sleep helps consolidate memories and protect them from subsequent interference (Ellenbogen, Hulbert, Stickgold, Dinges, & Thompson-Schill, 2006). We asked whether sleep can also repair memories that have already been damaged by interference. Using a perceptual learning paradigm, we induced interference either before or after a consolidation period. We varied brain states during consolidation by comparing active wake, quiet wake, and naps with either non-rapid eye movement sleep (NREM), or both NREM and REM sleep. When interference occurred after consolidation, sleep and wake both produced learning. However, interference prior to consolidation impaired memory, with retroactive interference showing more disruption than proactive interference. Sleep rescued learning damaged by interference. Critically, only naps that contained REM sleep were able to rescue learning that was highly disrupted by retroactive interference. Furthermore, the magnitude of rescued learning was correlated with the amount of REM sleep. We demonstrate the first evidence of a process by which the brain can rescue and consolidate memories damaged by interference, and that this process requires REM sleep. We explain these results within a theoretical model that considers how interference during encoding interacts with consolidation processes to predict which memories are retained or lost.

  5. REM sleep rescues learning from interference

    PubMed Central

    McDevitt, Elizabeth A.; Duggan, Katherine A.; Mednick, Sara C.

    2015-01-01

    Classical human memory studies investigating the acquisition of temporally-linked events have found that the memories for two events will interfere with each other and cause forgetting (i.e., interference; Wixted, 2004). Importantly, sleep helps consolidate memories and protect them from subsequent interference (Ellenbogen, Hulbert, Stickgold, Dinges, & Thompson-Schill, 2006). We asked whether sleep can also repair memories that have already been damaged by interference. Using a perceptual learning paradigm, we induced interference either before or after a consolidation period. We varied brain states during consolidation by comparing active wake, quiet wake, and naps with either non-rapid eye movement sleep (NREM), or both NREM and REM sleep. When interference occurred after consolidation, sleep and wake both produced learning. However, interference prior to consolidation impaired memory, with retroactive interference showing more disruption than proactive interference. Sleep rescued learning damaged by interference. Critically, only naps that contained REM sleep were able to rescue learning that was highly disrupted by retroactive interference. Furthermore, the magnitude of rescued learning was correlated with the amount of REM sleep. We demonstrate the first evidence of a process by which the brain can rescue and consolidate memories damaged by interference, and that this process requires REM sleep. We explain these results within a theoretical model that considers how interference during encoding interacts with consolidation processes to predict which memories are retained or lost. PMID:25498222

  6. All-weather capability for rescue helicopters

    NASA Astrophysics Data System (ADS)

    Kreitmair-Steck, Wolfgang; Haisch, Stefan

    2001-08-01

    In Germany as well as in numerous other countries the air rescue system has been extended significantly since the first operation of the rescue helicopter Christoph 1. The primary target of the air rescue system was to guarantee fast and efficient emergency medical services for victims of accidents. During the years, the scope of the helicopter operations has been extended not only to other types of emergency medical services, but also to secondary medical services like the displacement of patients from hospitals to special service hospitals. While in general the displacement of patients is operated from well known and registered helipads, the primary rescue service currently has to rely on available onboard systems only. Those operations are risky and challenging for the pilots because of time pressure and the danger of obstacles in the environment of the helicopter. In addition, reduced visibility due to fog, rainfall or low light levels can further increase the risks or can make the services unavailable at all. Almost one decade ago, Eurocopter started the investigation of technologies and systems that could help the pilots to perform their tasks with reduced workload and risk, and to allow for a 24 h operation of helicopters irrespective of the weather conditions. After a number of preliminary studies, in 1995 the research program 'All-weather helicopter' has been started as a joint effort of Eurocopter and the supplier industry in Europe. The first phase of the program has been successfully completed in 1999 and the second phase is currently in progress.

  7. Combat Search and Rescue - Military Stepchild

    DTIC Science & Technology

    1988-04-01

    development of combat search and rescue has dwindled to the point of being inconsequential. The corporate knowledge gained in Vietnam is over twenty years old... comnat conditions was then, and remains now, an important but dangerous one. The problems of successfully performing it remain as prevalent today as

  8. Emergency Medical Rescue in a Radiation Environment

    SciTech Connect

    Briesmeister, L.; Ellington, Y.; Hollis, R.; Kunzman, J.; McNaughton, M.; Ramsey, G.; Somers, B.; Turner, A.; Finn, J.

    1999-09-14

    Previous experience with emergency medical rescues in the presence of radiation or contamination indicates that the training provided to emergency responders is not always appropriate. A new course developed at Los Alamos includes specific procedures for emergency response in a variety of radiological conditions.

  9. Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: modulation by the Nrf2/Trx axis.

    PubMed

    Niso-Santano, Mireia; González-Polo, Rosa A; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Lastres-Becker, Isabel; Ortiz-Ortiz, Miguel A; Soler, Germán; Morán, José M; Cuadrado, Antonio; Fuentes, José M

    2010-05-15

    Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases. Copyright 2010 Elsevier Inc. All rights reserved.

  10. 46 CFR 199.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... nor its stowage arrangements will interfere with the operation of any survival craft at any other...) Each rescue boat must have a means provided for recharging the rescue boat batteries from the...

  11. ISS Update: Orion Recovery and Rescue Lead Tom Walker

    NASA Image and Video Library

    NASA Public Affairs Officer Brandi Dean talks with Tom Walker, Orion Recovery and Rescue Lead, about how the Neutral Buoyancy Laboratory (NBL) is being used to train rescue and recovery personnel f...

  12. Point-of-care ultrasonography during rescue operations on board a Polish Medical Air Rescue helicopter

    PubMed Central

    Gałązkowski, Robert; Sobczyk, Dorota; Żyła, Zbigniew; Drwiła, Rafał

    2014-01-01

    Point-of-care ultrasound examination has been increasingly widely used in pre-hospital care. The use of ultrasound in rescue medicine allows for a quick differential diagnosis, identification of the most important medical emergencies and immediate introduction of targeted treatment. Performing and interpreting a pre-hospital ultrasound examination can improve the accuracy of diagnosis and thus reduce mortality. The authors’ own experiences are presented in this paper, which consist in using a portable, hand-held ultrasound apparatus during rescue operations on board a Polish Medical Air Rescue helicopter. The possibility of using an ultrasound apparatus during helicopter rescue service allows for a full professional evaluation of the patient's health condition and enables the patient to be brought to a center with the most appropriate facilities for their condition. PMID:26674604

  13. Point-of-care ultrasonography during rescue operations on board a Polish Medical Air Rescue helicopter.

    PubMed

    Darocha, Tomasz; Gałązkowski, Robert; Sobczyk, Dorota; Żyła, Zbigniew; Drwiła, Rafał

    2014-12-01

    Point-of-care ultrasound examination has been increasingly widely used in pre-hospital care. The use of ultrasound in rescue medicine allows for a quick differential diagnosis, identification of the most important medical emergencies and immediate introduction of targeted treatment. Performing and interpreting a pre-hospital ultrasound examination can improve the accuracy of diagnosis and thus reduce mortality. The authors' own experiences are presented in this paper, which consist in using a portable, hand-held ultrasound apparatus during rescue operations on board a Polish Medical Air Rescue helicopter. The possibility of using an ultrasound apparatus during helicopter rescue service allows for a full professional evaluation of the patient's health condition and enables the patient to be brought to a center with the most appropriate facilities for their condition.

  14. [Medical rescue of China National Earthquake Disaster Emergency Search and Rescue Team in Lushan earthquake].

    PubMed

    Liu, Ya-hua; Yang, Hui-ning; Liu, Hui-liang; Wang, Fan; Hu, Li-bin; Zheng, Jing-chen

    2013-05-01

    To summarize and analyze the medical mission of China National Earthquake Disaster Emergency Search and Rescue Team (CNESAR) in Lushan earthquake, to promote the medical rescue effectiveness incorporated with search and rescue. Retrospective analysis of medical work data by CNESAR from April 21th, 2013 to April 27th during Lushan earthquake rescue, including the medical staff dispatch and the wounded case been treated. The reasonable medical corps was composed by 22 members, including 2 administrators, 11 doctors [covering emergency medicine, orthopedics (joints and limbs, spinal), obstetrics and gynecology, gastroenterology, cardiology, ophthalmology, anesthesiology, medical rescue, health epidemic prevention, clinical laboratory of 11 specialties], 1 ultrasound technician, 5 nurses, 1 pharmacist, 1 medical instrument engineer and 1 office worker for propaganda. There were two members having psychological consultants qualifications. The medical work were carried out in seven aspects, including medical care assurance for the CNESAR members, first aid cooperation with search and rescue on site, clinical work in refugees' camp, medical round service for scattered village people, evacuation for the wounded, mental intervention, and the sanitary and anti-epidemic work. The medical work covered 24 small towns, and medical staff established 3 medical clinics at Taiping Town, Shuangshi Town of Lushan County and Baoxing County. Medical rescue, mental intervention for the old and kids, and sanitary and anti-epidemic were performed at the above sites. The medical corps had successful evacuated 2 severe wounded patients and treated the wounded over thousands. Most of the wounded were soft tissue injuries, external injury, respiratory tract infections, diarrhea, and heat stroke. Compared with the rescue action in 2008 Wenchuan earthquake, the aggregation and departure of rescue team in Lushan earthquake, the traffic control order in disaster area, the self-aid and buddy aid

  15. Minimum-fuel rescue trajectories for the Extravehicular Excursion Unit

    NASA Technical Reports Server (NTRS)

    Fowler, W. T.; Neff, J. M.

    1991-01-01

    The problem of determining minimum-fuel trajectories for rescuing astronauts or equipment which become separated from a Space Station is addressed. Using the Clohessy-Wiltshire equations of relative motion and assuming impulsive Delta-Vs, the minimum-fuel rescue problem is shown to be a parameter optimization problem. Minimum-fuel rescue trajectories are found for seventeen test cases using a recursive quadratic programming algorithm. The results are analyzed and general rules for astronaut rescue and equipment retrieval are developed.

  16. Safety in alpine helicopter rescue operations--minimal requirements of alpine skills for rescue personnel.

    PubMed

    Küpper, Thomas; Hillebrandt, David; Steffgen, Juergen; Schöffl, Volker

    2013-11-01

    There is a lack of data to establish minimal requirements for technical alpine climbing skills needed by rescue teams involved in alpine helicopter rescue operations to perform such operations safely. A year of rescue operations (N = 2731) were investigated for the technical difficulties of the terrain. The difficulties were graded according to the Union Internationale des Associations d'Alpinisme (UIAA) scale for rocky terrain and steepness for ice slopes. For 99.7% of the operations, the terrain could be accurately evaluated. In at least 30.7% of all rescue operations, personal advanced alpine climbing skills were required for the rescue personnel, and in 6.0%, the difficulties of the rocky terrain correspond to UIAA scale grade III with another 2.4% to UIAA grade IV or above. About 1.5% of all operations took place in ice faces steeper than 50°. To be able to manage 90% of all operations safely, all crew members, except the pilot, must be competent at climbing rock terrain of UIAA scale grade IV and ice of 50° steepness using appropriate rescue, rope, and belaying techniques. These recommendations include a technical safety margin for adverse conditions, such as bad weather.

  17. Resource Guide for Search and Rescue Training Materials.

    ERIC Educational Resources Information Center

    LaValla, Patrick

    The bibliography about search and rescue training materials lists booklets, books, manuals, films, papers, periodicals, and pamphlets that treat many aspects of search and rescue situations: general, cave, disaster, and mountain rescues; strategy tactics; communications; knots and ropes; outdoor living; dogs; tracking; map and compass; survival;…

  18. Maryland Fire-Rescue Education and Training System. Organizational Design.

    ERIC Educational Resources Information Center

    Maryland Fire-Rescue Education and Training Commission.

    This is a description of the Maryland system which was created to evaluate local fire-rescue education and training needs and capabilities and to assist local authorities with fire-rescue education and training. In the first of four parts, an historical presentation is used to identify and describe in general terms the state fire, rescue, and…

  19. 47 CFR 80.1125 - Search and rescue coordinating communications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Search and rescue coordinating communications...) Operating Procedures for Distress and Safety Communications § 80.1125 Search and rescue coordinating... responsible for controlling a search and rescue operation will also coordinate the distress traffic relating...

  20. 47 CFR 80.1125 - Search and rescue coordinating communications.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Search and rescue coordinating communications...) Operating Procedures for Distress and Safety Communications § 80.1125 Search and rescue coordinating... responsible for controlling a search and rescue operation will also coordinate the distress traffic relating...

  1. 47 CFR 80.1125 - Search and rescue coordinating communications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Search and rescue coordinating communications...) Operating Procedures for Distress and Safety Communications § 80.1125 Search and rescue coordinating... responsible for controlling a search and rescue operation will also coordinate the distress traffic relating...

  2. 30 CFR 49.8 - Training for mine rescue teams.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Training for mine rescue teams. 49.8 Section 49.8 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Metal and Nonmetal Mines § 49.8 Training for...

  3. 30 CFR 49.8 - Training for mine rescue teams.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Training for mine rescue teams. 49.8 Section 49.8 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Metal and Nonmetal Mines § 49.8 Training for...

  4. 30 CFR 49.8 - Training for mine rescue teams.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Training for mine rescue teams. 49.8 Section 49.8 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Metal and Nonmetal Mines § 49.8 Training for...

  5. 30 CFR 49.2 - Availability of mine rescue teams.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Availability of mine rescue teams. 49.2 Section 49.2 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS § 49.2 Availability of mine rescue teams. (a) Except where...

  6. 30 CFR 49.12 - Availability of mine rescue teams.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Availability of mine rescue teams. 49.12 Section 49.12 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.12 Availability of mine...

  7. 30 CFR 49.8 - Training for mine rescue teams.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Training for mine rescue teams. 49.8 Section 49.8 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS § 49.8 Training for mine rescue teams. (a) Prior to serving on a mine...

  8. 30 CFR 49.5 - Mine rescue station.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Mine rescue station. 49.5 Section 49.5 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS § 49.5 Mine rescue station. (a) Except where alternative compliance is permitted, every...

  9. 30 CFR 49.5 - Mine rescue station.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Mine rescue station. 49.5 Section 49.5 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS § 49.5 Mine rescue station. (a) Except where alternative compliance is permitted, every...

  10. 30 CFR 49.18 - Training for mine rescue teams.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Training for mine rescue teams. 49.18 Section 49.18 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING MINE RESCUE TEAMS Mine Rescue Teams for Underground Coal Mines § 49.18 Training for mine...

  11. 46 CFR 131.855 - Lifeboats and rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Lifeboats and rescue boats. 131.855 Section 131.855... Markings for Fire Equipment and Emergency Equipment § 131.855 Lifeboats and rescue boats. (a) The following must be plainly marked or painted on each side of the bow of each lifeboat and rescue boat in block...

  12. 46 CFR 131.855 - Lifeboats and rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Lifeboats and rescue boats. 131.855 Section 131.855... Markings for Fire Equipment and Emergency Equipment § 131.855 Lifeboats and rescue boats. (a) The following must be plainly marked or painted on each side of the bow of each lifeboat and rescue boat in block...

  13. 46 CFR 199.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Stowage of rescue boats. 199.140 Section 199.140... boats. (a) General. Rescue boats must be stowed— (1) To be ready for launching in not more than 5 minutes. (2) In a position suitable for launching and recovery; (3) In a way that neither the rescue boat...

  14. 46 CFR 199.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Stowage of rescue boats. 199.140 Section 199.140... boats. (a) General. Rescue boats must be stowed— (1) To be ready for launching in not more than 5 minutes. (2) In a position suitable for launching and recovery; (3) In a way that neither the rescue boat...

  15. 46 CFR 131.855 - Lifeboats and rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Lifeboats and rescue boats. 131.855 Section 131.855... Markings for Fire Equipment and Emergency Equipment § 131.855 Lifeboats and rescue boats. (a) The following must be plainly marked or painted on each side of the bow of each lifeboat and rescue boat in block...

  16. 46 CFR 131.855 - Lifeboats and rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Lifeboats and rescue boats. 131.855 Section 131.855... Markings for Fire Equipment and Emergency Equipment § 131.855 Lifeboats and rescue boats. (a) The following must be plainly marked or painted on each side of the bow of each lifeboat and rescue boat in block...

  17. 46 CFR 199.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Stowage of rescue boats. 199.140 Section 199.140... boats. (a) General. Rescue boats must be stowed— (1) To be ready for launching in not more than 5 minutes. (2) In a position suitable for launching and recovery; (3) In a way that neither the rescue boat...

  18. 46 CFR 199.140 - Stowage of rescue boats.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Stowage of rescue boats. 199.140 Section 199.140... boats. (a) General. Rescue boats must be stowed— (1) To be ready for launching in not more than 5 minutes. (2) In a position suitable for launching and recovery; (3) In a way that neither the rescue boat...

  19. 46 CFR 131.855 - Lifeboats and rescue boats.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Lifeboats and rescue boats. 131.855 Section 131.855... Markings for Fire Equipment and Emergency Equipment § 131.855 Lifeboats and rescue boats. (a) The following must be plainly marked or painted on each side of the bow of each lifeboat and rescue boat in block...

  20. 47 CFR 80.1125 - Search and rescue coordinating communications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Search and rescue coordinating communications...) Operating Procedures for Distress and Safety Communications § 80.1125 Search and rescue coordinating... Coordination Center responsible for controlling a search and rescue operation will also coordinate the...

  1. Resource Guide for Search and Rescue Training Materials.

    ERIC Educational Resources Information Center

    LaValla, Patrick

    The bibliography about search and rescue training materials lists booklets, books, manuals, films, papers, periodicals, and pamphlets that treat many aspects of search and rescue situations: general, cave, disaster, and mountain rescues; strategy tactics; communications; knots and ropes; outdoor living; dogs; tracking; map and compass; survival;…

  2. 46 CFR 108.565 - Stowage of rescue boats.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Stowage of rescue boats. 108.565 Section 108.565 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Lifesaving Equipment § 108.565 Stowage of rescue boats. (a) Rescue boats must be...

  3. Psychiatric disorders after terrorist bombings among rescue workers and bombing survivors in Nairobi and rescue workers in Oklahoma City.

    PubMed

    Zhang, Gus; Pfefferbaum, Betty; Narayanan, Pushpa; Lee, Sungkyu; Thielman, Samuel; North, Carol S

    2016-02-01

    To examine the prevalence of psychopathology in 52 male rescue workers responding to the 1998 U.S. Embassy bombing in Nairobi, Kenya, comparing them with 176 male rescue workers responding to the 1995 Oklahoma City, Oklahoma, bombing and with 105 directly exposed male civilian survivors of the Nairobi bombing. The Diagnostic Interview Schedule/Disaster Supplement assessed pre-disaster and post-disaster psychiatric disorders and variables related to demographics, exposure, disaster perceptions, and coping in all 3 disaster subgroups. The most prevalent post-disaster disorders were posttraumatic stress disorder (PTSD) (22%) and major depressive disorder (MDD) (27%) among Nairobi rescue workers, which were more than 2 and 4 times higher, respectively, than among Oklahoma City rescue workers. Alcohol use disorder was the most prevalent pre- and post-disaster disorder among Oklahoma City rescue workers. Nairobi rescue workers had a prevalence of PTSD and MDD not significantly different from Nairobi civilian survivors. Nairobi rescue workers were more symptomatic than Oklahoma City rescue workers and were as symptomatic as Nairobi civilian survivors. The vulnerability of Nairobi rescue workers to psychological sequelae may be a reflection of their volunteer, rather than professional, status. These findings contribute to understanding rescue worker mental health, especially among volunteer rescue workers, with potential implications for the importance of professional status of rescue workers in conferring protection from adverse mental health outcomes.

  4. Microtubule-binding protein doublecortin-like kinase 1 (DCLK1) guides kinesin-3-mediated cargo transport to dendrites.

    PubMed

    Lipka, Joanna; Kapitein, Lukas C; Jaworski, Jacek; Hoogenraad, Casper C

    2016-02-01

    In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule-binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin-3 (KIF1) and kinesin-4 (KIF21) subfamily that can also target dendrites. We found that microtubule-binding protein doublecortin-like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1-dependent dense-core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule-binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport. © 2016 The Authors.

  5. Downregulation of Polo-like kinase 1 induces cellular senescence in human primary cells through a p53-dependent pathway.

    PubMed

    Kim, Hee-Jin; Cho, Jung Hee; Kim, Jae-Ryong

    2013-10-01

    Polo-like kinase 1 (PLK1) plays a key role in various stages of mitosis from entry into M phase to exit from mitosis. However, its role in cellular senescence remains to be determined. Therefore, the effects of PLK1 on cellular senescence in human primary cells were investigated. We found that expression of PLK1 decreased in human dermal fibroblasts and human umbilical vein endothelial cells under replicative senescence and premature senescence induced by adriamycin. PLK1 knockdown with PLK1 small interfering RNAs in young cells induced premature senescence. In contrast, upregulation of PLK1 in old cells partially reversed senescence phenotypes. Cellular senescence by PLK1 inhibition was observed in p16 knockdown cells but not in p53 knockdown cells. Our data suggest that PLK1 repression might result in cellular senescence in human primary cells via a p53-dependent pathway.

  6. Receptor for Activated C Kinase 1 (RACK1) Promotes Dishevelled Protein Degradation via Autophagy and Antagonizes Wnt Signaling.

    PubMed

    Cheng, Minzhang; Xue, Hua; Cao, Weipeng; Li, Wenxia; Chen, Hua; Liu, Bofeng; Ma, Benyu; Yan, Xiaohua; Chen, Ye-Guang

    2016-06-10

    Wnt signaling plays a critical role in embryonic development, tissue homeostasis, and cancer development. Dishevelled (Dvl) is an essential and central component in Wnt signaling, and its stability and activity is tightly regulated. It has been shown that Dvl can be degraded via both the proteasome and autophagy-lysosome pathways. Here we report that receptor for activated C kinase 1 (RACK1) negatively regulates Dishevelled stability and Wnt signaling. RACK1 interacts with Dvl proteins and promotes their lysosomal degradation, and this effect is enhanced by autophagy induction. RACK1 also interacts with LC3 and enhances the association of LC3 with Dvl2, thereby leading to degradation of Dvl proteins through autophagy. These findings reveal a novel regulatory function of RACK1 in Wnt signaling by modulating Dvl stability. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Activation of serum/glucocorticoid-induced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy

    PubMed Central

    Andres-Mateos, Eva; Brinkmeier, Heinrich; Burks, Tyesha N; Mejias, Rebeca; Files, Daniel C; Steinberger, Martin; Soleimani, Arshia; Marx, Ruth; Simmers, Jessica L; Lin, Benjamin; Finanger Hedderick, Erika; Marr, Tom G; Lin, Brian M; Hourdé, Christophe; Leinwand, Leslie A; Kuhl, Dietmar; Föller, Michael; Vogelsang, Silke; Hernandez-Diaz, Ivan; Vaughan, Dana K; Alvarez de la Rosa, Diego; Lang, Florian; Cohn, Ronald D

    2013-01-01

    Maintaining skeletal muscle mass is essential for general health and prevention of disease progression in various neuromuscular conditions. Currently, no treatments are available to prevent progressive loss of muscle mass in any of these conditions. Hibernating mammals are protected from muscle atrophy despite prolonged periods of immobilization and starvation. Here, we describe a mechanism underlying muscle preservation and translate it to non-hibernating mammals. Although Akt has an established role in skeletal muscle homeostasis, we find that serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates muscle mass maintenance via downregulation of proteolysis and autophagy as well as increased protein synthesis during hibernation. We demonstrate that SGK1 is critical for the maintenance of skeletal muscle homeostasis and function in non-hibernating mammals in normal and atrophic conditions such as starvation and immobilization. Our results identify a novel therapeutic target to combat loss of skeletal muscle mass associated with muscle degeneration and atrophy. PMID:23161797

  8. Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

    SciTech Connect

    Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M.

    2014-10-02

    Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

  9. Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7.

    PubMed

    Chia, Ruth; Haddock, Sara; Beilina, Alexandra; Rudenko, Iakov N; Mamais, Adamantios; Kaganovich, Alice; Li, Yan; Kumaran, Ravindran; Nalls, Michael A; Cookson, Mark R

    2014-12-15

    LRRK2, a gene relevant to Parkinson's disease, encodes a scaffolding protein with both GTPase and kinase activities. LRRK2 protein is itself phosphorylated and therefore is subject to regulation by cell signalling; however, the kinase(s) responsible for this event have not been definitively identified. Here using an unbiased siRNA kinome screen, we identify and validate casein kinase 1α (CK1α) as being responsible for LRRK2 phosphorylation, including in the adult mouse striatum. We further show that LRRK2 recruitment to TGN46-positive Golgi-derived vesicles is modulated by constitutive LRRK2 phosphorylation by CK1α. These effects are mediated by differential protein interactions of LRRK2 with a guanine nucleotide exchange factor, ARHGEF7. These pathways are therefore likely involved in the physiological maintenance of the Golgi in cells, which may play a role in the pathogenesis of Parkinson's disease.

  10. Comparative phosphoproteomics to identify targets of the clock-relevant casein kinase 1 in C. reinhardtii Flagella.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2014-01-01

    In the green biflagellate alga Chlamydomonas reinhardtii different clock-relevant components have been identified that are involved in maintaining phase, period, and amplitude of circadian rhythms. It became evident that several of them are interconnected to flagellar function such as CASEIN KINASE1 (CK1). CK1 is involved in keeping the period. But it is also relevant for the formation of flagella, where it is physically located, and it controls the swimming velocity. In this chapter, we describe (1) how the flagellar sub-proteome is purified, (2) how phosphopeptides from this organelle are enriched, (3) how in vivo phosphorylation sites are determined, and (4) how direct and indirect flagellar targets of CK1 can be found using a specific inhibitor. Such a procedure can also be employed with other clock-relevant kinases if specific inhibitors or mutants are available.

  11. Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to Induce Mitotic Arrest in Tumor Cells.

    PubMed

    Qin, Tan; Chen, Fangjin; Zhuo, Xiaolong; Guo, Xiao; Yun, Taikangxiang; Liu, Ying; Zhang, Chuanmao; Lai, Luhua

    2016-08-11

    Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 μM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

  12. Protein expression of phospho-lim kinase-1 in patients and an experimental rat model with intractable temporal lobe epilepsy

    PubMed Central

    Huang, Hao; Wang, Heng; Yuan, Jinxian; Wu, Xuling; Huang, Yunyi; Zhou, Xin; Chen, Yangmei

    2015-01-01

    Lim kinase-1 (LIMK1) plays a critical role in dendritic spine morphogenesis and brain function. The protein expression pattern of phospho-LIMK1 (p-LIMK1), the active form of LIMK1, in intractable temporal lobe epilepsy (TLE), however, is unknown. Here we measured p-LIMK1 protein expression in thirty temporal neocortex tissue samples from intractable TLE patients, fifteen histologically normal temporal neocortex tissue samples from trauma patients without epilepsy, in the hippocampi of lithium chloride/pilocarpine-induced TLE rats, and in controls. We found that p-LIMK1 was expressed mainly in the cytoplasm of neurons. The protein expression of p-LIMK1 was significantly higher in the TLE patients and rats than in the control groups. Our results suggest that p-LIMK1 might be involved in the pathogenesis of intractable TLE. PMID:25785037

  13. Mechanism of fibrotic cardiomyopathy in mice expressing truncated Rho-associated coiled-coil protein kinase 1

    PubMed Central

    Yang, Xiangsheng; Li, Qi; Lin, Xi; Ma, Yanlin; Yue, Xiaojing; Tao, Zhenyin; Wang, Fen; Mckeehan, Wallace L.; Wei, Lei; Schwartz, Robert J.; Chang, Jiang

    2012-01-01

    We have previously found that in failing human hearts, Rho-associated coiled-coil protein kinase 1 (ROCK1) is processed by caspase-3 into an active isoform, ROCKΔ1. The purpose of the current investigation was to elucidate the pathological consequences of truncated ROCK1 accumulation in the heart, the associated molecular mechanism of ROCKΔ1-mediated cardiac phenotype, and the molecular signaling between Rho kinase activation in cardiomyocytes and extracellular matrix response. We generated transgenic mice expressing ROCKΔ1 in cardiomyocytes to mimic the situation observed in human heart disease, whereas an additional kinase-deficient mouse was generated as a control. The ROCKΔ1 transgenic mice developed fibrotic cardiomyopathy with diastolic dysfunction. Transgenic hearts displayed activated TGFβ1 and NF-κB signaling and a release of a subset of cytokines and were susceptible to angiotensin II stress. Treatment with a Rho kinase inhibitor attenuated the fibrotic phenotype. Cardiac fibroblasts differentiated into myofibroblasts when cocultured with transgenic cardiomyocytes but not with wild-type cardiomyocytes. Inhibitors of Rho kinase as well as TGFβR1 and NF-κB decreased these effects. The serum response factor-dependent TGFβ1 regulation was shown to be responsible for the Rho kinase-mediated activation of TGFβ1 signaling. We conclude that ROCKΔ1 is a novel fibrotic factor. Activation of TGFβ1 and NF-κB signaling contributes to the Rho kinase-mediated pathological fibrosis.—Yang, X., Li, Q., Lin, X., Ma, Y., Yue, X., Tao, Z., Wang, F., Mckeehan, W. L., Wei, L., Schwartz, R. J., Chang, J. Mechanism of fibrotic cardiomyopathy in mice expressing truncated Rho-associated coiled-coil protein kinase 1. PMID:22278938

  14. Salt-inducible kinase 1 (SIK1) is induced by gastrin and inhibits migration of gastric adenocarcinoma cells.

    PubMed

    Selvik, Linn-Karina M; Rao, Shalini; Steigedal, Tonje S; Haltbakk, Ildri; Misund, Kristine; Bruland, Torunn; Prestvik, Wenche S; Lægreid, Astrid; Thommesen, Liv

    2014-01-01

    Salt-inducible kinase 1 (SIK1/Snf1lk) belongs to the AMP-activated protein kinase (AMPK) family of kinases, all of which play major roles in regulating metabolism and cell growth. Recent studies have shown that reduced levels of SIK1 are associated with poor outcome in cancers, and that this involves an invasive cellular phenotype with increased metastatic potential. However, the molecular mechanism(s) regulated by SIK1 in cancer cells is not well explored. The peptide hormone gastrin regulates cellular processes involved in oncogenesis, including proliferation, apoptosis, migration and invasion. The aim of this study was to examine the role of SIK1 in gastrin responsive adenocarcinoma cell lines AR42J, AGS-GR and MKN45. We show that gastrin, known to signal through the Gq/G11-coupled CCK2 receptor, induces SIK1 expression in adenocarcinoma cells, and that transcriptional activation of SIK1 is negatively regulated by the Inducible cAMP early repressor (ICER). We demonstrate that gastrin-mediated signalling induces phosphorylation of Liver Kinase 1B (LKB1) Ser-428 and SIK1 Thr-182. Ectopic expression of SIK1 increases gastrin-induced phosphorylation of histone deacetylase 4 (HDAC4) and enhances gastrin-induced transcription of c-fos and CRE-, SRE-, AP1- and NF-κB-driven luciferase reporter plasmids. We also show that gastrin induces phosphorylation and nuclear export of HDACs. Next we find that siRNA mediated knockdown of SIK1 increases migration of the gastric adenocarcinoma cell line AGS-GR. Evidence provided here demonstrates that SIK1 is regulated by gastrin and influences gastrin elicited signalling in gastric adenocarcinoma cells. The results from the present study are relevant for the understanding of molecular mechanisms involved in gastric adenocarcinomas.

  15. Role of serum- and glucocorticoid-inducible kinase-1 in regulating torsion-induced apoptosis in rats

    PubMed Central

    Cho, Y-M; Pu, H-F; Huang, W J; Ho, L-T; Wang, S-W; Wang, P S

    2011-01-01

    Serum- and glucocorticoid-inducible kinase-1 (SGK1) is a serine/threonine protein kinase that responds to various stimuli and mediates cell survival. Although it is known that testicular torsion leads to testicular damage and male infertility, the role of SGK1 in torsion remains unclear. This study investigated whether torsion-induced apoptosis is associated with changes in phosphoinositide-dependent protein kinase-1 (PDK1), SGK1 and forkhead transcription factor FOXO3a expression and/or phosphorylation in rats. Sprague-Dawley rats were divided into four groups: sham (control), 1, 2 and 4 h of unilateral torsion. Bilateral testes, testicular interstitial fluid (TIF) and blood samples were collected immediately after torsion. Our results revealed that SGK1 protein and mRNA were abundantly present in testes and were induced by 2 h of torsion, but that phosphorylation of SGK1, PDK1 and FOXO3a decreased simultaneously. After 2 h of torsion, the testosterone secretion capacity of the primary Leydig cells and testicular interstitial cells (TICs) was impaired and apoptotic spermatogonia and TICs were observed; in addition, the mean seminiferous tubular diameter was decreased. Torsion increased plasma corticosterone levels, but decreased plasma luteinizing hormone and testosterone levels. However, the testosterone levels of the TIF in the ipsilateral testes were significantly enhanced after 2 h of torsion, but suppressed in the contralateral testes. This animal study suggests that PDK1, SGK1 and FOXO3a are involved in torsion-induced apoptosis and that medical therapy should be performed as early as 2 h after the occurrence of torsion to prevent further damage. PMID:20738430

  16. Targeting Polo-Like Kinase 1 Enhances Radiation Efficacy for Head-and-Neck Squamous Cell Carcinoma

    SciTech Connect

    Gerster, Kate; Shi Wei; Ng, Benjamin; Yue Shijun; Ito, Emma; Waldron, John; Gilbert, Ralph; Liu Feifei

    2010-05-01

    Purpose: To investigate the efficacy of targeting polo-like kinase 1 (Plk1) combined with ionizing radiotherapy (RT) for head-and-neck squamous cell carcinoma (HNSCC). Methods and Materials: Polo-like kinase 1 messenger ribonucleic acid (mRNA) was targeted by small interfering RNA (siRNA) transfection into the FaDu HNSCC cell line; reduction was confirmed using quantitative real-time polymerase chain reaction. The cellular effects were assessed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium], clonogenic, flow cytometric, and caspase assays. In vivo efficacy of siPlk1 was evaluated using mouse xenograft models. Results: Small interfering Plk1 significantly decreased Plk1 mRNA expression, while also increasing cyclin B1 and p21(Waf1/CIP1) mRNA levels after 24 h. This depletion resulted in a time-dependent increase in FaDu cytotoxicity, which was enhanced by the addition of RT. Flow cytometric and caspase assays demonstrated progressive apoptosis, DNA double-strand breaks (gamma-H2AX), G2/M arrest, and activation of caspases 3 and 7. Implantation of siPlk1-treated FaDu cells in severe combined immunodeficient mice delayed tumor formation, and systemic administration of siPlk1 inhibited tumor growth enhanced by RT. Conclusions: These data demonstrate the suitability of Plk1 as a potential therapeutic target for HNSCC, because Plk1 depletion resulted in significant cytotoxicity in vitro and abrogated tumor-forming potential in vivo. The effects of Plk1 depletion were enhanced with the addition of RT, indicating that Plk1 represents an important potential radiation sensitizer for HNSCC.

  17. Issues in intelligent robots for search and rescue

    NASA Astrophysics Data System (ADS)

    Casper, Jennifer L.; Micire, Mark; Murphy, Robin R.

    2000-07-01

    Since the 1995 Oklahoma City bombing and Kobe, Japan, earthquake, robotics researchers have been considering search and rescue as a humanitarian research domain. The recent devastation in Turkey and Taiwan, compounded with the new Robocup Rescue and AAAI Urban Search and Rescue robot competition, may encourage more research. However, roboticists generally go not have access to domain experts: the emergency workers or first providers. This paper shares our understanding of urban search and rescue, based on our active research in this area and training sessions with rescue workers from the Hillsborough County (Florida) Fire Departments. The paper is intended to be a stepping stone for roboticists entering the field.

  18. Rescue intracytoplasmic sperm injection: a systematic review.

    PubMed

    Beck-Fruchter, Ronit; Lavee, Michal; Weiss, Amir; Geslevich, Yoel; Shalev, Eliezer

    2014-03-01

    To assess the feasibility, efficacy, and safety of rescue intracytoplasmic sperm injection (ICSI) in cases of fertilization failure, using a scientific literature search. Systematic review. Centers for reproductive care. Infertility patients with total or partial fertilization failure during an IVF cycle. An electronic literature search was performed in PubMed from 1992 through May 2013. The search was then expanded by using listed references from selected articles. Pregnancy rate. The secondary outcome measures were fertilization rate, normal fertilization rate, cleavage rate, birth rate, and malformation rate. Thirty-eight studies including 1,863 patients were included. The pooled pregnancy rate was 14.4%; 194 babies were delivered. Rescue ICSI can result in the delivery of a healthy newborn, although the pregnancy rates are low. The clinical evidence did not indicate an elevated rate of malformations, although the data are limited and incomplete. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  19. Skylab rescue space vehicle flight readiness test

    NASA Technical Reports Server (NTRS)

    Jevitt, S. J.

    1973-01-01

    A Skylab Rescue Space Vehicle flight readiness test is described which ensures that space vehicle systems are in a state of flight readiness and are compatible with associated ground support equipment. The functions of propellant loading, umbilical ejection, ignition, holddown arm release, liftoff, and service arm and tail service mast retraction are simulated. The test outline is presented along with a list of references, intercommunications information, operations interface control chart, and flight test.

  20. Combat Search and Rescue in Desert Storm

    DTIC Science & Technology

    2006-09-01

    literally thou- sands of airmen. It extensively documented what is now con- sidered the “ golden age” of rescue. This work is meant to follow in those...Bergan 23 Norwich 21 Tbird 56 Caesar 44 Jackal 11 Quicksand 12 Sunliner 403 Stroke 65 Clap 74 Newport 15 Corvette 03 Slate 46Stamford 01 Wolf 01...covered with AAA.52 CDR Bob Stumpf in the strike package remembered the scene. Seeing the glow stretching across the horizon and the “ golden BBs

  1. Rescuing Downed Aircrews: The Value of Time

    DTIC Science & Technology

    2015-01-01

    Warning and Control System C2 command and control CAS close air support CDF cumulative density function CSAR combat search and rescue FROG free...destroying Iraqi FROG (free rocket over ground) missile batteries. The aircraft was hit by a missile while engaging enemy forces and crashed into the...members were soon captured. 8. January 18, 1991, USMC OV-10: The aircraft was flying in Kuwait searching for targets, such as Iraqi artillery and FROG

  2. Lidar techniques for search and rescue

    SciTech Connect

    Cabral, W.L.

    1985-01-01

    Four techniques for using LIDAR in Search and Rescue Operations will be discussed. The topic will include laser retroreflection, laser-induced fluorescence in the visible, laser-induced fluorescence during daylight hours, and laser-induced fluorescence in the uv. These techniques use high-repetition rate lasers at a variety of frequencies to induce either fluorescence in dye markers or retroreflection from plastic corner cubes on life preservers and other emergency markers.

  3. Medical considerations in the use of helicopters in mountain rescue.

    PubMed

    Tomazin, Iztok; Kovacs, Tim

    2003-01-01

    The outcome of patient care can be dramatically improved by bringing rapid rescue and medical care to the mountain rescue scene and by rapid transport to a medical facility. The use of a helicopter for these purposes is common. It is necessary when it has clear advantages for victims in comparison with ground rescue and transport. Helicopters should work within the existing emergency medical system and must be staffed by appropriate mountain rescue and medically trained personnel. Activation time should be as short as possible. Activation of a helicopter for a mountain rescue should primarily include indication and assessment of flight and safety conditions. No other mediators or delaying factors should be permitted. The main safety criteria are appropriate mountain rescue and flight training, competence of air and ground crews, radio communication between the air and ground crews, and mission briefing before the rescue. Criteria for a helicopter used for mountain rescue are proper medical and rescue equipment, load capacity, adequate space, and others. There are two main groups of indications for use of a helicopter for mountain rescue: the patient's condition and the circumstances at the site of the accident. All persons responsible for the activation of the helicopter rescue operation should be aware of specific problems in the mountains or wilderness.

  4. Monte Carlo simulations within avalanche rescue

    NASA Astrophysics Data System (ADS)

    Reiweger, Ingrid; Genswein, Manuel; Schweizer, Jürg

    2016-04-01

    Refining concepts for avalanche rescue involves calculating suitable settings for rescue strategies such as an adequate probing depth for probe line searches or an optimal time for performing resuscitation for a recovered avalanche victim in case of additional burials. In the latter case, treatment decisions have to be made in the context of triage. However, given the low number of incidents it is rarely possible to derive quantitative criteria based on historical statistics in the context of evidence-based medicine. For these rare, but complex rescue scenarios, most of the associated concepts, theories, and processes involve a number of unknown "random" parameters which have to be estimated in order to calculate anything quantitatively. An obvious approach for incorporating a number of random variables and their distributions into a calculation is to perform a Monte Carlo (MC) simulation. We here present Monte Carlo simulations for calculating the most suitable probing depth for probe line searches depending on search area and an optimal resuscitation time in case of multiple avalanche burials. The MC approach reveals, e.g., new optimized values for the duration of resuscitation that differ from previous, mainly case-based assumptions.

  5. Big Data Cognition for City Emergency Rescue

    NASA Astrophysics Data System (ADS)

    Zhang, Xin; Chen, Yongxin; Wang, Weisheng

    2016-11-01

    There are many kinds of data produced in the city daily life, which operates as an elementary component of the citizen life support system. The city unexpected incidents occurs in a seemingly unpredictable patterns. With the Big Data analysis the emergency rescue can be carried out efficiently. In this paper, the Big Data cognition for city emergency rescue is studied from four perspectives. From the data volume perspective, the spatial data analysis technology is divided into two parts, the indoor data and the outdoor data. From the data velocity perspective, the big data is collected from the eyes in the sky and objects on-the-ground networks, together with demographic data. From the data variety analysis perspective, the population distribution data, the socio-economic data and model estimates are included. From the data value mining perspective, the crime model estimates are studied. In the end, the application in the big public venues emergency rescue is introduced, which is located in Urumqi, Xinjiang, China.

  6. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility (SLF), the KSC response team removes a crew member from a mock Shuttle. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to drop emergency equipment and fire/rescue workers to prepare the 'crew' for preliminary triage. The helicopters later are used to remove the crew five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise concluded with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  7. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    In the simulated rescue mission, the KSC response team takes part in the unlikely scenario of a Shuttle mishap at the Shuttle Landing Facility. The Mode 7 simulation of an astronaut rescue exercised all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to drop emergency equipment and fire/rescue workers to prepare the 'crew' for preliminary triage. The helicopters later are used to remove the crew five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise concluded with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.'

  8. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility, the KSC response team removes a Shuttle 'crew' member from the mock orbiter. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to drop emergency equipment and fire/rescue workers to prepare the 'crew' for preliminary triage. The helicopters later are used to remove the crew five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise concluded with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  9. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility, the KSC response team removes a Shuttle 'crew' member from the mock orbiter. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to drop emergency equipment and fire/rescue workers to prepare the 'crew' for preliminary triage. The helicopters later are used to remove the crew five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise concluded with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  10. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility (SLF), the KSC response team removes a crew member from a mock Shuttle. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to drop emergency equipment and fire/rescue workers to prepare the 'crew' for preliminary triage. The helicopters later are used to remove the crew five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise concluded with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  11. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    In the simulated rescue mission, the KSC response team takes part in the unlikely scenario of a Shuttle mishap at the Shuttle Landing Facility. The Mode 7 simulation of an astronaut rescue exercised all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to drop emergency equipment and fire/rescue workers to prepare the 'crew' for preliminary triage. The helicopters later are used to remove the crew five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise concluded with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.'

  12. Avalanche Survival After Rescue With the RECCO Rescue System: A Case Report.

    PubMed

    Grasegger, Katharina; Strapazzon, Giacomo; Procter, Emily; Brugger, Hermann; Soteras, Inigo

    2016-06-01

    We report a case of survival of a completely buried avalanche victim after being located with the radar-based RECCO Rescue System. In the winter of 2015, 2 off-piste skiers were completely buried in an avalanche near the secured ski area in Baqueira Beret, Spain. The first victim was located with the RECCO Rescue System in less than 35 minutes and was alive and conscious at extrication. This system emits radio waves and requires a specific reflector. It is a portable device that is used by more than 600 rescue organizations worldwide, especially in secured ski areas. The device should be brought to the avalanche site together with electronic avalanche transceivers, a probing team, and avalanche dogs. In the hands of experienced professionals, the device may allow rapid location of victims not carrying an electronic avalanche transceiver. Although it is not the first successful extrication of a victim with the RECCO Rescue System, it is the first case published in the medical literature and is intended to encourage data collection and to increase our understanding of the effectiveness of this device in avalanche rescue.

  13. Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1.

    PubMed

    Child, Matthew A; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A; Boothroyd, John C; Reese, Michael L; Bogyo, Matthew

    2017-02-28

    Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these

  14. A novel role of sphingosine kinase-1 in the invasion and angiogenesis of VHL mutant clear cell renal cell carcinoma

    PubMed Central

    Salama, Mohamed F.; Carroll, Brittany; Adada, Mohamad; Pulkoski-Gross, Michael; Hannun, Yusuf A.; Obeid, Lina M.

    2015-01-01

    Sphingosine kinase 1 (SK1), the enzyme responsible for sphingosine 1-phosphate (S1P) production, is overexpressed in many human solid tumors. However, its role in clear cell renal cell carcinoma (ccRCC) has not been described previously. ccRCC cases are usually associated with mutations in von Hippel-Lindau (VHL) and subsequent normoxic stabilization of hypoxia-inducible factor (HIF). We previously showed that HIF-2α up-regulates SK1 expression during hypoxia in glioma cells. Therefore, we hypothesized that the stabilized HIF in ccRCC cells will be associated with increased SK1 expression. Here, we demonstrate that SK1 is overexpressed in 786-0 renal carcinoma cells lacking functional VHL, with concomitant high S1P levels that appear to be HIF-2α mediated. Moreover, examining the TCGA RNA seq database shows that SK1 expression was ∼2.7-fold higher in solid tumor tissue from ccRCC patients, and this was associated with less survival. Knockdown of SK1 in 786-0 ccRCC cells had no effect on cell proliferation. On the other hand, this knockdown resulted in an ∼3.5-fold decrease in invasion, less phosphorylation of focal adhesion kinase (FAK), and an ∼2-fold decrease in angiogenesis. Moreover, S1P treatment of SK1 knockdown cells resulted in phosphorylation of FAK and invasion, and this was mediated by S1P receptor 2. These results suggest that higher SK1 and S1P levels in VHL-defective ccRCC could induce invasion in an autocrine manner and angiogenesis in a paracrine manner. Accordingly, targeting SK1 could reduce both the invasion and angiogenesis of ccRCC and therefore improve the survival rate of patients.—Salama, M. F., Carroll, B., Adada, M., Pulkoski-Gross, M., Hannun, Y. A., Obeid, L. M. A novel role of sphingosine kinase-1 in the invasion and angiogenesis of VHL mutant clear cell renal cell carcinoma. PMID:25805832

  15. Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1)

    PubMed Central

    Eylenstein, Anja; Gehring, Eva-Maria; Heise, Nicole; Shumilina, Ekaterina; Schmidt, Sebastian; Szteyn, Kalina; Münzer, Patrick; Nurbaeva, Meerim K.; Eichenmüller, Melanie; Tyan, Leonid; Regel, Ivonne; Föller, Michael; Kuhl, Dietmar; Soboloff, Jonathan; Penner, Reinhold; Lang, Florian

    2011-01-01

    Ca2+ signaling includes store-operated Ca2+ entry (SOCE) following depletion of endoplasmic reticulum (ER) Ca2+ stores. On store depletion, the ER Ca2+ sensor STIM1 activates Orai1, the pore-forming unit of Ca2+-release-activated Ca2+ (CRAC) channels. Here, we show that Orai1 is regulated by serum- and glucocorticoid-inducible kinase 1 (SGK1), a growth factor-regulated kinase. Membrane Orai1 protein abundance, ICRAC, and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active S422DSGK1 (by+81, +378, and+136%, respectively) but not by inactive K127NSGK1. Coexpression of the ubiquitin ligase Nedd4-2, an established negatively regulated SGK1 target, down-regulated SOCE (by −48%) and ICRAC (by −60%), an effect reversed by expression of S422DSGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1-knockout (sgk1−/−) mice (by −37% and −52%, respectively) than in mast cells from wild-type (sgk1+/+) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca2+-ATPase-inhibitor thapsigargin (2 μM) stimulated migration, an effect significantly higher (by +306%) in S422DSGK1-expressing than in K127NSGK1-expressing HEK293 cells, and also significantly higher (by +108%) in sgk1+/+ than in sgk1−/− mast cells. SGK1 is thus a novel key player in the regulation of SOCE.—Eylenstein, A., Gehring, E.-M., Heise, N., Shumilina, E., Schmidt, S., Szteyn, K., Münzer, P., Nurbaeva, M. K., Eichenmüller, M., Tyan, L., Regel, I., Föller, M., Kuhl, D., Soboloff, J., Penner, R., Lang, R. Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1). PMID:21385992

  16. Depressed Myocardial Contractility: Can It Be Rescued?

    PubMed

    Weber, Karl T

    2016-10-01

    Current dogma suggests patients with advanced systolic heart failure have an irreversible depression in myocardial contractility. Recent experience with improved ventricular function during continuous flow ventricular assist devices used as destination therapy would suggest otherwise. Herein, cellular and molecular signaling involved in reversing depressed myocardial contractility would be addressed. This includes cardiomyocyte thyroid hormone signaling responsible for the reexpression of fetal gene program that preserves cell efficiency (work and energy consumed) and the rescue of an endogenous population of atrophic myocytes bordering on microdomains of fibrosis to improve contractile mass. Copyright © 2016 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

  17. National Search and Rescue Manual. Addendum.

    DTIC Science & Technology

    1979-08-01

    1 . Purpose: This notice provides a change to subject addendum. S 2 . Discussion: This change provides the following revisions ...1933 U. S . COAST GUARD ADDENDUM TO THE NATIONAL SEARCH AND RESCUE MANUAL " AUG I 2 1978 CG-308 * LETTER OF PROMULGATION 1 . Prpose The Coast Guard...NOT above 500 feet or 180 not. cc S " (CO M10) 1 - 2 C4-7 011.4135 TARGET 3 4 SWIND FIGURE 2 STANDARD DROP PATTERN FOR FREE FALL DELIVERY (Upwind

  18. Search and rescue with serpentine robots

    NASA Astrophysics Data System (ADS)

    Choset, Howie M.; Hull, Douglas; Luntz, Jonathan E.; Shammas, Ellie; Rached, Tarek; Dent, Christina C.

    2000-07-01

    Serpentine robots offer advantages over traditional mobile robots and robot arms because they have enhanced flexibility and reachability; especially in convoluted environments. These mechanisms are especially well suited for search and rescue operations where making contact with surviving victims trapped in a collapsed building is essential. The same flexibility that makes serpentine robots incredibly useful also makes them difficult to design and control. This paper will describe the current status of serpentine robot design and path planning underway in our research group and point towards future directions of research.

  19. Nicotine Shifts the Temporal Activation of Hippocampal Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2 to Enhance Long-Term, but not Short-term, Hippocampus-Dependent Memory

    PubMed Central

    Gould, Thomas J.; Wilkinson, Derek S.; Yildirim, Emre; Poole, Rachel L. F.; Leach, Prescott T.; Simmons, Steven J.

    2014-01-01

    Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular regulated signaling kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 hours but not 2 hours post-training, delineating time points for STM (2 hours) and LTM (4 hours and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 hours but not 2 hours post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories. PMID:24457151

  20. Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

    PubMed

    Gould, Thomas J; Wilkinson, Derek S; Yildirim, Emre; Poole, Rachel L F; Leach, Prescott T; Simmons, Steven J

    2014-03-01

    Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility (SLF), the KSC response team practices lifting an injured crew member to an Air Force HH-60 helicopter for transport to a local hospital. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to reach and prepare the 'crew' five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker -- for preliminary triage. The exercise will conclude with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  2. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    In the woods next to the Shuttle Landing Facility (SLF), the KSC response team takes part in training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to drop emergency equipment and fire/rescue workers to prepare the 'crew' for preliminary triage. The helicopters later are used to remove the crew five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise concluded with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  3. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility (SLF), the KSC response team practices carrying an injured crew member to an Air Force HH-60 helicopter for transport to a local hospital. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to reach the site, drop emergency equipment and later remove the 'crew' five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise will conclude with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  4. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility (SLF), the KSC response team practices carrying an injured crew member to an Air Force HH-60 helicopter for transport to a local hospital. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to reach the site, drop emergency equipment and later remove the 'crew' five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker. The exercise will conclude with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  5. The KSC response team takes part in simulated rescue mission.

    NASA Technical Reports Server (NTRS)

    1999-01-01

    During a simulated rescue mission in the woods near the Shuttle Landing Facility (SLF), the KSC response team practices lifting an injured crew member to an Air Force HH-60 helicopter for transport to a local hospital. The response team is training for the unlikely scenario of a Shuttle mishap at the SLF. The Mode 7 simulation of an astronaut rescue exercises all aspects of command and control, search and rescue, and medical procedures required for a successful rescue. The remote location of the mock-up prevents a totally land-based crew rescue, and calls on a NASA UH-1 helicopter to locate the site and four Air Force HH-60 helicopters to reach and prepare the 'crew' five astronaut candidates, one representative from the Vehicle Integration Test office, and one fire/rescue worker -- for preliminary triage. The exercise will conclude with airlifted 'patients' arriving safely in the emergency rooms of participating area hospitals.

  6. Survey of space escape/rescue/survivability capabilities.

    NASA Technical Reports Server (NTRS)

    Fleisig, R.; Bolger, P. H.; Heath, G. W.

    1971-01-01

    Discussion of preventive or remedial systems to achieve safer space flight operations. Escape, rescue, and survival systems are defined by categories: on board, prepositioned aid, and earth-launched concepts. The survey considers separable escape or survival capsules; standby escape or rescue systems; and earth-launched manned and unmanned rescue systems. Reports covering such systems are listed, and the contents are classified as to scope of investigation, space mission, and design approach. Mission classes considered are earth orbit, lunar, and interplanetary. Results of the space escape, rescue, and survivability investigations are summarized in terms of system features and performance, including apparent voids or limitations in rescue capability. Recovery requirements and resources for space rescue are discussed.

  7. Serum Soluble Fms-Like Tyrosine Kinase 1 (sFlt-1) Predicts the Severity of Acute Pancreatitis

    PubMed Central

    Dumnicka, Paulina; Sporek, Mateusz; Mazur-Laskowska, Małgorzata; Ceranowicz, Piotr; Kuźniewski, Marek; Drożdż, Ryszard; Ambroży, Tadeusz; Olszanecki, Rafał; Kuśnierz-Cabala, Beata

    2016-01-01

    Organ failure is the most important determinant of the severity of acute pancreatitis (AP). Soluble fms-like tyrosine kinase 1 (sFlt-1) is positively associated with organ failure in sepsis. Our aim was to evaluate the diagnostic utility of automated sFlt-1 measurements for early prediction of AP severity. Adult patients (66) with AP were recruited, including 46 with mild (MAP), 15 with moderately-severe (MSAP) and 5 with severe AP (SAP). Serum and urine samples were collected twice. Serum sFlt-1 was measured with automated electrochemiluminescence immunoassay. Serum concentrations of sFlt-1 were significantly higher in patients with MSAP and SAP as compared to MAP. SAP patients had the highest concentrations. At 24 and 48 h, sFlt-1 positively correlated with inflammatory markers (leukocyte count, C-reactive protein), kidney function (creatinine, urea, cystatin C, serum and urine neutrophil gelatinase-associated lipocalin, urine albumin/creatinine ratio), D-dimer and angiopoietin-2. sFlt-1 positively correlated with the bedside index of severity in AP (BISAP) score and the duration of hospital stay. Serum sFlt-1 above 139 pg/mL predicted more severe AP (MSAP + SAP). In the early phase of AP, sFlt-1 is positively associated with the severity of AP and predicts organ failure, in particular kidney failure. Serum sFlt-1 may be a practical way to improve early assessment of AP severity. PMID:27929426

  8. Secretion of adenylate kinase 1 is required for extracellular ATP synthesis in C2C12 myotubes

    PubMed Central

    Choo, Hyo-Jung; Kim, Bong-Woo; Kwon, Oh-Bong; Lee, Chang Seok; Choi, Jong-Soon

    2008-01-01

    Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase β during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase β in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1β was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes. PMID:18446060

  9. FHL-2 suppresses VEGF-induced phosphatidylinositol 3-kinase/Akt activation via interaction with sphingosine kinase-1.

    PubMed

    Hayashi, Hiroki; Nakagami, Hironori; Takami, Yoichi; Koriyama, Hiroshi; Mori, Masaki; Tamai, Katsuto; Sun, Jianxin; Nagao, Kaori; Morishita, Ryuichi; Kaneda, Yasufumi

    2009-06-01

    In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

  10. Label-free, ITO-based immunosensor for the detection of a cancer biomarker: Receptor for Activated C Kinase 1.

    PubMed

    Bahadır, E B; Sezgintürk, M K

    2016-10-07

    A new, quite sensitive disposable immunosensor, based on the anti-RACK1 antibody, was developed for the determination of Receptor for Activated C Kinase 1 (RACK1) for the first time. Moreover, indium tin oxide (ITO) flexible sheets were modified by using aldehyde ended silane 11-(triethoxysilyl)undecanal (TESU) self-assembled monolayers (SAMs) for immobilizing the anti-RACK1 antibody via covalent bonds. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV) methods were used for characterizing the immobilization steps and for determining the RACK1 concentration. To obtain a successful immunosensor, experimental factors were optimized. This quite sensitive biosensor was able to detect concentrations as low as fg mL(-1). The detection principle was based on the change in charge transfer resistance and the Nyquist plot diameter and these changes were monitored by using electrochemical impedance spectroscopy (EIS). This impedimetric immunosensor had perfect repeatability and reproducibility. We proved that the new silane agent, TESU, performs well in biosensor applications. The feasibility of the fabricated immunosensor was tested by detecting RACK1 in artificial and real human serum samples.

  11. Identification of TGF-β-activated kinase 1 as a possible novel target for renal cell carcinoma intervention

    SciTech Connect

    Meng, Fandong; Li, Yan; Tian, Xin; Fu, Liye; Yin, Yuanqin; Sui, Chengguang; Ma, Ping; Jiang, Youhong

    2014-10-10

    Highlights: • Inhibition of TAK1 kinase activity suppresses NF-κB activation and RCC cell survival. • TAK1 inhibitors induces apoptotic cytotoxicity against RCC cells. • RCC cells with TAK1 depletion show reduced cell viability and increased apoptosis. • TAK1 and p-NF-κB are both over-expressed in human RCC tissues. • Inhibition or depletion of TAK1 enhances the activity of vinblastine sulfate. - Abstract: Renal cell carcinoma (RCC) is common renal malignancy within poor prognosis. TGF-β-activated kinase 1 (TAK1) plays vital roles in cell survival, apoptosis-resistance and carcinogenesis through regulating nuclear factor-κB (NF-κB) and other cancer-related pathways. Here we found that TAK1 inhibitors (LYTAK1, 5Z-7-oxozeanol (5Z) and NG-25) suppressed NF-κB activation and RCC cell (786-O and A489 lines) survival. TAK1 inhibitors induced apoptotic cytotoxicity against RCC cells, which was largely inhibited by the broad or specific caspase inhibitors. Further, shRNA-mediated partial depletion of TAK1 reduced 786-O cell viability whiling activating apoptosis. Significantly, TAK1 was over-expressed in human RCC tissues, and its level was correlated with phosphorylated NF-κB. Finally, kinase inhibition or genetic depletion of TAK1 enhanced the activity of vinblastine sulfate (VLB) in RCC cells. Together, these results suggest that TAK1 may be an important oncogene or an effective target for RCC intervention.

  12. Nuclear pool of phosphatidylinositol 4 phosphate 5 kinase 1α is modified by polySUMO-2 during apoptosis

    SciTech Connect

    Chakrabarti, Rajarshi; Bhowmick, Debajit; Bhargava, Varsha; Bhar, Kaushik; Siddhanta, Anirban

    2013-09-20

    Highlights: •Nuclear pool of PIP5K is SUMOylated. •Enhancement of SUMOylated nuclear PIP5K during apoptosis. •Nuclear PIP5K is modified by polySUMO-1 during apoptosis. •Nuclear PIP5K is modified by polySUMO-2 chain during apoptosis. -- Abstract: Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis.

  13. Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

    PubMed Central

    Child, Matthew A.; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A.; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A.; Boothroyd, John C.; Reese, Michael L.

    2017-01-01

    ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. PMID:28246362

  14. Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

    PubMed

    Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

    2015-08-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis.

  15. Identification of a novel Polo-like kinase 1 inhibitor that specifically blocks the functions of Polo-Box domain

    PubMed Central

    Li, Dongsheng; Jiang, Jiandong; Wang, Yanchang; Si, Shuyi

    2017-01-01

    Polo-like kinase 1 (Plk1) is a promising target for cancer therapy due to its essential role in cell division. In addition to a highly conserved kinase domain, Plk1 also contains a Polo-Box domain (PBD), which is essential for Plk1's subcellular localization and mitotic functions. We adopted a fluorescence polarization assay and identified a new Plk1 PBD inhibitor T521 from a small-molecule compound library. T521 specifically inhibits the PBD of Plk1, but not those of Plk2-3. T521 exhibits covalent binding to some lysine residues of Plk1 PBD, which causes significant changes in the secondary structure of Plk1 PBD. Using a cell-based assay, we showed that T521 impedes the interaction between Plk1 and Bub1, a mitotic checkpoint protein. Moreover, HeLa cells treated with T521 exhibited dramatic mitotic defects. Importantly, T521 suppresses the growth of A549 cells in xenograft nude mice. Taken together, we have identified a novel Plk1 inhibitor that specifically disrupts the functions of Plk1 PBD and shows anticancer activity. PMID:27902479

  16. Tank-Binding Kinase 1 (TBK1) Gene and Open-Angle Glaucomas (An American Ophthalmological Society Thesis).

    PubMed

    Fingert, John H; Robin, Alan L; Scheetz, Todd E; Kwon, Young H; Liebmann, Jeffrey M; Ritch, Robert; Alward, Wallace L M

    2016-08-01

    To investigate the role of TANK-binding kinase 1 (TBK1) gene copy-number variations (ie, gene duplications and triplications) in the pathophysiology of various open-angle glaucomas. In previous studies, we discovered that copy-number variations in the TBK1 gene are associated with normal-tension glaucoma. Here, we investigated the prevalence of copy-number variations in cohorts of patients with other open-angle glaucomas-juvenile-onset open-angle glaucoma (n=30), pigmentary glaucoma (n=209), exfoliation glaucoma (n=225), and steroid-induced glaucoma (n=79)-using a quantitative polymerase chain reaction assay. No TBK1 gene copy-number variations were detected in patients with juvenile-onset open-angle glaucoma, pigmentary glaucoma, or steroid-induced glaucoma. A TBK1 gene duplication was detected in one (0.44%) of the 225 exfoliation glaucoma patients. TBK1 gene copy-number variations (gene duplications and triplications) have been previously associated with normal-tension glaucoma. An exploration of other open-angle glaucomas detected a TBK1 copy-number variation in a patient with exfoliation glaucoma, which is the first example of a TBK1 mutation in a glaucoma patient with a diagnosis other than normal-tension glaucoma. A broader phenotypic range may be associated with TBK1 copy-number variations, although mutations in this gene are most often detected in patients with normal-tension glaucoma.

  17. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    PubMed

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

  18. Overproduction of the membrane-bound receptor-like protein kinase 1, RPK1, enhances abiotic stress tolerance in Arabidopsis.

    PubMed

    Osakabe, Yuriko; Mizuno, Shinji; Tanaka, Hidenori; Maruyama, Kyonoshin; Osakabe, Keishi; Todaka, Daisuke; Fujita, Yasunari; Kobayashi, Masatomo; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2010-03-19

    RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H(2)O(2)-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.

  19. Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP PNP.

    PubMed

    Zhao, Baoguang; Lehr, Ruth; Smallwood, Angela M; Ho, Thau F; Maley, Kathleen; Randall, Tanya; Head, Martha S; Koretke, Kristin K; Schnackenberg, Christine G

    2007-12-01

    Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 A crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP-PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The alphaC helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a beta-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel beta-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase.

  20. Metabolomic analysis of human oral cancer cells with adenylate kinase 2 or phosphorylate glycerol kinase 1 inhibition

    PubMed Central

    Ji, Eoon Hye; Cui, Li; Yuan, Xiaoqing; Cheng, Siliangyu; Messadi, Diana; Yan, Xinmin; Hu, Shen

    2017-01-01

    The purpose of this study was to use liquid chromatography-mass spectrometry (LC-MS) with XCMS for a quantitative metabolomic analysis of UM1 and UM2 oral cancer cells after knockdown of metabolic enzyme adenylate kinase 2 (AK2) or phosphorylate glycerol kinase 1 (PGK1). UM1 and UM2 cells were initially transfected with AK2 siRNA, PGK1 siRNA or scrambled control siRNA, and then analyzed with LC-MS for metabolic profiles. XCMS analysis of the untargeted metabolomics data revealed a total of 3200-4700 metabolite features from the transfected UM1 or UM2 cancer cells and 369-585 significantly changed metabolites due to AK2 or PGK1 suppression. In addition, cluster analysis showed that a common group of metabolites were altered by AK2 knockdown or by PGK1 knockdown between the UM1 and UM2 cells. However, the set of significantly changed metabolites due to AK2 knockdown was found to be distinct from those significantly changed by PGK1 knockdown. Our study has demonstrated that LC-MS with XCMS is an efficient tool for metabolomic analysis of oral cancer cells, and knockdown of different genes results in distinct changes in metabolic phenotypes in oral cancer cells. PMID:28243334