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Sample records for activate downstream targets

  1. NF-kB activation and its downstream target genes expression after heavy ions exposure

    NASA Astrophysics Data System (ADS)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  2. Fn14, a Downstream Target of the TGF-β Signaling Pathway, Regulates Fibroblast Activation

    PubMed Central

    Yang, Min; Lai, Wen; Ye, Litong; Chen, Jing; Hou, Xinghua; Ding, Hong; Zhang, Wenwei; Wu, Yueheng; Liu, Xiaoying; Huang, Shufang; Yu, Xiyong; Xiao, Dingzhang

    2015-01-01

    Fibrosis, the hallmark of human injuries and diseases such as serious burns, is characterized by excessive collagen synthesis and myofibroblast accumulation. Transforming growth factor-β (TGF-β), a potent inducer of collagen synthesis, has been implicated in fibrosis in animals. In addition to TGF-β, fibroblast growth factor-inducible molecule 14 (Fn14) has been reported to play an important role in fibrotic diseases, such as cardiac fibrosis. However, the function and detailed regulatory mechanism of Fn14 in fibrosis are unclear. Here, we investigated the effect of Fn14 on the activation of human dermal fibroblasts. In normal dermal fibroblasts, TGF-β signaling increased collagen production and Fn14 expression. Furthermore, Fn14 siRNA blocked extracellular matrix gene expression; even when TGF-β signaling was activated by TGF-β1, fibroblast activation remained blocked in the presence of Fn14 siRNA. Overexpressing Fn14 increased extracellular matrix gene expression. In determining the molecular regulatory mechanism, we discovered that SMAD4, an important TGF-β signaling co-mediator, bound to the Fn14 promoter and activated Fn14 transcription. Taken together, these results indicate that the TGF-β signaling pathway activates Fn14 expression through the transcription factor SMAD4 and that activated Fn14 expression increases extracellular matrix synthesis and fibroblast activation. Therefore, Fn14 may represent a promising approach to preventing the excessive accumulation of collagen or ECM in skin fibrosis. PMID:26625141

  3. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    PubMed Central

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  4. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells.

    PubMed

    Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

    2016-08-26

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. PMID:27329811

  5. Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

    PubMed Central

    Sattu, Kamaraj; Hochgräfe, Falko; Wu, Jianmin; Umapathy, Ganesh; Schönherr, Christina; Ruuth, Kristina; Chand, Damini; Witek, Barbara; Fuchs, James; Li, Pui-Kai; Hugosson, Fredrik; Daly, Roger J; Palmer, Ruth H; Hallberg, Bengt

    2013-01-01

    Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule-associated protein-like 4–ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma. PMID:23889739

  6. Targeting pathways downstream of KRAS in lung adenocarcinoma.

    PubMed

    Zhu, Zehua; Golay, Hadrien G; Barbie, David A

    2014-08-01

    Oncogenic KRAS activation is responsible for the most common genetic subtype of lung cancer. Although many of the major downstream signaling pathways that KRAS engages have been defined, these discoveries have yet to translate into effective targeted therapy. Much of the current focus has been directed at inhibiting the activation of RAF/MAPK and PI3K/AKT signaling, but clinical trials combining multiple different agents that target these pathways have failed to show significant activity. In this article, we will discuss the evidence for RAF and PI3K as key downstream RAS effectors, as well as the RAL guanine exchange factor, which is equally essential for transformation. Furthermore, we will delineate alternative pathways, including cytokine activation and autophagy, which are co-opted by oncogenic RAS signaling and also represent attractive targets for therapy. Finally, we will present strategies for combining inhibitors of these downstream KRAS signaling pathways in a rational fashion, as multitargeted therapy will be required to achieve a cure. PMID:25303301

  7. Targeting pathways downstream of KRAS in lung adenocarcinoma

    PubMed Central

    Zhu, Zehua; Golay, Hadrien G; Barbie, David A

    2014-01-01

    Oncogenic KRAS activation is responsible for the most common genetic subtype of lung cancer. Although many of the major downstream signaling pathways that KRAS engages have been defined, these discoveries have yet to translate into effective targeted therapy. Much of the current focus has been directed at inhibiting the activation of RAF/MAPK and PI3K/AKT signaling, but clinical trials combining multiple different agents that target these pathways have failed to show significant activity. In this article, we will discuss the evidence for RAF and PI3K as key downstream RAS effectors, as well as the RAL guanine exchange factor, which is equally essential for transformation. Furthermore, we will delineate alternative pathways, including cytokine activation and autophagy, which are co-opted by oncogenic RAS signaling and also represent attractive targets for therapy. Finally, we will present strategies for combining inhibitors of these downstream KRAS signaling pathways in a rational fashion, as multitargeted therapy will be required to achieve a cure. PMID:25303301

  8. Adiponectin, a downstream target gene of peroxisome proliferator-activated receptor {gamma}, controls hepatitis B virus replication

    SciTech Connect

    Yoon, Sarah; Jung, Jaesung; Kim, Taeyeung; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

    2011-01-20

    In this study, HepG2-hepatitis B virus (HBV)-stable cells that did not overexpress HBx and HBx-deficient mutant-transfected cells were analyzed for their expression of HBV-induced, upregulated adipogenic and lipogenic genes. The mRNAs of CCAAT enhancer binding protein {alpha} (C/EBP{alpha}), peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), adiponectin, liver X receptor {alpha} (LXR{alpha}), sterol regulatory element binding protein 1c (SREBP1c), and fatty acid synthase (FAS) were expressed at higher levels in HepG2-HBV and lamivudine-treated stable cells and HBx-deficient mutant-transfected cells than in the HepG2 cells. Lamivudine treatment reduced the mRNA levels of PPAR{gamma} and C/EBP{alpha}. Conversely, HBV replication was upregulated by adiponectin and PPAR{gamma} agonist rosiglitazone treatments and was downregulated by adiponectin siRNAs. Collectively, our results demonstrate that HBV replication and/or protein expression, even in the absence of HBx, upregulated adipogenic or lipogenic genes, and that the control of adiponectin might prove useful as a therapeutic modality for the treatment of chronic hepatitis B.

  9. Phytocomponent 4-hydroxy-3-methoxycinnamaldehyde ablates T-cell activation by targeting protein kinase C-θ and its downstream pathways.

    PubMed

    Akber, Uroos; Na, Bo-Ra; Ko, Yoo-Seung; Ko, You-Seung; Lee, Hyun-Su; Kim, Hye-Ran; Kwon, Min-Sung; Park, Zee-Yong; Choi, Eun-Ju; Han, Weon-Cheol; Lee, Seung-Ho; Oh, Hyun-Mee; Jun, Chang-Duk

    2015-03-01

    Autoreactive T-cell responses have a crucial role in the pathology and clinical course of autoimmune diseases. Therefore, controlling the activation of these cells is an important strategy for developing therapies and therapeutics. Here, we identified that 4-hydroxy-3-methoxycinnamaldehyde (4H3MC) has a therapeutic potential for T-cell activation by modulating protein kinase C-θ (PKCθ) and its downstream pathways. Pre- and post-treatment with 4H3MC prevented IL-2 release from human transformed and untransformed T cells at the micromolar concentrations without any cytotoxic effects, in fact more efficiently than its structural analogue 4-hydroxycinnamic acid-a previously reported T-cell inhibitor. In silico analysis showed that 4H3MC is a potential inhibitor of PKC isotypes, including PKCθ-a crucial PKC isotype in T cells. Consistently, 4H3MC significantly blocked PKC activity in vitro and also inhibited the phosphorylation of PKCθ in T cells. 4H3MC had no effect on TCR-mediated membrane-proximal-signalling events such as phosphorylation of Zap70. Instead, it attenuated the phosphorylation of mitogen-activated protein kinases (ERK and p38) and promoter activities of NF-κB, AP-1 and NFAT. Taken together, our results provide the evidences that 4H3MC may have curative potential as a novel immune modulator in a broad range of immunopathological disorders by modulating PKCθ activity. PMID:25637768

  10. Commissioning The Darht-II Accelerator Downstream Transport And Target

    SciTech Connect

    Ekdahl, Carl; Schulze, Martin E

    2008-01-01

    The DARHT-II accelerator produces a 2-kA, 17-MeV beam in a 1600-ns pulse. After exiting the accelerator, the pulse is sliced into four short pulses by a kicker and quadrupole septum and then transported for several meters to a tantalum target for conversion to x-rays for radiography. They describe the commissioning of the kicker, septum, transport, and multi-pulse converter target. The results of beam measurements made during the commissioning of the downstream transport are described.

  11. Turbulence decay downstream of an active grid

    NASA Astrophysics Data System (ADS)

    Bewley, Gregory; Bodenschatz, Eberhard

    2015-11-01

    A grid in a wind tunnel stirs up turbulence that has a certain large-scale structure. The moving parts in a so-called ``active grid'' can be programmed to produce different structures. We use a special active grid in which each of 129 paddles on the grid has its own position-controlled servomotor that can move independently of the others. We observe among other things that the anisotropy in the amplitude of the velocity fluctuations and in the correlation lengths can be set and varied with an algorithm that oscillates the paddles in a specified way. The variation in the anisotropies that we observe can be explained by our earlier analysis of anisotropic ``soccer ball'' turbulence (Bewley, Chang and Bodenschatz 2012, Phys. Fluids). We define the influence of this variation in structure on the downstream evolution of the turbulence. with Eberhard Bodenschatz and others.

  12. The MSX1 homeobox transcription factor is a downstream target of PHOX2B and activates the Delta-Notch pathway in neuroblastoma

    SciTech Connect

    Revet, Ingrid; Huizenga, Gerda; Chan, Alvin; Koster, Jan; Volckmann, Richard; Sluis, Peter van; Ora, Ingrid; Versteeg, Rogier; Geerts, Dirk

    2008-02-15

    Neuroblastoma is an embryonal tumour of the peripheral sympathetic nervous system (SNS). One of the master regulator genes for peripheral SNS differentiation, the homeobox transcription factor PHOX2B, is mutated in familiar and sporadic neuroblastomas. Here we report that inducible expression of PHOX2B in the neuroblastoma cell line SJNB-8 down-regulates MSX1, a homeobox gene important for embryonic neural crest development. Inducible expression of MSX1 in SJNB-8 caused inhibition of both cell proliferation and colony formation in soft agar. Affymetrix micro-array and Northern blot analysis demonstrated that MSX1 strongly up-regulated the Delta-Notch pathway genes DLK1, NOTCH3, and HEY1. In addition, the proneural gene NEUROD1 was down-regulated. Western blot analysis showed that MSX1 induction caused cleavage of the NOTCH3 protein to its activated form, further confirming activation of the Delta-Notch pathway. These experiments describe for the first time regulation of the Delta-Notch pathway by MSX1, and connect these genes to the PHOX2B oncogene, indicative of a role in neuroblastoma biology. Affymetrix micro-array analysis of a neuroblastic tumour series consisting of neuroblastomas and the more benign ganglioneuromas showed that MSX1, NOTCH3 and HEY1 are more highly expressed in ganglioneuromas. This suggests a block in differentiation of these tumours at distinct developmental stages or lineages.

  13. EVALUATING THE EFFECT OF UPSTREAM WATERSHED ACTIVITIES TO DOWNSTREAM STREAMFLOW

    EPA Science Inventory

    Linking the impacts of upstream activities such as urban development to changes in downstream streamflow is critical to achieving a balance between economic development and environmental protection as a basis for sustainable watershed development. This paper presents a modeling a...

  14. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  15. Alternative Activation Mechanisms of Protein Kinase B Trigger Distinct Downstream Signaling Responses.

    PubMed

    Balzano, Deborah; Fawal, Mohamad-Ali; Velázquez, Jose V; Santiveri, Clara M; Yang, Joshua; Pastor, Joaquín; Campos-Olivas, Ramón; Djouder, Nabil; Lietha, Daniel

    2015-10-01

    Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation, and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity toward specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif are essential for high PKB activity in general. On the other hand, we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial hydrophobic motif phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects. PMID:26286748

  16. Slug is a novel downstream target of MyoD. Temporal profiling in muscle regeneration.

    PubMed

    Zhao, Po; Iezzi, Simona; Carver, Ethan; Dressman, Devin; Gridley, Thomas; Sartorelli, Vittorio; Hoffman, Eric P

    2002-08-16

    Temporal expression profiling was utilized to define transcriptional regulatory pathways in vivo in a mouse muscle regeneration model. Potential downstream targets of MyoD were identified by temporal expression, promoter data base mining, and gel shift assays; Slug and calpain 6 were identified as novel MyoD targets. Slug, a member of the snail/slug family of zinc finger transcriptional repressors critical for mesoderm/ectoderm development, was further shown to be a downstream target by using promoter/reporter constructs and demonstration of defective muscle regeneration in Slug null mice. PMID:12023284

  17. Targeting the cis-dimerization of LINGO-1 with low MW compounds affects its downstream signalling

    PubMed Central

    Cobret, L; De Tauzia, M L; Ferent, J; Traiffort, E; Hénaoui, I; Godin, F; Kellenberger, E; Rognan, D; Pantel, J; Bénédetti, H; Morisset-Lopez, S

    2015-01-01

    Background and Purpose The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. Experimental Approach LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein–protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. Key Results LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Conclusions and Implications Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery. PMID:25257685

  18. Human muscle fibre type-specific regulation of AMPK and downstream targets by exercise

    PubMed Central

    Kristensen, Dorte E; Albers, Peter H; Prats, Clara; Baba, Otto; Birk, Jesper B; Wojtaszewski, Jørgen F P

    2015-01-01

    AMP-activated protein kinase (AMPK) is a regulator of energy homeostasis during exercise. Studies suggest muscle fibre type-specific AMPK expression. However, fibre type-specific regulation of AMPK and downstream targets during exercise has not been demonstrated. We hypothesized that AMPK subunits are expressed in a fibre type-dependent manner and that fibre type-specific activation of AMPK and downstream targets is dependent on exercise intensity. Pools of type I and II fibres were prepared from biopsies of vastus lateralis muscle from healthy men before and after two exercise trials: (1) continuous cycling (CON) for 30 min at 69 ± 1% peak rate of O2 consumption () or (2) interval cycling (INT) for 30 min with 6 × 1.5 min high-intensity bouts peaking at 95 ± 2% . In type I vs. II fibres a higher β1 AMPK (+215%) and lower γ3 AMPK expression (−71%) was found. α1, α2, β2 and γ1 AMPK expression was similar between fibre types. In type I vs. II fibres phosphoregulation after CON was similar (AMPKThr172, ACCSer221, TBC1D1Ser231 and GS2+2a) or lower (TBC1D4Ser704). Following INT, phosphoregulation in type I vs. II fibres was lower (AMPKThr172, TBC1D1Ser231, TBC1D4Ser704 and ACCSer221) or higher (GS2+2a). Exercise-induced glycogen degradation in type I vs. II fibres was similar (CON) or lower (INT). In conclusion, a differentiated response to exercise of metabolic signalling/effector proteins in human type I and II fibres was evident during interval exercise. This could be important for exercise type-specific adaptations, i.e. insulin sensitivity and mitochondrial density, and highlights the potential for new discoveries when investigating fibre type-specific signalling. PMID:25640469

  19. RNA Interference Mediated Interleukin-1β Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses

    PubMed Central

    Ortved, Kyla F.; Austin, Bethany S.; Scimeca, Michael S.; Nixon, Alan J.

    2016-01-01

    Posttraumatic activation of the catabolic cascade plays a major role in degradation of cartilage. Interleukin-1β (IL-1β), a primary instigator in the catabolic axis, is upregulated in chondrocytes following injury. IL-1β activates key degradative enzymes, including MMPs and aggrecanases, and other proinflammatory mediators such as PGE2 which contribute to ECM breakdown. Posttranscriptional silencing of IL-1β by RNA interference (RNAi) may drive a reduction in IL-1β. We hypothesized that transduction of chondrocytes using rAAV2 expressing a short hairpin RNAi motif targeting IL-1β (shIL-1β) would significantly decrease IL-1β expression and, in turn, decrease expression of other catabolic enzymes. Chondrocyte cultures were transduced with rAAV2-tdT-shIL-1β in serum-free media. The fluorescent protein, tdTomato, was used to determine transduction efficiency via flow cytometry and fluorescent microscopy. Cells were stimulated with lipopolysaccharide (LPS) 48 hours following transduction. After 24-hour stimulation, supernatants were collected for cytokine analysis, and cells lysed for gene expression analysis. IL-1β knockdown led to significantly decreased expression of IL-1β, TNF-α, and ADAMTS5. PGE2 synthesis was also significantly downregulated. Overall, effective silencing of IL-1β using rAAV2 vector expressing a short hairpin IL-1β knockdown sequence was shown. Additionally, significant downstream effects were evident, including decreased expression of TNF-α and ADAMTS5. Targeted silencing of catabolic cytokines may provide a promising treatment avenue for osteoarthritic (OA) joints. PMID:27073697

  20. RNA Interference Mediated Interleukin-1β Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses.

    PubMed

    Ortved, Kyla F; Austin, Bethany S; Scimeca, Michael S; Nixon, Alan J

    2016-01-01

    Posttraumatic activation of the catabolic cascade plays a major role in degradation of cartilage. Interleukin-1β (IL-1β), a primary instigator in the catabolic axis, is upregulated in chondrocytes following injury. IL-1β activates key degradative enzymes, including MMPs and aggrecanases, and other proinflammatory mediators such as PGE2 which contribute to ECM breakdown. Posttranscriptional silencing of IL-1β by RNA interference (RNAi) may drive a reduction in IL-1β. We hypothesized that transduction of chondrocytes using rAAV2 expressing a short hairpin RNAi motif targeting IL-1β (shIL-1β) would significantly decrease IL-1β expression and, in turn, decrease expression of other catabolic enzymes. Chondrocyte cultures were transduced with rAAV2-tdT-shIL-1β in serum-free media. The fluorescent protein, tdTomato, was used to determine transduction efficiency via flow cytometry and fluorescent microscopy. Cells were stimulated with lipopolysaccharide (LPS) 48 hours following transduction. After 24-hour stimulation, supernatants were collected for cytokine analysis, and cells lysed for gene expression analysis. IL-1β knockdown led to significantly decreased expression of IL-1β, TNF-α, and ADAMTS5. PGE2 synthesis was also significantly downregulated. Overall, effective silencing of IL-1β using rAAV2 vector expressing a short hairpin IL-1β knockdown sequence was shown. Additionally, significant downstream effects were evident, including decreased expression of TNF-α and ADAMTS5. Targeted silencing of catabolic cytokines may provide a promising treatment avenue for osteoarthritic (OA) joints. PMID:27073697

  1. MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

    SciTech Connect

    Wang, Yelin; Hu, Chen; Cheng, Jun; Chen, Binquan; Ke, Qinghong; Lv, Zhen; Wu, Jian; Zhou, Yanfeng

    2014-04-18

    Highlights: • MiR-145 expression is down-regulated in HCC tissues and inversely related with IRS1 levels. • MiR-145 directly targets IRS1 in HCC cells. • Restored expression of miR-145 suppressed HCC cell proliferation and growth. • MiR-145 induced IRS1 under-expression potentially reduced downstream AKT signaling. - Abstract: Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

  2. Investment prioritization based on broadscale spatial budgeting to meet downstream targets for suspended sediment loads

    NASA Astrophysics Data System (ADS)

    Lu, Hua; Moran, C. J.; Prosser, Ian P.; Derose, Ronald

    2004-09-01

    On the basis of a spatially distributed sediment budget across a large basin, costs of achieving certain sediment reduction targets in rivers were estimated. A range of investment prioritization scenarios were tested to identify the most cost-effective strategy to control suspended sediment loads. The scenarios were based on successively introducing more information from the sediment budget. The relationship between spatial heterogeneity of contributing sediment sources on cost effectiveness of prioritization was investigated. Cost effectiveness was shown to increase with sequential introduction of sediment budget terms. The solution which most decreased cost was achieved by including spatial information linking sediment sources to the downstream target location. This solution produced cost curves similar to those derived using a genetic algorithm formulation. Appropriate investment prioritization can offer large cost savings because the magnitude of the costs can vary by several times depending on what type of erosion source or sediment delivery mechanism is targeted. Target settings which only consider the erosion source rates can potentially result in spending more money than random management intervention for achieving downstream targets. Coherent spatial patterns of contributing sediment emerge from the budget model and its many inputs. The heterogeneity in these patterns can be summarized in a succinct form. This summary was shown to be consistent with the cost difference between local and regional prioritization for three of four test catchments. To explain the effect for the fourth catchment, the detail of the individual sediment sources needed to be taken into account.

  3. Analysis of plausible downstream target genes of Hoxc8 in F9 teratocarcinoma cells. Putative downstream target genes of Hoxc8.

    PubMed

    Kwon, Yunjeong; Ko, Jeong Heon; Byung-Gyu, Kim; Kim, Myoung Hee; Kim, Byungkyu

    2003-09-01

    Although Hox genes are known to mediate developmental decisions involved in pattern formation during embryogenesis, it is still not well understood what Hox regulates. In order to analyze Hoxc8 downstream target genes, a stable cell line overexpressing Hoxc8 was established using F9 murine teratocarcinoma cells, proteom samples were analyzed by 2-DE, and compared with controls. The protein spots having differences more than 4 fold in intensity were selected, analyzed by MALDI-TOF, and grouped in terms of putative function; cytoskeleton and motility (vimentin, gamma-actin, tropomyosin, and tubulin beta-5 chain); folding, modification and degradation of protein (GRP78, proteasome subunit alpha type 5, 26S proteasome regulatory subunit p27 protein, and PDIR); metabolism (ATP synthase beta subunit, Pgam1, and CAII); transcription/translation factors and general nucleic acid binding proteins (RbAp46, PCNA, eEF-1-beta, and nucleophosmin). Although it may not be significant, 50% of the genes were located on chromosomes 2 and 3, suggesting the possibility of a non-random distribution of Hox downstream genes. Almost 50% of the genes analyzed showed some relation with Hox protein directly or indirectly; i.e., tubulin beta 5, EF-1 beta and PCNA have been reported to contain putative Hox binding regulatory sites and genes like vimentin, pgam1 and nucleophosmin to be regulated by RA, a potent modulator of Hox expression. These results altogether imply that proteom analysis could be a possible tool for the analysis of the potent Hox realizator genes, which provides a new insight into the function of Hox on pattern formation during embryogenesis. PMID:12974468

  4. Using mechanistic Bayesian networks to identify downstream targets of the Sonic Hedgehog pathway

    PubMed Central

    2009-01-01

    Background The topology of a biological pathway provides clues as to how a pathway operates, but rationally using this topology information with observed gene expression data remains a challenge. Results We introduce a new general-purpose analytic method called Mechanistic Bayesian Networks (MBNs) that allows for the integration of gene expression data and known constraints within a signal or regulatory pathway to predict new downstream pathway targets. The MBN framework is implemented in an open-source Bayesian network learning package, the Python Environment for Bayesian Learning (PEBL). We demonstrate how MBNs can be used by modeling the early steps of the sonic hedgehog pathway using gene expression data from different developmental stages and genetic backgrounds in mouse. Using the MBN approach we are able to automatically identify many of the known downstream targets of the hedgehog pathway such as Gas1 and Gli1, along with a short list of likely targets such as Mig12. Conclusions The MBN approach shown here can easily be extended to other pathways and data types to yield a more mechanistic framework for learning genetic regulatory models. PMID:20021670

  5. Pharmacologically targeting the primary defect and downstream pathology in Duchenne muscular dystrophy.

    PubMed

    Fairclough, Rebecca J; Perkins, Kelly J; Davies, Kay E

    2012-06-01

    DMD is a devastatingly progressive muscle wasting disorder of childhood that significantly shortens life expectancy. Despite efforts to develop an effective therapy that dates back over a century, clinical interventions are still restricted to management of symptoms rather than a cure. The rationale to develop effective therapies changed in 1986 with the discovery of the dystrophin gene. Since then extensive research into both the molecular basis and pathophysiology of DMD has paved the way not only for development of strategies which aim to correct the primary defect, but also towards the identification of countless therapeutic targets with the potential to alleviate the downstream pathology. In addition to gene and cell-based therapies, which aim to deliver the missing gene and/or protein, an exciting spectrum of pharmacological approaches aimed at modulating therapeutic targets within DMD muscle cells through the use of small drugs are also being developed. This review presents promising pharmacological approaches aimed at targeting the primary defect, including suppression of nonsense mutations and functional compensation by upregulation of the dystrophin homologue, utrophin. Downstream of the primary membrane fragility, inflammation and fibrosis are reduced by blocking NF-κB, TGF-α and TGF-β, and free radical damage has been targeted using antioxidants and dietary/nutritional supplements. There are new hopes that ACE and PDE5 inhibitors can protect against skeletal as well as cardiac pathology, and modulating Ca2+ influx, NO, BMP, protein degradation and the mitochondrial permeability pore hold further promise in tackling the complex pathogenesis of this multifaceted disorder. PMID:22571500

  6. Upstream Regulators and Downstream Effectors of NADPH Oxidases as Novel Therapeutic Targets for Diabetic Kidney Disease

    PubMed Central

    Gorin, Yves; Wauquier, Fabien

    2015-01-01

    Oxidative stress has been linked to the pathogenesis of diabetic nephropathy, the complication of diabetes in the kidney. NADPH oxidases of the Nox family, and in particular the homologue Nox4, are a major source of reactive oxygen species in the diabetic kidney and are critical mediators of redox signaling in glomerular and tubulointerstitial cells exposed to the diabetic milieu. Here, we present an overview of the current knowledge related to the understanding of the role of Nox enzymes in the processes that control mesangial cell, podocyte and tubulointerstitial cell injury induced by hyperglycemia and other predominant factors enhanced in the diabetic milieu, including the renin-angiotensin system and transforming growth factor-β. The nature of the upstream modulators of Nox enzymes as well as the downstream targets of the Nox NADPH oxidases implicated in the propagation of the redox processes that alter renal biology in diabetes will be highlighted. PMID:25824546

  7. Transcription factor network downstream of protease activated receptors (PARs) modulating mouse bladder inflammation

    PubMed Central

    Saban, Ricardo; Simpson, Cindy; Davis, Carole A; Dozmorov, Igor; Maier, Julie; Fowler, Ben; Ihnat, Michael A; Hurst, Robert E; Wershil, Barry K; Saban, Marcia R

    2007-01-01

    Background All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders. Methods For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kitw/Kitw-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB. Results TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kitw/Kitw-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kitw/Kitw-v mice. Conclusion This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders. PMID:17705868

  8. Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

    SciTech Connect

    Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong; Chung, Hyun Joo; Kim, Myoung Hee

    2010-02-19

    Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna was similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.

  9. A long downstream probe-based platform for multiplex target capture.

    PubMed

    Wang, Ping; Huang, Jianqin; Xu, Yingwu

    2015-12-15

    A simple and rapid detection platform was established for multiplex target capture through generating single-strand long downstream probe (ssLDP), which was integrated with the ligase detection reaction (LDR) method for the purpose of multiplicity and high specificity. To increase sensitivity, the ladder-like polymerase chain reaction (PCR) amplicons were generated by using universal primers that complement ligated products. Each of the amplicons contained a stuffer sequence with a defined yet variable length. Thus, the length of the amplicon is an index of the specific suppressor, allowing its identification via electrophoresis. The multiplexed diagnostic platform was optimized using standard plasmids and validated by using potato virus suppressors as a detection model. This technique can detect down to 1.2 × 10(3) copies for single or two mixed target plasmids. When compared with microarray results, the electrophoresis showed 98.73-100% concordance rates for the seven suppressors in the 79 field samples. This strategy could be applied to detect a large number of targets in field and clinical surveillance. PMID:26344895

  10. Identification of Aurora Kinase B and Wee1-Like Protein Kinase as Downstream Targets of V600EB-RAF in Melanoma

    PubMed Central

    Sharma, Arati; Madhunapantula, SubbaRao V.; Gowda, Raghavendra; Berg, Arthur; Neves, Rogerio I.; Robertson, Gavin P.

    2014-01-01

    BRAF is the most mutated gene in melanoma, with approximately 50% of patients containing V600E mutant protein. V600EB-RAF can be targeted using pharmacological agents, but resistance develops in patients by activating other proteins in the signaling pathway. Identifying downstream members in this signaling cascade is important to design strategies to avoid the development of resistance. Unfortunately, downstream proteins remain to be identified and therapeutic potential requires validation. A kinase screen was undertaken to identify downstream targets in the V600EB-RAF signaling cascade. Involvement of aurora kinase B (AURKB) and Wee1-like protein kinase (WEE1) as downstream proteins in the V600EB-RAF pathway was validated in xenografted tumors, and mechanisms of action were characterized in size- and time-matched tumors. Levels of only AURKB and WEE1 decreased in melanoma cells, when V600EB-RAF, mitogen-activated protein kinase 1/2, or extracellular signal–regulated kinase 1/2 protein levels were reduced using siRNA compared with other identified kinases. AURKB and WEE1 were expressed in tumors of patients with melanoma at higher levels than observed in normal human melanocytes. Targeting these proteins reduced tumor development by approximately 70%, similar to that observed when inhibiting V600EB-RAF. Furthermore, protein or activity levels of AURKB and WEE1 decreased in melanoma cells when pharmacological agents targeting upstream V600EB-RAF or mitogen-activated protein kinase were used to inhibit the V600EB-RAF pathway. Thus, AURKB and WEE1 are targets and biomarkers of therapeutic efficacy, lying downstream of V600EB-RAF in melanomas. PMID:23416158

  11. Identification of novel Hoxa1 downstream targets regulating hindbrain, neural crest and inner ear development

    PubMed Central

    Makki, Nadja; Capecchi, Mario R.

    2011-01-01

    Hoxgenes play a crucial role during embryonic patterning and organogenesis. Of the 39 Hox genes, Hoxa1 is the first to be expressed during embryogenesis and the only anterior Hox gene linked to a human syndrome. Hoxa1 is necessary for proper development of the brainstem, inner ear and heart in humans and mice; however, almost nothing is known about the molecular downstream targets through which it exerts its function. To gain insight into the transcriptional network regulated by this protein, we performed microarray analysis on tissue microdissected from the prospective rhombomere 3–5 region of Hoxa1 null and wild type embryos. Due to the very early and transient expression of this gene, dissections were performed on early somite stage embryos during an eight-hour time window of development. Our array yielded a list of around 300 genes differentially expressed between the two samples. Many of the identified genes play a role in a specific developmental or cellular process. Some of the validated targets regulate early neural crest induction and specification. Interestingly, three of these genes, Zic1, Hnf1b and Foxd3, were down-regulated in the posterior hindbrain, where cardiac neural crest cells arise, which pattern the outflow tract of the heart. Other targets are necessary for early inner ear development, e.g. Pax8 and Fgfr3 or are expressed in specific hindbrain neurons regulating respiration, e.g. Lhx5. These findings allow us to propose a model where Hoxa1 acts in a genetic cascade upstream of genes controlling specific aspects of embryonic development, thereby providing insight into possible mechanisms underlying the human HoxA1-syndrome. PMID:21784065

  12. PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase

    PubMed Central

    Leenders, Frauke; Möpert, Kristin; Schmiedeknecht, Anett; Santel, Ansgar; Czauderna, Frank; Aleku, Manuela; Penschuck, Silke; Dames, Sibylle; Sternberger, Maria; Röhl, Thomas; Wellmann, Axel; Arnold, Wolfgang; Giese, Klaus; Kaufmann, Jörg; Klippel, Anke

    2004-01-01

    Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K. PMID:15282551

  13. Target activated frame capture

    NASA Astrophysics Data System (ADS)

    Roberts, G. Marlon; Fitzgerald, James; McCormack, Michael; Steadman, Robert

    2008-04-01

    Over the past decade, technological advances have enabled the use of increasingly intelligent systems for battlefield surveillance. These systems are triggered by a combination of external devices including acoustic and seismic sensors. Such products are mainly used to detect vehicles and personnel. These systems often use infra-red imagery to record environmental information, but Textron Defense Systems' Terrain Commander is one of a small number of systems which analyze these images for the presence of targets. The Terrain Commander combines acoustic, infrared, magnetic, seismic, and visible spectrum sensors to detect nearby targets in military scenarios. When targets are detected by these sensors, the cameras are triggered and images are captured in the infrared and visible spectrum. In this paper we discuss a method through which such systems can perform target tracking in order to record and transmit only the most pertinent surveillance images. This saves bandwidth which is crucial because these systems often use communication systems with throughputs below 2400bps. This method is expected to be executable on low-power processors at frame rates exceeding 10HZ. We accomplish this by applying target activated frame capture algorithms to infra-red video data. The target activated frame capture algorithms combine edge detection and motion detection to determine the best frames to be transmitted to the end user. This keeps power consumption and bandwidth requirements low. Finally, the results of the algorithm are analyzed.

  14. ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination.

    PubMed

    Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K; Ucher, Anna J; Leus, Niek G J; Ogilvie, Colin; Lou, Zhenkun; Schrader, Carol E; Stavnezer, Janet

    2014-05-15

    Activation-induced cytidine deaminase (AID) initiates Ab class-switch recombination (CSR) in activated B cells resulting in exchanging the IgH C region and improved Ab effector function. During CSR, AID instigates DNA double-strand break (DSB) formation in switch (S) regions located upstream of C region genes. DSBs are necessary for CSR, but improper regulation of DSBs can lead to chromosomal translocations that can result in B cell lymphoma. The protein kinase ataxia telangiectasia mutated (ATM) is an important proximal regulator of the DNA damage response (DDR), and translocations involving S regions are increased in its absence. ATM phosphorylates H2AX, which recruits other DNA damage response (DDR) proteins, including mediator of DNA damage checkpoint 1 (Mdc1) and p53 binding protein 1 (53BP1), to sites of DNA damage. As these DDR proteins all function to promote repair and recombination of DSBs during CSR, we examined whether mouse splenic B cells deficient in these proteins would show alterations in S region DSBs when undergoing CSR. We find that in atm(-/-) cells Sμ DSBs are increased, whereas DSBs in downstream Sγ regions are decreased. We also find that mutations in the unrearranged Sγ3 segment are reduced in atm(-/-) cells. Our data suggest that ATM increases AID targeting and activity at downstream acceptor S regions during CSR and that in atm(-/-) cells Sμ DSBs accumulate as they lack a recombination partner. PMID:24729610

  15. Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin

    PubMed Central

    Law, Ivy Ka Man; Jensen, Dane; Bunnett, Nigel W.; Pothoulakis, Charalabos

    2016-01-01

    Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH. PMID:26902265

  16. Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin.

    PubMed

    Law, Ivy Ka Man; Jensen, Dane; Bunnett, Nigel W; Pothoulakis, Charalabos

    2016-01-01

    Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH. PMID:26902265

  17. Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway

    PubMed Central

    Leon-Reyes, Antonio; Van der Does, Dieuwertje; De Lange, Elvira S.; Delker, Carolin; Wasternack, Claus; Van Wees, Saskia C. M.; Ritsema, Tita

    2010-01-01

    Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway. PMID:20839007

  18. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

    PubMed

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul Mk

    2015-11-12

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism

  19. RORγ directly regulates the circadian expression of clock genes and downstream targets in vivo.

    PubMed

    Takeda, Yukimasa; Jothi, Raja; Birault, Veronique; Jetten, Anton M

    2012-09-01

    In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP-Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ-/- liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes. PMID:22753030

  20. Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

    PubMed Central

    Bauer, Andrea; Mylroie, Hayley; Thornton, C. Clare; Calay, Damien; Birdsey, Graeme M.; Kiprianos, Allan P.; Wilson, Garrick K.; Soares, Miguel P.; Yin, Xiaoke; Mayr, Manuel; Randi, Anna M.; Mason, Justin C.

    2016-01-01

    Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1−/− mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis. PMID:27388959

  1. Downstream Effect of Ramping Neuronal Activity through Synapses with Short-Term Plasticity.

    PubMed

    Wei, Wei; Wang, Xiao-Jing

    2016-04-01

    Ramping neuronal activity refers to spiking activity with a rate that increases quasi-linearly over time. It has been observed in multiple cortical areas and is correlated with evidence accumulation processes or timing. In this work, we investigated the downstream effect of ramping neuronal activity through synapses that display short-term facilitation (STF) or depression (STD). We obtained an analytical result for a synapse driven by deterministic linear ramping input that exhibits pure STF or STD and numerically investigated the general case when a synapse displays both STF and STD. We show that the analytical deterministic solution gives an accurate description of the averaging synaptic activation of many inputs converging onto a postsynaptic neuron, even when fluctuations in the ramping input are strong. Activation of a synapse with STF shows an initial cubical increase with time, followed by a linear ramping similar to a synapse without STF. Activation of a synapse with STD grows in time to a maximum before falling and reaching a plateau, and this steady state is independent of the slope of the ramping input. For a synapse displaying both STF and STD, an increase in the depression time constant from a value much smaller than the facilitation time constant τ(F) to a value much larger than τ(F) leads to a transition from facilitation dominance to depression dominance. Therefore, our work provides insights into the impact of ramping neuronal activity on downstream neurons through synapses that display short-term plasticity. In a perceptual decision-making process, ramping activity has been observed in the parietal and prefrontal cortices, with a slope that decreases with task difficulty. Our work predicts that neurons downstream from such a decision circuit could instead display a firing plateau independent of the task difficulty, provided that the synaptic connection is endowed with short-term depression. PMID:26890350

  2. Uncovering potential downstream targets of oncogenic GRPR overexpression in prostate carcinomas harboring ETS rearrangements.

    PubMed

    Santos, Joana; Mesquita, Diana; Barros-Silva, João D; Jerónimo, Carmen; Henrique, Rui; Morais, António; Paulo, Paula; Teixeira, Manuel R

    2015-01-01

    Gastrin-releasing peptide receptor (GRPR) is known to be overexpressed in several human malignancies, including prostate cancer, and has been implicated in multiple important neoplastic signaling pathways. We recently have shown that GRPR is an ERG and ETV1 target gene in prostate cancer, using a genome-wide scale and exon-level expression microarray platform. Due to its cellular localization, the relevance of its function and the availability of blocking agents, GRPR seems to be a promising candidate as therapeutic target. Our present work shows that effective knockdown of GRPR in LNCaP and VCaP cells attenuates their malignant phenotype by decreasing proliferation, invasion and anchorage-independent growth, while increasing apoptosis. Using an antibody microarray we were able to validate known and identify new targets of GRPR pathway, namely AKT1, PKCε, TYK2 and MST1. Finally, we show that overexpression of these GRPR targets is restricted to prostate carcinomas harboring ERG and/or ETV1 rearrangements, establishing their potential as therapeutic targets for these particular molecular subsets of the disease. PMID:26097883

  3. Uncovering potential downstream targets of oncogenic GRPR overexpression in prostate carcinomas harboring ETS rearrangements

    PubMed Central

    Santos, Joana; Mesquita, Diana; Barros-Silva, João D.; Jerónimo, Carmen; Henrique, Rui; Morais, António; Paulo, Paula; Teixeira, Manuel R.

    2015-01-01

    Gastrin-releasing peptide receptor (GRPR) is known to be overexpressed in several human malignancies, including prostate cancer, and has been implicated in multiple important neoplastic signaling pathways. We recently have shown that GRPR is an ERG and ETV1 target gene in prostate cancer, using a genome-wide scale and exon-level expression microarray platform. Due to its cellular localization, the relevance of its function and the availability of blocking agents, GRPR seems to be a promising candidate as therapeutic target. Our present work shows that effective knockdown of GRPR in LNCaP and VCaP cells attenuates their malignant phenotype by decreasing proliferation, invasion and anchorage-independent growth, while increasing apoptosis. Using an antibody microarray we were able to validate known and identify new targets of GRPR pathway, namely AKT1, PKCε, TYK2 and MST1. Finally, we show that overexpression of these GRPR targets is restricted to prostate carcinomas harboring ERG and/or ETV1 rearrangements, establishing their potential as therapeutic targets for these particular molecular subsets of the disease. PMID:26097883

  4. Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation.

    PubMed

    Wang, Chunhe; Mooney, Jeffery L; Meza-Romero, Roberto; Chou, Yuan K; Huan, Jianya; Vandenbark, Arthur A; Offner, Halina; Burrows, Gregory G

    2003-08-15

    Recombinant TCR ligands (RTLs) consisting of covalently linked alpha(1) and beta(1) domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP(72-89)) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP(72-89) peptide-specific CD4(+) T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP(72-89)-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP(72-89) peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3zeta p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3epsilon treatment produced stronger activation of these cellular events with additional activation of NF-kappaB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-kappaB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4(+) T cells. PMID:12902496

  5. Requirement of Mammalian Target of Rapamycin Complex 1 Downstream Effectors in Cued Fear Memory Reconsolidation and Its Persistence

    PubMed Central

    Huynh, Thu N.; Santini, Emanuela

    2014-01-01

    Memory retrieval, often termed reconsolidation, can render previously consolidated memories susceptible to manipulation that can lead to alterations in memory strength. Although it is known that reconsolidation requires mammalian target of rapamycin complex 1 (mTORC1)-dependent translation, the specific contributions of its downstream effectors in reconsolidation are unclear. Using auditory fear conditioning in mice, we investigated the role of eukaryotic translation initiation factor 4E (eIF4E)–eIF4G interactions and p70 S6 kinase polypeptide 1 (S6K1) in reconsolidation. We found that neither 4EGI-1 (2-[(4-(3,4-dichlorophenyl)-thiazol-2-ylhydrazono)-3-(2-nitrophenyl)]propionic acid), an inhibitor of eFI4E–eIF4G interactions, nor PF-4708671 [2-((4-(5-ethylpyrimidin-4-yl)piperazin-1-yl)methyl)-5-(trifluoromethyl)-1H-benzo[d]imidazole], an inhibitor of S6K1, alone blocked the reconsolidation of auditory fear memory. In contrast, using these drugs in concert to simultaneously block eIF4E–eIF4G interactions and S6K1 immediately after memory reactivation significantly attenuated fear memory reconsolidation. Moreover, the combination of 4EGI-1 and PF-4708671 further destabilized fear memory 10 d after memory reactivation, which was consistent with experiments using rapamycin, an mTORC1 inhibitor. Furthermore, inhibition of S6K1 immediately after retrieval resulted in memory destabilization 10 d after reactivation, whereas inhibition of eIF4E–eIF4G interactions did not. These results indicate that the reconsolidation of fear memory requires concomitant association of eIF4E to eIF4G as well as S6K1 activity and that the persistence of memory at longer intervals after memory reactivation also requires mTORC1-dependent processes that involve S6K1. These findings suggest a potential mechanism for how mTORC1-dependent translation is fine tuned to alter memory persistence. PMID:24990923

  6. GSK3β, But Not GSK3α, Inhibits the Neuronal Differentiation of Neural Progenitor Cells As a Downstream Target of Mammalian Target of Rapamycin Complex1

    PubMed Central

    Ahn, Jyhyun; Jang, Jiwon; Choi, Jinyong; Lee, Junsub; Oh, Seo-Ho; Lee, Junghun; Yoon, Keejung

    2014-01-01

    Glycogen synthase kinase 3 (GSK3) acts as an important regulator during the proliferation and differentiation of neural progenitor cells (NPCs), but the roles of the isoforms of this molecule (GSK3α and GSK3β) have not been clearly defined. In this study, we investigated the functions of GSK3α and GSK3β in the context of neuronal differentiation of murine NPCs. Treatment of primary NPCs with a GSK3 inhibitor (SB216763) resulted in an increase in the percentage of TuJ1-positive immature neurons, suggesting an inhibitory role of GSK3 in embryonic neurogenesis. Downregulation of GSK3β expression increased the percentage of TuJ1-positive cells, while knock-down of GSK3α seemed to have no effect. When primary NPCs were engineered to stably express either isoform of GSK3 using retroviral vectors, GSK3β, but not GSK3α, inhibited neuronal differentiation and helped the cells to maintain the characteristics of NPCs. Mutant GSK3β (Y216F) failed to suppress neuronal differentiation, indicating that the kinase activity of GSK3β is important for this regulatory function. Similar results were obtained in vivo when a retroviral vector expressing GSK3β was delivered to E9.5 mouse brains using the ultrasound image-guided gene delivery technique. In addition, SB216763 was found to block the rapamycin-mediated inhibition of neuronal differentiation of NPCs. Taken together, our results demonstrate that GSK3β, but not GSK3α, negatively controls the neuronal differentiation of progenitor cells and that GSK3β may act downstream of the mammalian target of rapamycin complex1 signaling pathway. PMID:24397546

  7. C. elegans sym-1 is a downstream target of the hunchback-like-1 developmental timing transcription factor

    PubMed Central

    Niwa, Ryusuke; Hada, Kazumasa; Moliyama, Kouichi; Ohniwa, Ryosuke L.; Tan, Yi-Meng; Olsson-Carter, Katherine; Chi, Woo; Reinke, Valerie; Slack, Frank J.

    2010-01-01

    In the nematode Caenorhabditis elegans, the let-7 microRNA (miRNA) and its family members control the timing of key developmental events in part by directly regulating expression of hunchback-like-1 (hbl-1). C. elegans hbl-1 mutants display multiple developmental timing deficiencies, including cell cycle defects during larval development. While hbl-1 is predicted to encode a transcriptional regulator, downstream targets of HBL-1 have not been fully elucidated. Here we report using microarray analysis to uncover genes downstream of HBL-1. We established a transgenic strain that overexpresses hbl-1 under the control of a heat shock promoter. Heat shock-induced hbl-1 overexpression led to retarded hypodermal structures at the adult stage, opposite to the effect seen in loss of function (lf) hbl-1 mutants. The microarray screen identified numerous potential genes that are upregulated or downregulated by HBL-1, including sym-1, which encodes a leucine-rich repeat protein with a signal sequence. We found an increase in sym-1 transcription in the heat shock-induced hbl-1 overexpression strain, while loss of hbl-1 function caused a decrease in sym-1 expression levels. Furthermore, we found that sym-1(lf) modified the hypodermal abnormalities in hbl-1 mutants. Given that SYM-1 is a protein secreted from hypodermal cells to the surrounding cuticle, we propose that the adult-specific cuticular structures may be under the temporal control of HBL-1 through regulation of sym-1 transcription. PMID:19923914

  8. Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

    SciTech Connect

    Springer, Patricia S

    2010-07-12

    The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes that encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.

  9. Nek7 is an essential mediator of NLRP3 activation downstream of potassium efflux

    PubMed Central

    He, Yuan; Zeng, Melody Y.; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-01-01

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases1. To date, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-182,3. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases4,5 as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations6,7. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by multiple stimuli8,9. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. We report here the identification of Nek7, a member of the family of mammalian NIMA-related kinases (Neks)10, as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of Nek7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasome. NLRP3-activating stimuli promoted the NLRP3-Nek7 interaction in a process dependent on potassium efflux. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-Nek7 complex, which along with ASC oligomerization and ASC speck formation were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed that Nek7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that Nek7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome

  10. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.

    PubMed

    He, Yuan; Zeng, Melody Y; Yang, Dahai; Motro, Benny; Núñez, Gabriel

    2016-02-18

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases. So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins), as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7(-/-) or Nlrp3(-/-) haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to

  11. Identification of Tox chromatin binding properties and downstream targets by DamID-Seq.

    PubMed

    de Jesus Domingues, António Miguel; Artegiani, Benedetta; Dahl, Andreas; Calegari, Federico

    2016-03-01

    In recent years, DNA adenine methyltransferase identification (DamID) has emerged as a powerful tool to profile protein-DNA interaction on a genome-wide scale. While DamID has been primarily combined with microarray analyses, which limits the spatial resolution and full potential of this technique, our group was the first to combine DamID with sequencing (DamID-Seq) for characterizing the binding loci and properties of a transcription factor (Tox) (sequencing data available at NCBI's Gene Expression Omnibus under the accession number GSE64240). Our approach was based on the combination and optimization of several bioinformatics tools that are here described in detail. Analysis of Tox proximity to transcriptional start sites, profiling on enhancers and binding motif has allowed us to identify this transcription factor as an important new regulator of neural stem cells differentiation and newborn neurons maturation during mouse cortical development. Here we provide a valuable resource to study the role of Tox as a novel key determinant of mammalian somatic stem cells during development of the nervous and lymphatic system, in which this factor is known to be active, and describe a useful pipeline to perform DamID-Seq analyses for any other transcription factor. PMID:26981424

  12. Rbfox proteins regulate microRNA biogenesis by sequence-specific binding to their precursors and target downstream Dicer

    PubMed Central

    Chen, Yu; Zubovic, Lorena; Yang, Fan; Godin, Katherine; Pavelitz, Tom; Castellanos, Javier; Macchi, Paolo; Varani, Gabriele

    2016-01-01

    Rbfox proteins regulate tissue-specific splicing by targeting a conserved GCAUG sequence within pre-mRNAs. We report here that sequence-specific binding of the conserved Rbfox RRM to miRNA precursors containing the same sequence motif in their terminal loops, including miR-20b and miR-107, suppresses their nuclear processing. The structure of the complex between precursor miR-20b and Rbfox RRM shows the molecular basis for recognition, and reveals changes in the stem-loop upon protein binding. In mammalian cells, Rbfox2 downregulates mature miR-20b and miR-107 levels and increases the expression of their downstream targets PTEN and Dicer, respectively, suggesting that Rbfox2 indirectly regulates many more cellular miRNAs. Thus, some of the widespread cellular functions of Rbfox2 protein are attributable to regulation of miRNA biogenesis, and might include the mis-regulation of miR-20b and miR-107 in cancer and neurodegeneration. PMID:27001519

  13. Rbfox proteins regulate microRNA biogenesis by sequence-specific binding to their precursors and target downstream Dicer.

    PubMed

    Chen, Yu; Zubovic, Lorena; Yang, Fan; Godin, Katherine; Pavelitz, Tom; Castellanos, Javier; Macchi, Paolo; Varani, Gabriele

    2016-05-19

    Rbfox proteins regulate tissue-specific splicing by targeting a conserved GCAUG sequence within pre-mRNAs. We report here that sequence-specific binding of the conserved Rbfox RRM to miRNA precursors containing the same sequence motif in their terminal loops, including miR-20b and miR-107, suppresses their nuclear processing. The structure of the complex between precursor miR-20b and Rbfox RRM shows the molecular basis for recognition, and reveals changes in the stem-loop upon protein binding. In mammalian cells, Rbfox2 downregulates mature miR-20b and miR-107 levels and increases the expression of their downstream targets PTEN and Dicer, respectively, suggesting that Rbfox2 indirectly regulates many more cellular miRNAs. Thus, some of the widespread cellular functions of Rbfox2 protein are attributable to regulation of miRNA biogenesis, and might include the mis-regulation of miR-20b and miR-107 in cancer and neurodegeneration. PMID:27001519

  14. Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors

    PubMed Central

    Ma, Gary S.; Aznar, Nicolas; Kalogriopoulos, Nicholas; Midde, Krishna K.; Lopez-Sanchez, Inmaculada; Sato, Emi; Dunkel, Ying; Gallo, Richard L.; Ghosh, Pradipta

    2015-01-01

    In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV’s C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV’s GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions. PMID:25926659

  15. Deciphering downstream gene targets of PI3K/mTOR/p70S6K pathway in breast cancer

    PubMed Central

    Heinonen, Henna; Nieminen, Anni; Saarela, Matti; Kallioniemi, Anne; Klefström, Juha; Hautaniemi, Sampsa; Monni, Outi

    2008-01-01

    Background The 70 kDa ribosomal protein S6 kinase (RPS6KB1), located at 17q23, is amplified and overexpressed in 10–30% of primary breast cancers and breast cancer cell lines. p70S6K is a serine/threonine kinase regulated by PI3K/mTOR pathway, which plays a crucial role in control of cell cycle, growth and survival. Our aim was to determine p70S6K and PI3K/mTOR/p70S6K pathway dependent gene expression profiles by microarrays using five breast cancer cell lines with predefined gene copy number and gene expression alterations. The p70S6K dependent profiles were determined by siRNA silencing of RPS6KB1 in two breast cancer cell lines overexpressing p70S6K. These profiles were further correlated with gene expression alterations caused by inhibition of PI3K/mTOR pathway with PI3K inhibitor Ly294002 or mTOR inhibitor rapamycin. Results Altogether, the silencing of p70S6K altered the expression of 109 and 173 genes in two breast cancer cell lines and 67 genes were altered in both cell lines in addition to RPS6KB1. Furthermore, 17 genes including VTCN1 and CDKN2B showed overlap with genes differentially expressed after PI3K or mTOR inhibition. The gene expression signatures responsive to both PI3K/mTOR pathway and p70S6K inhibitions revealed previously unidentified genes suggesting novel downstream targets for PI3K/mTOR/p70S6K pathway. Conclusion Since p70S6K overexpression is associated with aggressive disease and poor prognosis of breast cancer patients, the potential downstream targets of p70S6K and the whole PI3K/mTOR/p70S6K pathway identified in our study may have diagnostic value. PMID:18652687

  16. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling.

    PubMed

    Tee, Andrew R; Fingar, Diane C; Manning, Brendan D; Kwiatkowski, David J; Cantley, Lewis C; Blenis, John

    2002-10-15

    Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products hamartin and tuberin, respectively. Here, we show that hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets. PMID:12271141

  17. Suppression or Induction of Apoptosis by Opposing Pathways Downstream from Calcium-Activated Calcineurin

    NASA Astrophysics Data System (ADS)

    Lotem, Joseph; Kama, Rachel; Sachs, Leo

    1999-10-01

    Ca2+-mobilizing compounds such as the Ca2+ ionophore A23187 or the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin can suppress or induce apoptosis in the same cells. The use of different calcineurin inhibitors has shown that both suppression and induction of apoptosis by the Ca2+-mobilizing compounds were mediated by calcineurin activation. Ca2+-mobilizing compounds activated p38 and p44/42 mitogen-activated protein kinases (MAPKs). Induction of apoptosis by the Ca2+-mobilizing compounds was suppressed by an inhibitor of p38 MAPK but not by an inhibitor of p44/42 MAPK. These MAPK inhibitors did not suppress apoptosis induction by wild-type p53 or by withdrawal of IL-6 from IL-6-dependent cells that are mediated by calcineurin-independent pathways. These MAPK inhibitors also did not affect the ability of Ca2+-mobilizing compounds to suppress apoptosis. The results indicate that (i) Ca2+-mobilizing compounds activate different and opposing pathways that diverge downstream from calcineurin activation that can either suppress or induce apoptosis in the same cells; (ii) p38 MAPK but not p44/42 MAPK is involved in induction of apoptosis but not in its suppression by the Ca2+-mobilizing compounds; and (iii) neither p38 nor p44/42 MAPKs mediate induction of apoptosis by some calcineurin-independent pathways.

  18. Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

    PubMed Central

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

    2015-01-01

    Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

  19. Salicylic Acid Suppresses Jasmonic Acid Signaling Downstream of SCFCOI1-JAZ by Targeting GCC Promoter Motifs via Transcription Factor ORA59[C][W][OA

    PubMed Central

    Van der Does, Dieuwertje; Leon-Reyes, Antonio; Koornneef, Annemart; Van Verk, Marcel C.; Rodenburg, Nicole; Pauwels, Laurens; Goossens, Alain; Körbes, Ana P.; Memelink, Johan; Ritsema, Tita; Van Wees, Saskia C.M.; Pieterse, Corné M.J.

    2013-01-01

    Antagonism between the defense hormones salicylic acid (SA) and jasmonic acid (JA) plays a central role in the modulation of the plant immune signaling network, but the molecular mechanisms underlying this phenomenon are largely unknown. Here, we demonstrate that suppression of the JA pathway by SA functions downstream of the E3 ubiquitin-ligase Skip-Cullin-F-box complex SCFCOI1, which targets JASMONATE ZIM-domain transcriptional repressor proteins (JAZs) for proteasome-mediated degradation. In addition, neither the stability nor the JA-induced degradation of JAZs was affected by SA. In silico promoter analysis of the SA/JA crosstalk transcriptome revealed that the 1-kb promoter regions of JA-responsive genes that are suppressed by SA are significantly enriched in the JA-responsive GCC-box motifs. Using GCC:GUS lines carrying four copies of the GCC-box fused to the β-glucuronidase reporter gene, we showed that the GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Using plants overexpressing the GCC-box binding APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors ERF1 or ORA59, we found that SA strongly reduces the accumulation of ORA59 but not that of ERF1. Collectively, these data indicate that the SA pathway inhibits JA signaling downstream of the SCFCOI1-JAZ complex by targeting GCC-box motifs in JA-responsive promoters via a negative effect on the transcriptional activator ORA59. PMID:23435661

  20. Targeting TRAP1 as a downstream effector of BRAF cytoprotective pathway: A novel strategy for human BRAF-driven colorectal carcinoma

    PubMed Central

    Sisinni, Lorenza; Lettini, Giacomo; Matassa, Danilo Swann; Piscazzi, Annamaria; Palladino, Giuseppe; Amoroso, Maria Rosaria; Esposito, Franca; Landriscina, Matteo

    2015-01-01

    The HSP90 chaperone TRAP1 is translational regulator of BRAF synthesis/ubiquitination, since BRAF down-regulation, ERK signaling inhibition and delay of cell cycle progression occur upon TRAP1 silencing/inhibition. Since TRAP1 is upregulated in human colorectal carcinomas (CRCs) and involved in protection from apoptosis and as human BRAF-driven CRCs are poorly responsive to anticancer therapies, the relationship between TRAP1 regulation of mitochondrial apoptotic pathway and BRAF antiapoptotic signaling has been further evaluated. This study reports that BRAF cytoprotective signaling involves TRAP1-dependent inhibition of the mitochondrial apoptotic pathway. It is worth noting that BRAF and TRAP1 interact and that the activation of BRAF signaling results in enhanced TRAP1 serine-phosphorylation, a condition associated with resistance to apoptosis. Consistently, a BRAF dominant-negative mutant prevents TRAP1 serine phosphorylation and restores drug sensitivity in BRAFV600E CRC drug-resistant cells with high TRAP1 levels. In addition, TRAP1 targeting by the mitochondria-directed HSP90 chaperones inhibitor gamitrinib induces apoptosis and inhibits colony formation in BRAF-driven CRC cells. Thus, TRAP1 is a downstream effector of BRAF cytoprotective pathway in mitochondria and TRAP1 targeting may represent a novel strategy to improve the activity of proapoptotic agents in BRAF-driven CRC cells. PMID:26084290

  1. Arsenic-induced mitochondrial oxidative damage is mediated by decreased PGC-1α expression and its downstream targets in rat brain.

    PubMed

    Prakash, Chandra; Kumar, Vijay

    2016-08-25

    The present study was carried out to investigate the molecular mechanism of arsenic-induced mitochondrial oxidative damage and its relation to biogenesis in rat brain. Chronic sodium arsenite (25 ppm, orally) administration for 12 weeks decreased mitochondrial complexes activities and mRNA expression of selective complexes subunits. The expression of mitochondrial biogenesis regulator PGC-1α, and its downstream targets NRF-1, NRF-2 and Tfam were decreased significantly both at mRNA and protein levels suggesting impaired biogenesis following chronic arsenic-exposure. In addition to this, protein expression analysis also revealed activation of Bax and caspase-3, leading to translocation of cytochrome c from mitochondria to cytosol suggesting induction of apoptotic pathway under oxidative stress. This was further confirmed by electron microscopy study which depicted morphological changes in mitochondria in terms of altered nuclear and mitochondrial shape and chromatin condensation in arsenic-treated rats. The immunohistochemical studies showed both nuclear and cytosolic localization of NRF-1 and NRF-2 in arsenic-exposed rat brain further suggesting regulatory role of these transcription factors under arsenic neurotoxicity. The results of present study indicate that arsenic-induced mitochondrial oxidative damage is associated with decreased mitochondrial biogenesis in rat brain that may present as important target to reveal the mechanism for arsenic-induced neurotoxicity. PMID:27425645

  2. Epigenetic regulation of miRNA-124 and multiple downstream targets is associated with treatment response in myeloid malignancies

    PubMed Central

    Liu, Hongbin; Pattie, Phillip; Chandrasekara, Sahan; Spencer, Andrew; Dear, Anthony E.

    2016-01-01

    Epigenetic regulation of microRNA (miRNA) expression has recently been implicated in the pathogenesis of myelodysplastic syndrome (MDS). Particular interest has focused on miRNA-124 expression, which is inhibited in MDS and has recently been demonstrated to be upregulated in response to epigenetic treatment (EGT). Previous studies have determined the in vitro and in vivo expression of miRNA-124 and several molecular targets, including cyclin-dependent kinase (CDK) 4, CDK6 and enhancer of zeste homolog 2 (EZH2), in order to elucidate the molecular mechanisms associated with the miRNA-124-mediated therapeutic response to EGT in MDS and identify additional potential biomarkers of early EGT treatment response in myeloid malignancies. In vitro studies in the HL60 leukemic cell line identified upregulation of miRNA-124 expression in response to single-agent EGT with either azacytidine (AZA) or the histone deacetylase inhibitor panobinostat (LBH589). Combination EGT with AZA and LBH589 resulted in significant additive induction of miRNA-124 expression. Expression of downstream targets of miRNA-124, including CDK4, CDK6 and EZH2, in response to single agent and combined EGT was determined in HL60 cells. Single and combination EGT treatment resulted in inhibition of CDK4, CDK6 and EZH2 expression with combination EGT resulting in a significant and additive inhibitory effect. In vivo studies using peripheral blood mononuclear cells from patients receiving combination EGT for high risk MDS or acute myeloid leukemia demonstrated significant induction of miRNA-124 and inhibition CDK4 and CDK6 messenger (m)RNA expression in patients that responded to combination EGT. A trend to inhibited EZH2 mRNA expression was also identified in response to combination EGT. Overall, the present observations identify a potential molecular mechanism for miRNA-124-mediated response to EGT involving regulation of CDK4, CDK6 and EZH2 expression. In addition, the present findings further qualify mi

  3. Transglutaminase inhibition protects against oxidative stress-induced neuronal death downstream of pathological ERK activation

    PubMed Central

    Basso, Manuela; Berlin, Jill; Li, Xia; Sleiman, Sama F.; Ko, Brendan; Haskew-Layton, Renee; Kim, Eunhee; Antonyak, Marc A.; Cerione, Richard A.; Iismaa, Siiri E.; Willis, Dianna; Cho, Sunghee; Ratan, Rajiv R.

    2012-01-01

    Molecular deletion of transglutaminase 2 (TG2) has been shown to improve function and survival in a host of neurological conditions including stroke, Huntington’s disease, and Parkinson’s disease. However, unifying schemes by which these crosslinking or polyaminating enzymes participate broadly in neuronal death have yet to be presented. Unexpectedly, we found that in addition to TG2, TG1 gene expression level is significantly induced following stroke in vivo or due to oxidative stress in vitro. Forced expression of TG1 or TG2 proteins is sufficient to induce neuronal death in Rattus novergicus cortical neurons in vitro. Accordingly, molecular deletion of TG2 alone is insufficient to protect Mus musculus neurons from oxidative death. By contrast, structurally diverse inhibitors used at concentrations that inhibit TG1 and TG2 simultaneously are neuroprotective. These small molecules inhibit increases in neuronal transamidating activity induced by oxidative stress; they also protect neurons downstream of pathological ERK activation when added well after the onset of the death stimulus. Together, these studies suggest that multiple TG isoforms, not only TG2, participate in oxidative stress-induced cell death signaling; and that isoform non-selective inhibitors of TG will be most efficacious in combating oxidative death in neurological disorders. PMID:22573678

  4. Egr1 protein acts downstream of estrogen-leukemia inhibitory factor (LIF)-STAT3 pathway and plays a role during implantation through targeting Wnt4.

    PubMed

    Liang, Xiao-Huan; Deng, Wen-Bo; Li, Ming; Zhao, Zhen-Ao; Wang, Tong-Song; Feng, Xu-Hui; Cao, Yu-Jing; Duan, En-Kui; Yang, Zeng-Ming

    2014-08-22

    Embryo implantation is a highly synchronized process between an activated blastocyst and a receptive uterus. Successful implantation relies on the dynamic interplay of estrogen and progesterone, but the key mediators underlying embryo implantation are not fully understood. Here we show that transcription factor early growth response 1 (Egr1) is regulated by estrogen as a downstream target through leukemia inhibitory factor (LIF) signal transducer and activator of transcription 3 (STAT3) pathway in mouse uterus. Egr1 is localized in the subluminal stromal cells surrounding the implanting embryo on day 5 of pregnancy. Estrogen rapidly, markedly, and transiently enhances Egr1 expression in uterine stromal cells, which fails in estrogen receptor α knock-out mouse uteri. STAT3 is phosphorylated by LIF and subsequently recruited on Egr1 promoter to induce its expression. Our results of Egr1 expression under induced decidualization in vivo and in vitro show that Egr1 is rapidly induced after deciduogenic stimulus. Egr1 knockdown can inhibit in vitro decidualization of cultured uterine stromal cells. Chromatin immunoprecipitation data show that Egr1 is recruited to the promoter of wingless-related murine mammary tumor virus integration site 4 (Wnt4). Collectively, our study presents for the first time that estrogen regulates Egr1 expression through LIF-STAT3 signaling pathway in mouse uterus, and Egr1 functions as a critical mediator of stromal cell decidualization by regulating Wnt4. PMID:25012664

  5. ZBTB16 as a Downstream Target Gene of Osterix Regulates Osteoblastogenesis of Human Multipotent Mesenchymal Stromal Cells.

    PubMed

    Onizuka, Satoru; Iwata, Takanori; Park, Sung-Joon; Nakai, Kenta; Yamato, Masayuki; Okano, Teruo; Izumi, Yuichi

    2016-10-01

    Human multipotent mesenchymal stromal cells (hMSCs) possess the ability to differentiate into osteoblasts, and they can be utilized as a source for bone regenerative therapy. Osteoinductive pretreatment, which induces the osteoblastic differentiation of hMSCs in vitro, has been widely used for bone tissue engineering prior to cell transplantation. However, the molecular basis of osteoblastic differentiation induced by osteoinductive medium (OIM) is still unknown. Therefore, we used a next-generation sequencer to investigate the changes in gene expression during the osteoblastic differentiation of hMSCs. The hMSCs used in this study possessed both multipotency and self-renewal ability. Whole-transcriptome analysis revealed that the expression of zinc finger and BTB domain containing 16 (ZBTB16) was significantly increased during the osteoblastogenesis of hMSCs. ZBTB16 mRNA and protein expression was enhanced by culturing the hMSCs with OIM. Small interfering RNA (siRNA)-mediated gene silencing of ZBTB16 decreased the activity of alkaline phosphatase (ALP); the expression of osteogenic genes, such as osteocalcin (OCN) and bone sialoprotein (BSP), and the mineralized nodule formation induced by OIM. siRNA-mediated gene silencing of Osterix (Osx), which is known as an essential regulator of osteoblastic differentiation, markedly downregulated the expression of ZBTB16. In addition, chromatin immunoprecipitation (ChIP) assays showed that Osx associated with the ZBTB16 promoter region containing the GC-rich canonical Sp1 sequence, which is the specific Osx binding site. These findings suggest that ZBTB16 acts as a downstream transcriptional regulator of Osx and can be useful as a late marker of osteoblastic differentiation. J. Cell. Biochem. 117: 2423-2434, 2016. © 2016 Wiley Periodicals, Inc. PMID:27335174

  6. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5

    PubMed Central

    Nezich, Catherine L.; Wang, Chunxin; Fogel, Adam I.

    2015-01-01

    The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats–generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy. PMID:26240184

  7. Activation of p53 Facilitates the Target Search in DNA by Enhancing the Target Recognition Probability.

    PubMed

    Itoh, Yuji; Murata, Agato; Sakamoto, Seiji; Nanatani, Kei; Wada, Takehiko; Takahashi, Satoshi; Kamagata, Kiyoto

    2016-07-17

    Tumor suppressor p53 binds to the target in a genome and regulates the expression of downstream genes. p53 searches for the target by combining three-dimensional diffusion and one-dimensional sliding along the DNA. To examine the regulation mechanism of the target binding, we constructed the pseudo-wild type (pseudo-WT), activated (S392E), and inactive (R248Q) mutants of p53 and observed their target binding in long DNA using single-molecule fluorescence imaging. The pseudo-WT sliding along the DNA showed many pass events over the target and possessed target recognition probability (TRP) of 7±2%. The TRP increased to 18±2% for the activated mutant but decreased to 0% for the inactive mutant. Furthermore, the fraction of the target binding by the one-dimensional sliding among the total binding events increased from 63±9% for the pseudo-WT to 87±2% for the activated mutant. Control of TRP upon activation, as demonstrated here for p53, might be a general activation mechanism of transcription factors. PMID:27291286

  8. Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ).

    PubMed

    Gandhirajan, Rajesh Kumar; Jain, Manaswita; Walla, Benedikt; Johnsen, Marc; Bartram, Malte P; Huynh Anh, Minh; Rinschen, Markus M; Benzing, Thomas; Schermer, Bernhard

    2016-05-27

    Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway. PMID:27048650

  9. Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF.

    PubMed

    Bernard, Monique; Dieudé, Mélanie; Yang, Bing; Hamelin, Katia; Underwood, Katy; Hébert, Marie-Josée

    2014-01-01

    Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation. PMID:25495560

  10. Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF

    PubMed Central

    Bernard, Monique; Dieudé, Mélanie; Yang, Bing; Hamelin, Katia; Underwood, Katy; Hébert, Marie-Josée

    2015-01-01

    Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation. PMID:25495560

  11. Involvement of PI3K/Akt Signaling Pathway and Its Downstream Intracellular Targets in the Antidepressant-Like Effect of Creatine.

    PubMed

    Cunha, Mauricio P; Budni, Josiane; Ludka, Fabiana K; Pazini, Francis L; Rosa, Julia Macedo; Oliveira, Ágatha; Lopes, Mark W; Tasca, Carla I; Leal, Rodrigo B; Rodrigues, Ana Lúcia S

    2016-07-01

    Creatine has been proposed to exert beneficial effects in the management of depression, but the cell signaling pathways implicated in its antidepressant effects are not well established. This study investigated the involvement of PI3K/Akt signaling pathway and its downstream intracellular targets in the antidepressant-like effect of creatine. The acute treatment of mice with creatine (1 mg/kg, po) increased the Akt and P70S6K phosphorylation, and HO-1, GPx and PSD95 immunocontents. The pretreatment of mice with LY294002 (10 nmol/mouse, icv, PI3K inhibitor), wortmannin (0.1 μg/mouse, icv, PI3K inhibitor), ZnPP (10 μg/mouse, icv, HO-1 inhibitor), or rapamycin (0.2 nmol/mouse, icv, mTOR inhibitor) prevented the antidepressant-like effect of creatine (1 mg/kg, po) in the TST. In addition, the administration of subeffective dose of either the selective GSK3 inhibitor AR-A014418 (0.01 μg/mouse, icv), the nonselective GSK3 inhibitor lithium chloride (10 mg/kg, po), or the HO-1 inductor CoPP (0.01 μg/mouse, icv), in combination with a subeffective dose of creatine (0.01 mg/kg, po) reduced the immobility time in the TST as compared with either drug alone. No treatment caused significant changes in the locomotor activity of mice. These results indicate that the antidepressant-like effect of creatine in the TST depends on the activation of Akt, Nrf2/HO-1, GPx, and mTOR, and GSK3 inhibition. PMID:25943184

  12. Krüppel-like Factor 6 Is a Co-activator of NF-κB That Mediates p65-dependent Transcription of Selected Downstream Genes*

    PubMed Central

    Zhang, Yu; Lei, Cao-Qi; Hu, Yun-Hong; Xia, Tian; Li, Mi; Zhong, Bo; Shu, Hong-Bing

    2014-01-01

    The transcription factor NF-κB plays a pivotal role in a broad range of physiological and pathological processes, including development, inflammation, and immunity. How NF-κB integrates activating signals to expression of specific sets of target genes is of great interest. Here, we identified Krüppel-like factor 6 (KLF6) as a co-activator of NF-κB after TNFα and IL-1β stimulation. Overexpression of KLF6 enhanced TNFα- and IL-1β-induced activation of NF-κB and transcription of a subset of downstream genes, whereas knockdown of KLF6 had opposite effects. KLF6 interacted with p65 in the nucleus and bound to the promoters of target genes. Upon IL-1β stimulation, KLF6 was recruited to promoters of a subset of NF-κB target genes in a p65-dependent manner, which was in turn required for the optimal binding of p65 to the target gene promoters. Our findings thus identified KLF6 as a previously unknown but essential co-activator of NF-κB and provided new insight into the molecular regulation of p65-dependent gene expression. PMID:24634218

  13. CEBPA exerts a specific and biologically important proapoptotic role in pancreatic β cells through its downstream network targets.

    PubMed

    Barbagallo, Davide; Condorelli, Angelo Giuseppe; Piro, Salvatore; Parrinello, Nunziatina; Fløyel, Tina; Ragusa, Marco; Rabuazzo, Agata Maria; Størling, Joachim; Purrello, Francesco; Di Pietro, Cinzia; Purrello, Michele

    2014-08-15

    Transcription factor CEBPA has been widely studied for its involvement in hematopoietic cell differentiation and causal role in hematological malignancies. We demonstrate here that it also performs a causal role in cytokine-induced apoptosis of pancreas β cells. Treatment of two mouse pancreatic α and β cell lines (αTC1-6 and βTC1) with proinflammatory cytokines IL-1β, IFN-γ, and TNF-α at doses that specifically induce apoptosis of βTC1 significantly increased the amount of mRNA and protein encoded by Cebpa and its proapoptotic targets, Arl6ip5 and Tnfrsf10b, in βTC1 but not in αTC1-6. Cebpa knockdown in βTC1 significantly decreased cytokine-induced apoptosis, together with the amount of Arl6ip5 and Tnfrsf10b. Analysis of the network comprising CEBPA, its targets, their first interactants, and proteins encoded by genes known to regulate cytokine-induced apoptosis in pancreatic β cells (genes from the apoptotic machinery and from MAPK and NFkB pathways) revealed that CEBPA, ARL6IP5, TNFRSF10B, TRAF2, and UBC are the top five central nodes. In silico analysis further suggests TRAF2 as trait d'union node between CEBPA and the NFkB pathway. Our results strongly suggest that Cebpa is a key regulator within the apoptotic network activated in pancreatic β cells during insulitis, and Arl6ip5, Tnfrsf10b, Traf2, and Ubc are key executioners of this program. PMID:24943845

  14. FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

    PubMed

    Fan, Qipeng; Cai, Qingchun; Xu, Yan

    2015-09-29

    Lysophosphatidic acid (LPA), a prototypical ligand for G protein coupled receptors, and Forkhead box protein M1 (FOXM1), a transcription factor that regulates expression of a wide array of genes involved in cancer initiation and progression, are two important oncogenic signaling molecules in human epithelial ovarian cancers (EOC). We conducted in vitro mechanistic studies using pharmacological inhibitors, genetic forms of the signaling molecules, and RNAi-mediated gene knock-down to uncover the molecular mechanisms of how these two molecules interact in EOC cells. Additionally, in vivo mouse studies were performed to confirm the functional involvement of FOXM1 in EOC tumor formation and progression. We show for the first time that LPA up-regulates expression of active FOXM1 splice variants in a time- and dose-dependent manner in the human EOC cell lines OVCA433, CAOV3, and OVCAR5. Gi-PI3K-AKT and G12/13-Rho-YAP signaling pathways were both involved in the LPA receptor (LPA1-3) mediated up-regulation of FOXM1 at the transcriptional level. In addition, down-regulation of FOXM1 in CAOV3 xenografts significantly reduced tumor and ascites formation, metastasis, and expression of FOXM1 target genes involved in cell proliferation, migration, or invasion. Collectively, our data link the oncolipid LPA, the oncogene YAP, and the central regulator of cell proliferation/mutagenesis FOXM1 in EOC cells. Moreover, these results provide further support for the importance of these pathways as potential therapeutic targets in EOC. PMID:26299613

  15. Centrosome movements in vivo correlate with specific neurite formation downstream of LIM homeodomain transcription factor activity.

    PubMed

    Andersen, Erica F; Halloran, Mary C

    2012-10-01

    Neurons must develop complex structure to form proper connections in the nervous system. The initiation of axons in defined locations on the cell body and their extension to synaptic targets are critical steps in neuronal morphogenesis, yet the mechanisms controlling axon formation in vivo are poorly understood. The centrosome has been implicated in multiple aspects of neuronal morphogenesis; however, its function in axon development is under debate. Conflicting results from studies of centrosome function in axonogenesis suggest that its role is context dependent and underscore the importance of studying centrosome function as neurons develop in their natural environment. Using live imaging of zebrafish Rohon-Beard (RB) sensory neurons in vivo, we discovered a spatiotemporal relationship between centrosome position and the formation of RB peripheral, but not central, axons. We tested centrosome function by laser ablation and found that centrosome disruption inhibited peripheral axon outgrowth. In addition, we show that centrosome position and motility are regulated by LIM homeodomain transcription factor activity, which is specifically required for the development of RB peripheral axons. Furthermore, we show a correlation between centrosome mislocalization and ectopic axon formation in bashful (laminin alpha 1) mutants. Thus, both intrinsic transcription factor activity and extracellular cues can influence centrosome position and axon formation in vivo. This study presents the first positive association between the centrosome and axon formation in vivo and suggests that the centrosome is important for differential neurite formation in neurons with complex axonal morphologies. PMID:22899847

  16. Stromal progesterone receptors mediate induction of Indian Hedgehog (IHH) in uterine epithelium and its downstream targets in uterine stroma.

    PubMed

    Simon, Liz; Spiewak, Kerry A; Ekman, Gail C; Kim, Jaeyeon; Lydon, John P; Bagchi, Milan K; Bagchi, Indrani C; DeMayo, Francesco J; Cooke, Paul S

    2009-08-01

    Uterine receptivity to embryo implantation depends on appropriate progesterone (P4) and estrogen stimulation. P4 rapidly stimulates production of the morphogen Indian hedgehog (IHH) in murine uterine epithelium as well as downstream molecules in the hedgehog pathway such as Patched homolog 1 (PTCH1) and nuclear receptor subfamily 2, group F, member 2 (NR2F2) in uterine stroma. Studies using IHH-null mice indicate that IHH is obligatory for the normal P4 response in the uterus. To determine whether IHH induction in uterine epithelium is mediated through P4 receptor (PR) in epithelium (E) and/or stroma (S), we produced tissue recombinants using uteri from neonatal PR knockout (ko) mice and wild-type (wt) mice containing PR in S and/or E or lacking PR altogether using a tissue recombinant methodology and assessed their response to P4. In tissue recombinants containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced Ihh mRNA expression at 6 h that was 6-fold greater than in oil-treated controls (P < 0.05; n = 6) in both types of tissue recombinants despite the absence of epithelial PR in wt-S + ko-E grafts. Conversely, Ihh mRNA expression was unaffected by P4 in ko-S + ko-E and ko-S + wt-E grafts despite epithelial PR expression in the latter. Nr2f2 and Ptch1 mRNA expression was similar in that it was stimulated by P4 only in recombinants containing stromal PR. These results indicate that stromal PR is both necessary and sufficient for P4 stimulation of epithelial IHH as well as downstream events such as PTCH1 and NR2F2 increases in stroma. PMID:19372202

  17. Neutrino Factory Downstream Systems

    SciTech Connect

    Zisman, Michael S.

    2009-12-23

    We describe the Neutrino Factory accelerator systems downstream from the target and capture area. These include the bunching and phase rotation, cooling, acceleration, and decay ring systems. We also briefly discuss the R&D program under way to develop these systems, and indicate areas where help from CERN would be invaluable.

  18. Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer

    PubMed Central

    Heinonen, Henna; Lepikhova, Tatiana; Sahu, Biswajyoti; Pehkonen, Henna; Pihlajamaa, Päivi; Louhimo, Riku; Gao, Ping; Wei, Gong‐Hong; Hautaniemi, Sampsa; Jänne, Olli A.

    2015-01-01

    HOXB7 encodes a transcription factor that is overexpressed in a number of cancers and encompasses many oncogenic functions. Previous results have shown it to promote cell proliferation, angiogenesis, epithelial–mesenchymal transition, DNA repair and cell survival. Because of its role in many cancers and tumorigenic processes, HOXB7 has been suggested to be a potential drug target. However, HOXB7 binding sites on chromatin and its targets are poorly known. The aim of our study was to identify HOXB7 binding sites on breast cancer cell chromatin and to delineate direct target genes located nearby these binding sites. We found 1,504 HOXB7 chromatin binding sites in BT‐474 breast cancer cell line that overexpresses HOXB7. Seventeen selected binding sites were validated by ChIP‐qPCR in several breast cancer cell lines. Furthermore, we analyzed expression of a large number of genes located nearby HOXB7 binding sites and found several new direct targets, such as CTNND2 and SCGB1D2. Identification of HOXB7 chromatin binding sites and target genes is essential to understand better the role of HOXB7 in breast cancer and mechanisms by which it regulates tumorigenic processes. PMID:26014856

  19. Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    VEGF is one of the most critical factors that induce angiogenesis, and has thus become an attractive target for anti-angiogenesis treatment. However, most of the current anti-VEGF agents that often cause side effects cannot be recommended for long term use. Identification of natural VEGF inhibitors...

  20. Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci

    PubMed Central

    Miller, Clint; Pjanic, Milos; Castano, Victor G.; Kim, Juyong B.; Salfati, Elias L.; Kundaje, Anshul B.; Bejerano, Gill; Assimes, Themistocles; Yang, Xia; Quertermous, Thomas

    2015-01-01

    To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including “growth factor binding,” “matrix interaction,” and “smooth muscle contraction.” We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology. PMID:26020271

  1. Target for optically activated seekers and trackers

    NASA Astrophysics Data System (ADS)

    Lakin, C. T.; Willett, N. F.

    1984-05-01

    This abstract discloses a target for optically activated seekers and trackers (TOAST) which provides for calibrated and variable target characteristics such as size, intensity, spatial position, color and interfering background. The TOAST has a first ilumination system providing a target light beam through an adjustable iris which controls image size. The target beam passes through a collimator lens which focuses the light at infinity. With the target beam focused at infinity, the motion of an elevation plate lengthens or shortens the distance from the collimator lens to a one motion mirror. The target beam is attenuated by a variable filter driven by a servo-motor, and a color selection process is provided by passing the beam through spectral filters. A background light beam with background imagery is provided to the beamsplitter mirror and mixed with the target image so as to simulate the target environment encountered by an operating optically activated seeker and tracker.

  2. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor1[OPEN

    PubMed Central

    Kim, Mi Jung

    2016-01-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15. However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. PMID:27246098

  3. Chick Lrrn2, a novel downstream effector of Hoxb1 and Shh, functions in the selective targeting of rhombomere 4 motor neurons

    PubMed Central

    Andreae, Laura C; Lumsden, Andrew; Gilthorpe, Jonathan D

    2009-01-01

    Background Capricious is a Drosophila adhesion molecule that regulates specific targeting of a subset of motor neurons to their muscle target. We set out to identify whether one of its vertebrate homologues, Lrrn2, might play an analogous role in the chick. Results We have shown that Lrrn2 is expressed from early development in the prospective rhombomere 4 (r4) of the chick hindbrain. Subsequently, its expression in the hindbrain becomes restricted to a specific group of motor neurons, the branchiomotor neurons of r4, and their pre-muscle target, the second branchial arch (BA2), along with other sites outside the hindbrain. Misexpression of the signalling molecule Sonic hedgehog (Shh) via in ovo electroporation results in upregulation of Lrrn2 exclusively in r4, while the combined expression of Hoxb1 and Shh is sufficient to induce ectopic Lrrn2 in r1/2. Misexpression of Lrrn2 in r2/3 results in axonal rerouting from the r2 exit point to the r4 exit point and BA2, suggesting a direct role in motor axon guidance. Conclusion Lrrn2 acts downstream of Hoxb1 and plays a role in the selective targeting of r4 motor neurons to BA2. PMID:19602272

  4. Active Targets For Capacitive Proximity Sensors

    NASA Technical Reports Server (NTRS)

    Jenstrom, Del T.; Mcconnell, Robert L.

    1994-01-01

    Lightweight, low-power active targets devised for use with improved capacitive proximity sensors described in "Capacitive Proximity Sensor Has Longer Range" (GSC-13377), and "Capacitive Proximity Sensors With Additional Driven Shields" (GSC-13475). Active targets are short-distance electrostatic beacons; they generate known alternating electro-static fields used for alignment and/or to measure distances.

  5. Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes.

    PubMed

    van Unen, Jakobus; Rashidfarrokhi, Ali; Hoogendoorn, Eelco; Postma, Marten; Gadella, Theodorus W J; Goedhart, Joachim

    2016-09-01

    Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves. PMID:27358232

  6. LRF is an essential downstream target of GATA1 in erythroid development and regulates BIM-dependent apoptosis.

    PubMed

    Maeda, Takahiro; Ito, Keisuke; Merghoub, Taha; Poliseno, Laura; Hobbs, Robin M; Wang, Guocan; Dong, Lin; Maeda, Manami; Dore, Louis C; Zelent, Arthur; Luzzatto, Lucio; Teruya-Feldstein, Julie; Weiss, Mitchell J; Pandolfi, Pier Paolo

    2009-10-01

    GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential antiapoptotic role during terminal erythroid differentiation. We find that loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late-stage erythroblasts. This programmed cell death is Arf and p53 independent and is instead mediated by upregulation of the proapoptotic factor Bim. We identify Lrf as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim loss delays the lethality of Lrf-deficient embryos and rescues their anemia phenotype. Thus, our data define a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF. PMID:19853566

  7. LRF is an essential downstream target of GATA1 in erythroid development and regulates BIM-dependent apoptosis

    PubMed Central

    Maeda, Takahiro; Ito, Keisuke; Merghoub, Taha; Poliseno, Laura; Hobbs, Robin M.; Wang, Guocan; Dong, Lin; Maeda, Manami; Dore, Louis C.; Zelent, Arthur; Luzzatto, Lucio; Teruya-Feldstein, Julie; Weiss, Mitchell J.; Pandolfi, Pier Paolo

    2011-01-01

    SUMMARY GATA-1-dependent transcription is essential for erythroid differentiation and maturation. Suppression of programmed cell death is also thought to be critical for this process; however, the link between these two features of erythropoiesis has remained elusive. Here, we show that the POZ-Krüppel family transcription factor, LRF (also known as Zbtb7a/Pokemon), is a direct target of GATA1 and plays an essential anti-apoptotic role during terminal erythroid differentiation. We find that loss of Lrf leads to lethal anemia in embryos, due to increased apoptosis of late stage erythroblasts. This programmed cell death is Arf- and p53-independent and is instead mediated by up-regulation of the pro-apoptotic factor Bim. We identify Lrf as a direct repressor of Bim transcription. In strong support of this mechanism, genetic Bim-loss delays the lethality of Lrf-deficient embryos and rescues their anemia-phenotype. Thus, our data defines a key transcriptional cascade for effective erythropoiesis, whereby GATA-1 suppresses BIM-mediated apoptosis via LRF. PMID:19853566

  8. Application of activated charcoal in the downstream processing of bacterial olefinic poly(3-hydroxyalkanoates).

    PubMed

    Wampfler, Bruno; Ramsauer, Thomas; Kehl, Karl; Zinn, Manfred; Thöny-Meyer, Linda

    2010-01-01

    Medium chain length poly(hydroxyalkanoates) (mcl-PHAs) are bacterial thermoplastic elastomers with a large potential in medical applications. The present study provides a novel process to isolate and purify poly([R]-3-hydroxy-omega-undecenoate-co-3-hydroxy-omega-nonenoate-co-3-hydroxy-omega-heptenoate) (PHUE) and poly([R]-3-hydroxy-omega-undecenoate-co-3-hydroxy-omega-nonenoate-co-3-hydroxyoctanoate-co-3-hydroxy-omega-heptenoate-co-3-hydroxyhexanoate) (PHOUE) from Pseudomonas putida species. Three different types of activated charcoal were compared with regard to their capability to selectively remove impurities. The product 'Charcoal activated, powder, pure' from Merck was found to be most suitable. Using ethyl acetate as solvent, the polyesters were extracted from freeze-dried biomass at room temperature and simultaneously purified by addition of activated charcoal at the beginning of the extraction. The period of extraction was one hour and the ratio solvent to biomass was 15:1 (vol/wt). After extraction, the solids were separated by pressure filtration through a metallic lace tissue. The filtrate was again passed through the previously accumulated filter cake, followed by a second filtration through a 0.45 microm membrane to remove finest coal particles. The resulting filtrate was concentrated, thus yielding polyesters whose quality and yield depended on the quantity of activated charcoal applied. For highly pure PHUE and PHOUE with low endotoxin levels, the optimum ratio of activated charcoal to solvent for extraction (V/V) was found to be 0.5 for PHUE and 0.25 for PHOUE. The yields with regard to the raw polymers amounted to 55 wt% for PHUE and 75 wt% for PHOUE, which are acceptable for polymers that can be used for medical applications. PMID:21197841

  9. Insulin-degrading enzyme as a downstream target of insulin receptor signaling cascade: implications for Alzheimer's disease intervention.

    PubMed

    Zhao, Lixia; Teter, Bruce; Morihara, Takashi; Lim, Giselle P; Ambegaokar, Surendra S; Ubeda, Oliver J; Frautschy, Sally A; Cole, Greg M

    2004-12-01

    Insulin-degrading enzyme (IDE) is one of the proteins that has been demonstrated to play a key role in degrading beta-amyloid (Abeta) monomer in vitro and in vivo, raising the possibility of upregulating IDE as an approach to reduce Abeta. Little is known, however, about the cellular and molecular regulation of IDE protein. Because one of the main functions of IDE is to degrade insulin, we hypothesized that there is a negative feedback mechanism whereby stimulation of insulin receptor-mediated signaling upregulates IDE to prevent chronic activation of the pathway. We show that treatment of primary hippocampal neurons with insulin increased IDE protein levels by approximately 25%. Insulin treatment also led to phosphatidylinositol-3 (PI3) kinase activation evidenced by Akt phosphorylation, which was blocked by PI3 kinase inhibitors, wortmannin and LY 294002. Inhibition of PI3 kinase abolished the IDE upregulation by insulin, indicating a cause-effect relationship between insulin signaling and IDE upregulation. Further support for this link was provided by the findings that deficient insulin signaling (decreased PI3 kinase subunit P85) was correlated with reduced IDE in Alzheimer's disease (AD) brains and in Tg2576 Swedish amyloid precursor protein transgenic mice fed a safflower oil-enriched ("Bad") diet used to accelerate pathogenesis. Consistent with IDE function in the degradation of Abeta monomer, the IDE decrease in the Bad diet-fed Tg2576 mice was associated with increased Abeta monomer levels. These in vitro and in vivo analyses validate the use of enhanced CNS insulin signaling as a potential strategy for AD intervention to correct the IDE defects occurring in AD. PMID:15590928

  10. Proteome analysis for downstream targets of oncogenic KRAS--the potential participation of CLIC4 in carcinogenesis in the lung.

    PubMed

    Okudela, Koji; Katayama, Akira; Woo, Tetsukan; Mitsui, Hideaki; Suzuki, Takehisa; Tateishi, Yoko; Umeda, Shigeaki; Tajiri, Michihiko; Masuda, Munetaka; Nagahara, Noriyuki; Kitamura, Hitoshi; Ohashi, Kenichi

    2014-01-01

    This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated. PMID:24503901

  11. Proteome Analysis for Downstream Targets of Oncogenic KRAS - the Potential Participation of CLIC4 in Carcinogenesis in the Lung

    PubMed Central

    Okudela, Koji; Katayama, Akira; Woo, Tetsukan; Mitsui, Hideaki; Suzuki, Takehisa; Tateishi, Yoko; Umeda, Shigeaki; Tajiri, Michihiko; Masuda, Munetaka; Nagahara, Noriyuki; Kitamura, Hitoshi; Ohashi, Kenichi

    2014-01-01

    This study investigated the proteome modulated by oncogenic KRAS in immortalized airway epithelial cells. Chloride intracellular channel protein 4 (CLIC4), S100 proteins (S100A2 and S100A11), tropomyosin 2, cathepsin L1, integrinsα3, eukaryotic elongation factor 1, vimentin, and others were discriminated. We here focused on CLIC4 to investigate its potential involvement in carcinogenesis in the lung because previous studies suggested that some chloride channels and chloride channel regulators could function as tumor suppressors. CILC4 protein levels were reduced in some lung cancer cell lines. The restoration of CLIC4 in lung cancer cell lines in which CLIC4 expression was reduced attenuated their growth activity. The immunohistochemical expression of the CLIC4 protein was weaker in primary lung cancer cells than in non-tumorous airway epithelial cells and was occasionally undetectable in some tumors. CLIC4 protein levels were significantly lower in a subtype of mucinous ADC than in others, and were also significantly lower in KRAS-mutated ADC than in EGFR-mutated ADC. These results suggest that the alteration in CLIC4 could be involved in restrictedly the development of a specific fraction of lung adenocarcinomas. The potential benefit of the proteome modulated by oncogenic KRAS to lung cancer research has been demonstrated. PMID:24503901

  12. Target activation by regulatory RNAs in bacteria

    PubMed Central

    Papenfort, Kai; Vanderpool, Carin K.

    2015-01-01

    Bacterial small regulatory RNAs (sRNAs) are commonly known to repress gene expression by base pairing to target mRNAs. In many cases, sRNAs base pair with and sequester mRNA ribosome-binding sites, resulting in translational repression and accelerated transcript decay. In contrast, a growing number of examples of translational activation and mRNA stabilization by sRNAs have now been documented. A given sRNA often employs a conserved region to interact with and regulate both repressed and activated targets. However, the mechanisms underlying activation differ substantially from repression. Base pairing resulting in target activation can involve sRNA interactions with the 5′ untranslated region (UTR), the coding sequence or the 3′ UTR of the target mRNAs. Frequently, the activities of protein factors such as cellular ribonucleases and the RNA chaperone Hfq are required for activation. Bacterial sRNAs, including those that function as activators, frequently control stress response pathways or virulence-associated functions required for immediate responses to changing environments. This review aims to summarize recent advances in knowledge regarding target mRNA activation by bacterial sRNAs, highlighting the molecular mechanisms and biological relevance of regulation. PMID:25934124

  13. Suramin blocks interaction between human FGF1 and FGFR2 D2 domain and reduces downstream signaling activity.

    PubMed

    Wu, Zong-Sian; Liu, Che Fu; Fu, Brian; Chou, Ruey-Hwang; Yu, Chin

    2016-09-01

    The extracellular portion of the human fibroblast growth factor receptor2 D2 domain (FGFR2 D2) interacts with human fibroblast growth factor 1 (hFGF1) to activate a downstream signaling cascade that ultimately affects mitosis and differentiation. Suramin is an antiparasiticdrug and a potent inhibitor of FGF-induced angiogenesis. Suramin has been shown to bind to hFGF1, and might block the interaction between hFGF1 and FGFR2 D2. Here, we titrated hFGF1 with FGFR2 D2 and suramin to elucidate their interactions using the detection of NMR. The docking results of both hFGF1-FGFR2 D2 domain and hFGF1-suramin complex were superimposed. The results indicate that suramin blocks the interaction between hFGF1 and FGFR2 D2. We used the PyMOL software to show the hydrophobic interaction of hFGF1-suramin. In addition, we used a Water-soluble Tetrazolium salts assay (WST1) to assess hFGF1 bioactivity. The results will be useful for the development of new antimitogenic activity drugs. PMID:27387234

  14. Improved algorithms in the CE-QUAL-W2 water-quality model for blending dam releases to meet downstream water-temperature targets

    USGS Publications Warehouse

    Rounds, Stewart A.; Buccola, Norman L.

    2015-01-01

    Water-quality models allow water resource professionals to examine conditions under an almost unlimited variety of potential future scenarios. The two-dimensional (longitudinal, vertical) water-quality model CE-QUAL-W2, version 3.7, was enhanced and augmented with new features to help dam operators and managers explore and optimize potential solutions for temperature management downstream of thermally stratified reservoirs. Such temperature management often is accomplished by blending releases from multiple dam outlets that access water of different temperatures at different depths. The modified blending algorithm in version 3.7 of CE-QUAL-W2 allows the user to specify a time-series of target release temperatures, designate from 2 to 10 floating or fixed-elevation outlets for blending, impose minimum and maximum head and flow constraints for any blended outlet, and set priority designations for each outlet that allow the model to choose which outlets to use and how to balance releases among them. The modified model was tested with a variety of examples and against a previously calibrated model of Detroit Lake on the North Santiam River in northwestern Oregon, and the results compared well. These updates to the blending algorithms will allow more complicated dam-operation scenarios to be evaluated somewhat automatically with the model, with decreased need for multiple model runs or preprocessing of model inputs to fully characterize the operational constraints.

  15. Minireview: Targeting GPCR Activated ERK Pathways for Drug Discovery

    PubMed Central

    Eishingdrelo, Haifeng; Kongsamut, Sathapana

    2013-01-01

    It has become clear in recent years that multiple signal transduction pathways are employed upon GPCR activation. One of the major cellular effectors activated by GPCRs is extracellular signal-regulated kinase (ERK). Both G-protein and β-arrestin mediated signaling pathways can lead to ERK activation. However, depending on activation pathway, the subcellular destination of activated ERK1/2 may be different. G-protein -dependent ERK activation results in the translocation of active ERK to the nucleus, whereas ERK activated via an arrestin-dependent mechanism remains largely in the cytoplasm. The subcellular location of activated ERK1/2 determines the downstream signaling cascade. Many substrates of ERK1/2 are found in the nucleus: nuclear transcription factors that participate in gene transcription, cell proliferation and differentiation. ERK1/2 substrates are also found in cytosol and other cellular organelles: they may play roles in translation, mitosis, apoptosis and cross-talk with other signaling pathways. Therefore, determining specific subcellular locations of activated ERK1/2 mediated by GPCR ligands would be important in correlating signaling pathways with cellular physiological functions. While GPCR-stimulated selective ERK pathway activation has been studied in several receptor systems, exploitation of these different signaling cascades for therapeutics has not yet been seriously pursued. Many old drug candidates were identified from screens based on G-protein signaling assays, and their activity on β-arrestin signaling pathways being mostly unknown, especially regarding their subcellular ERK pathways. With today’s knowledge of complicated GPCR signaling pathways, drug discovery can no longer rely on single-pathway approaches. Since ERK activation is an important signaling pathway and associated with many physiological functions, targeting the ERK pathway, especially specific subcellular activation pathways should provide new avenues for GPCR drug

  16. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    PubMed Central

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  17. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

    PubMed

    Kong, Qing; Sun, Tongjun; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-Ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-06-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  18. Video Guidance Sensors Using Remotely Activated Targets

    NASA Technical Reports Server (NTRS)

    Bryan, Thomas C.; Howard, Richard T.; Book, Michael L.

    2004-01-01

    Four updated video guidance sensor (VGS) systems have been proposed. As described in a previous NASA Tech Briefs article, a VGS system is an optoelectronic system that provides guidance for automated docking of two vehicles. The VGS provides relative position and attitude (6-DOF) information between the VGS and its target. In the original intended application, the two vehicles would be spacecraft, but the basic principles of design and operation of the system are applicable to aircraft, robots, objects maneuvered by cranes, or other objects that may be required to be aligned and brought together automatically or under remote control. In the first two of the four VGS systems as now proposed, the tracked vehicle would include active targets that would light up on command from the tracking vehicle, and a video camera on the tracking vehicle would be synchronized with, and would acquire images of, the active targets. The video camera would also acquire background images during the periods between target illuminations. The images would be digitized and the background images would be subtracted from the illuminated-target images. Then the position and orientation of the tracked vehicle relative to the tracking vehicle would be computed from the known geometric relationships among the positions of the targets in the image, the positions of the targets relative to each other and to the rest of the tracked vehicle, and the position and orientation of the video camera relative to the rest of the tracking vehicle. The major difference between the first two proposed systems and prior active-target VGS systems lies in the techniques for synchronizing the flashing of the active targets with the digitization and processing of image data. In the prior active-target VGS systems, synchronization was effected, variously, by use of either a wire connection or the Global Positioning System (GPS). In three of the proposed VGS systems, the synchronizing signal would be generated on, and

  19. Active Targets for Experiments with Rare Isotopes

    NASA Astrophysics Data System (ADS)

    Wiedenhoever, Ingo

    2014-09-01

    Experimental studies of un-bound nuclear states and nuclear reaction rates relevant for astrophysical processes are an important area of research with rare isotope beams. Both topics require the development of specialized experimental methods to study resonant reactions. The so-called active target approach, where the target material becomes part of the detection process, promises to combine high yields from thicker targets and low background with high resolution. This presentation will describe the implementation of the active-target technique in the ANASEN detector, which was developed by researchers from Louisiana State University and Florida State University. ANASEN was used in a number of stable and rare iosotope experiments in α- and proton scattering, as well as (α , p) and (d , p) reactions at FSU's in-flight radioactive beam facility RESOLUT. ANASEN also was used to perform the first experiment, proton scattering off a 37K beam at the ReA3 facility. Another active-target detector with a very different approach is found in the Active Target Time-Projection Chamber, which was developed by a collaboration between researchers from MSU, the University of Notre Dame, Western Michigan University, LLNL, LBNL, and St. Mary's University (Canada). First experiments with an AT-TPC prototype have been reported. The talk will summarize the results from the first experiments with these systems, describe further development and future research projects. Experimental studies of un-bound nuclear states and nuclear reaction rates relevant for astrophysical processes are an important area of research with rare isotope beams. Both topics require the development of specialized experimental methods to study resonant reactions. The so-called active target approach, where the target material becomes part of the detection process, promises to combine high yields from thicker targets and low background with high resolution. This presentation will describe the implementation of the

  20. Targeted activation in deterministic and stochastic systems

    NASA Astrophysics Data System (ADS)

    Eisenhower, Bryan; Mezić, Igor

    2010-02-01

    Metastable escape is ubiquitous in many physical systems and is becoming a concern in engineering design as these designs (e.g., swarms of vehicles, coupled building energetics, nanoengineering, etc.) become more inspired by dynamics of biological, molecular and other natural systems. In light of this, we study a chain of coupled bistable oscillators which has two global conformations and we investigate how specialized or targeted disturbance is funneled in an inverse energy cascade and ultimately influences the transition process between the conformations. We derive a multiphase averaged approximation to these dynamics which illustrates the influence of actions in modal coordinates on the coarse behavior of this process. An activation condition that predicts how the disturbance influences the rate of transition is then derived. The prediction tools are derived for deterministic dynamics and we also present analogous behavior in the stochastic setting and show a divergence from Kramers activation behavior under targeted activation conditions.

  1. Designing an Active Target Test Projection Chamber

    NASA Astrophysics Data System (ADS)

    Koci, James; Tan Ahn Collaboration, Dr.; Nicolas Dixneuf Collaboration

    2015-10-01

    The development of instrumentation in nuclear physics is crucial for advancing our ability to measure the properties of exotic nuclei. One limitation of the use of exotic nuclei in experiment is their very low production intensities. Recently, detectors, called active-target dectectors, have been developed to address this issue. Active-target detectors use a gas medium to image charged-particle tracks that are emitted in nuclear reactions. Last semester, I designed a vacuum chamber to be used in developing Micro-Pattern Gas detectors that will upgrade the capabilities of an active-target detector called the Prototype AT-TPC. With the exterior of the chamber complete, I have now been using an electric field modeling program, Garfield, developed by CERN to design a field cage to be placed within the vacuum chamber. The field cage will be a box-like apparatus consisting of two parallel metal plates connected with a resistor chain and attached to wires wrapped between them. The cage will provide a uniform electric field within the chamber to drift electrons from nuclear reactions down to the detector in the bottom of the chamber. These signals are then amplified by a proportional counter, and the data is sent to a computer. For the long term, we would like to incorporate a Micro-Pattern Gas Detectors in the interior of the chamber and eventually use the AT-TPC to examine various nuclei. Dr. Ahn is my advising professor.

  2. Sp and GATA factors are critical for Apolipoprotein AI downstream enhancer activity in human HepG2 cells.

    PubMed

    Ivanov, Gleb S; Kater, Jessie M; Jha, Shivkumar H; Stutius, Erica A; Sabharwal, Ravleen; Tricarico, Marisa D; Ginsburg, Geoffrey S; Ozer, Josef S

    2003-12-24

    The factors that bind to the hepatic-specific human apolipoprotein AI (apoAI) 48-bp downstream enhancer (DSE) were identified and characterized by electrophoretic mobility shift assays. A significant homology was shown between the histone 4 (H4) promoters and the hepatic-specific human apoAI DSE at Sp1 and H4TF2 binding sites. Human HepG2 nuclear extracts were used to form four specific complexes with the DSE (referred to as apoAI DSE-1, -2, -3, and -4). The apoAI DSE-1 and -2 complexes showed similar binding specificity to the Sp/H4TF1 consensus site within the apoAI DSE. The apoAI DSE-1 complex was predominantly recognized by anti-Sp1 and Sp3 sera in gel shift assays, indicating that the DSE was recognized by multiple Sp family members. Nuclear extracts that were prepared from retinoic acid treated HepG2 cells showed increased levels of Sp factors in gel shift and Western blot assays. The apoAI DSE-2 complex was identified as H4TF1 and formed in the absence of magnesium chloride. The apoAI DSE-3 complex bound to a consensus GATA element within the DSE that was recognized by recombinant human GATA-6 as well. The apoAI DSE-3 complex was completely disrupted by a GATA-4 antibody in EMSA. GATA-4 and -6 were detected in nuclear extracts prepared from retinoic acid treated HepG2 cells using Western blot assays. The highest apoAI DSE-3 levels were observed with retinoic acid treated HepG2 cell nuclear extracts in EMSA. ApoAI DSE-4 is a multi-factor complex that includes an Sp/H4TF1 factor and either H4TF2 or apoAI DSE-3. Because apoAI DSE mutations revealed transcription defects in transient transfection assays, we conclude that the entire DSE sequence is required for full apoAI transcriptional activity in HepG2 cells. PMID:14659877

  3. Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells.

    PubMed

    Tanaka, Maiko; Hiragun, Takaaki; Tsutsui, Tomoko; Yanase, Yuhki; Suzuki, Hidenori; Hide, Michihiro

    2008-06-15

    Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors. PMID:18339533

  4. The Activation of Nrf2 and Its Downstream Regulated Genes Mediates the Antioxidative Activities of Xueshuan Xinmaining Tablet in Human Umbilical Vein Endothelial Cells

    PubMed Central

    Xiong, Lingxin; Xie, Jingshu; Song, Chenxue; Liu, Jinping; Zheng, Jingtong; Liu, Chuangui; Zhang, Xiaotian; Li, Pingya; Wang, Fang

    2015-01-01

    Epidemiological studies have verified the critical role that antioxidative stress plays in protecting vascular endothelial cells. The aims of the present study were to investigate the antioxidative activities and differential regulation of nuclear erythroid-related factor 2- (Nrf2-) mediated gene expression by Xueshuan Xinmaining Tablet (XXT), a traditional Chinese medicine with the effect of treating cardiovascular diseases. The antioxidative activities of XXT were investigated using quantitative real-time PCR (qPCR), a PCR array, and western blotting. Our results indicated that XXT exhibited potent antioxidative activities by suppressing the levels of hydrogen peroxide- (H2O2-) induced reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs). We were also conscious of strong Nrf2-mediated antioxidant induction. XXT enhanced the expressions of Keap1, Nrf2, and Nrf2-mediated genes, such as glutamate-cysteine ligase modifier subunit (GCLM), NAD(P)H: quinine oxidoreductase 1 (NQO1), heme oxygenase 1 (HMOX1), and glutathione peroxidase (GPX) in HUVECs. In summary, XXT strongly activated Nrf2 and its downstream regulated genes, which may contribute to the antioxidative and vascular endothelial cell protective activities of XXT. PMID:26681964

  5. Characterising low molecular weight dissolved organic carbon compounds in subglacial systems; implications for subglacial metabolic activity and potential downstream export

    NASA Astrophysics Data System (ADS)

    Lawson, Emily; Wadham, Jemma; Lis, Grzegorz; Telling, Jon

    2010-05-01

    Glaciers and ice sheets represent ~10% of the contemporary global surface coverage, yet remain one of the least explored sectors of the Earth's biosphere. The basal regions of these ice masses, known as subglacial environments, are capable of harbouring a diverse range of microorganisms that are often metabolically active despite the lack of sunlight, the cold temperatures and nutrient scarcity. Here, we consider the potential for such environments to be active components of the Earth's biogeochemical cycles. Subglacial environments have traditionally been excluded from global carbon budgets because they were assumed to be predominantly abiotic. Organic carbon (OC) reservoirs and transformations were also believed to be limited. However, significant stores of bioavailable carbon are thought to be present in glacially-overridden material, providing a potential substrate for in situ microbial metabolism. We examine the molecular characteristics of dissolved OC in basal ice and subglacial runoff from two glacier/ice-sheet systems with contrasting organic carbon substrates; Russell/Leverett Glacier, Greenland ice sheet, and Engabreen, Norway, to determine the range of dissolved low molecular weight OC (LMWOC) compounds and their relative bioavailability. Overridden material beneath the Greenland ice sheet is relatively young and organic-rich, contrasting with the older crystalline bedrock/continental shield that was overridden during glaciation at Engabreen. We first utilise a combination of fluorescence spectroscopy and ion chromatography to identify and quantify volatile fatty acids, carbohydrates and amino acids in basal ice. Volatile fatty acids are key metabolic substrates and their provision is thought to be a primary control on subglacial metabolic activity. We then provide a temporal record of amino acids and carbohydrates in subglacial runoff from Leverett Glacier (June 23rd - August 18th 2009), and compare this with subglacial runoff from Engabreen (2008 melt

  6. Analysis of the effects of human activities on the hydromorphological evolution channel of the Saint-Maurice River downstream from La Gabelle dam (Quebec, Canada)

    NASA Astrophysics Data System (ADS)

    Vadnais, Marie-Ève; Assani, Ali A.; Landry, Raphaëlle; Leroux, Denis; Gratton, Denis

    2012-11-01

    During the first half of the twentieth century, many hydroelectric facilities were built in the Saint-Maurice River watershed, followed by other human activities in the second half of the century (pleasure boating, boom dismantling, urbanization, etc.). The goal of the study is to constrain the effects of these various types of human activities, particularly those of the many dams in the watershed, on the hydromorphological evolution of the Saint-Maurice River downstream from the La Gabelle (dam) power plant (43,000 km2). Comparison of specific discharge in this river with streamflow measured in a natural river setting reveals a significant decrease in seasonal maximum flows, aside from winter, when daily maximum flows increased significantly. Also, unlike natural rivers, the long-term trend in spring flows is not characterized by a significant change in mean downstream from the La Gabelle plant. These hydrological changes are linked to the inversion-type management mode of the reservoirs built downstream from the plant. As for the morphological evolution, the longitudinal variability of bankfull width downstream from the plant shows two significant shifts in mean: the first, which was quasi-abrupt, took place downstream of the des Forges rapid; and the second, which was gradual, occurred upstream from the confluence of the Saint-Maurice River with the St. Lawrence River, above the point where the Saint-Maurice splits into two branches. Comparison of aerial photographs taken at various times (1948, 1964, 1975, 1996, and 2008) reveals no significant change in the mean of bankfull width over time. However, a significant increase in the surface area of islets located at the confluence was observed, which is caused by sediment accumulation. These sediments were likely derived from local bank erosion resulting from anthropogenic changes.

  7. An Upstream By-product from Ester Activation via NHC-Catalysis Catalyzes Downstream Sulfonyl Migration Reaction.

    PubMed

    Han, Runfeng; He, Liwenze; Liu, Lin; Xie, Xingang; She, Xuegong

    2016-01-01

    A sequential reaction combining N-heterocyclic carbene (NHC) and N-hydroxyphthalimide (NHPI) catalysis allowed for the upstream by-product NHPI, which was generated in the NHC-catalyzed cycloaddition reaction, to act as the catalyst for a downstream nitrogen-to-carbon sulfonyl migration reaction. Enantiomeric excess of the major product in the cycloaddition reaction remained intact in the follow-up sulfonyl migration reaction. PMID:26522328

  8. Up-stream and Down-stream Events in the NF-kB Activation Cascade in Response to Sparsely and Densely Ionizing Radiation

    NASA Astrophysics Data System (ADS)

    Langen, Britta; Hellweg, Christine; Baumstark-Khan, Christa; Ruscher, Roland; Schmitz, Claudia; Arenz, Andrea; Lau, Patrick; Meier, Matthias M.; Testard, Isabelle; Reitz, Guenther

    Radiation is a potentially limiting factor for long term orbital and interplanetary missions. Long-term exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. As an antiapoptotic factor, if activated in human cells by exposure to components of cosmic rays, the transcription factor nuclear factor κB (NF- κB) could influence the cancer risk of astronauts exposed to cosmic radiation and improve cellular survival after exposure to high radiation doses. In previous studies using a screening assay for the detection of NF-κB-dependent gene induction (HEK-pNF-κB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown (Radiat. Res. 164: 527- 530, 2005). In this work, the events upstream of NF-κB attachment to its promoter and enhancer binding sites, and downstream expression of target genes were analysed. It is supposed that the ATM kinase mediates the signal from damaged DNA in the nucleus to kinases in the cytoplasm, such as NF-κB essential modulator (NEMO). For liberation of NF-κB and its nuclear translocation, the inhibitor of NF-κB (IκB) has to be degraded in the proteasom. In order to evaluate a role of NEMO in the radiation response, the survival of murine embryonic fibroblasts expressing wildtype NEMO and lacking NEMO in response to ionizing radiation was analyzed. Lack of NEMO impairs survival after X-ray exposure. The inhibition of ATM by KU-55933 suppresses the X-ray and heavy ion (13C, 35 MeV/u, LET 70 keV/µm) induced activation of NF-κB dependent gene expression, indicating the central position of ATM in radiation induced NF-κB activation. Short-term incubation with the proteasome inhibitor MG-132 also blocks NF-κB activation by radiation. These results suggest a role of

  9. Genomic Profiling of a Human Organotypic Model of AEC Syndrome Reveals ZNF750 as an Essential Downstream Target of Mutant TP63

    PubMed Central

    Zarnegar, Brian J.; Webster, Dan E.; Lopez-Pajares, Vanessa; Vander Stoep Hunt, Brook; Qu, Kun; Yan, Karen J.; Berk, David R.; Sen, George L.; Khavari, Paul A.

    2012-01-01

    The basis for impaired differentiation in TP63 mutant ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome is unknown. Human epidermis harboring AEC TP63 mutants recapitulated this impairment, along with downregulation of differentiation activators, including HOPX, GRHL3, KLF4, PRDM1, and ZNF750. Gene-set enrichment analysis indicated that disrupted expression of epidermal differentiation programs under the control of ZNF750 and KLF4 accounted for the majority of disrupted epidermal differentiation resulting from AEC mutant TP63. Chromatin immunoprecipitation (ChIP) analysis and ChIP-sequencing of TP63 binding in differentiated keratinocytes revealed ZNF750 as a direct target of wild-type and AEC mutant TP63. Restoring ZNF750 to AEC model tissue rescued activator expression and differentiation, indicating that AEC TP63-mediated ZNF750 inhibition contributes to differentiation defects in AEC. Incorporating disease-causing mutants into regenerated human tissue can thus dissect pathomechanisms and identify targets that reverse disease features. PMID:22922031

  10. Oligodendroglial Argonaute protein Ago2 associates with molecules of the Mbp mRNA localization machinery and is a downstream target of Fyn kinase

    PubMed Central

    Müller, Christina; Schäfer, Isabelle; Luhmann, Heiko J.; White, Robin

    2015-01-01

    Oligodendrocytes myelinate neuronal axons in the central nervous system (CNS) facilitating rapid transmission of action potentials by saltatory conduction. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination in the CNS of rodents. Mbp mRNA is not translated immediately after exit from the nucleus in the cytoplasm, but is transported to the plasma membrane in RNA transport granules in a translationally silenced state. We have previously identified the small non-coding RNA 715 (sncRNA715) as an inhibitor of Mbp translation associated with RNA granules. Argonaute (Ago) proteins and small RNAs form the minimal core of the RNA induced silencing complex and together recognize target mRNAs to be translationally inhibited or degraded. Recently, tyrosine phosphorylation of Ago2 was reported to be a regulator of small RNA binding. The oligodendroglial non-receptor tyrosine kinase Fyn is activated by neuronal signals and stimulates the translation of Mbp mRNA at the axon-glial contact site. Here we analyzed the expression of Ago proteins in oligodendrocytes, if they associate with Mbp mRNA transport granules and are tyrosine phosphorylated by Fyn. We show that all Ago proteins (Ago1-4) are expressed by oligodendrocytes and that Ago2 colocalizes with hnRNP A2 in granular cytoplasmic structures. Ago2 associates with hnRNP A2, Mbp mRNA, sncRNA715 and Fyn kinase and is tyrosine phosphorylated in response to Fyn activity. Our findings suggest an involvement of Ago2 in the translational regulation of Mbp. The identification of Ago proteins as Fyn targets will foster further research to understand in more molecular detail how Fyn activity regulates Mbp translation. PMID:26379499

  11. Oligodendroglial Argonaute protein Ago2 associates with molecules of the Mbp mRNA localization machinery and is a downstream target of Fyn kinase.

    PubMed

    Müller, Christina; Schäfer, Isabelle; Luhmann, Heiko J; White, Robin

    2015-01-01

    Oligodendrocytes myelinate neuronal axons in the central nervous system (CNS) facilitating rapid transmission of action potentials by saltatory conduction. Myelin basic protein (MBP) is an essential component of myelin and its absence results in severe hypomyelination in the CNS of rodents. Mbp mRNA is not translated immediately after exit from the nucleus in the cytoplasm, but is transported to the plasma membrane in RNA transport granules in a translationally silenced state. We have previously identified the small non-coding RNA 715 (sncRNA715) as an inhibitor of Mbp translation associated with RNA granules. Argonaute (Ago) proteins and small RNAs form the minimal core of the RNA induced silencing complex and together recognize target mRNAs to be translationally inhibited or degraded. Recently, tyrosine phosphorylation of Ago2 was reported to be a regulator of small RNA binding. The oligodendroglial non-receptor tyrosine kinase Fyn is activated by neuronal signals and stimulates the translation of Mbp mRNA at the axon-glial contact site. Here we analyzed the expression of Ago proteins in oligodendrocytes, if they associate with Mbp mRNA transport granules and are tyrosine phosphorylated by Fyn. We show that all Ago proteins (Ago1-4) are expressed by oligodendrocytes and that Ago2 colocalizes with hnRNP A2 in granular cytoplasmic structures. Ago2 associates with hnRNP A2, Mbp mRNA, sncRNA715 and Fyn kinase and is tyrosine phosphorylated in response to Fyn activity. Our findings suggest an involvement of Ago2 in the translational regulation of Mbp. The identification of Ago proteins as Fyn targets will foster further research to understand in more molecular detail how Fyn activity regulates Mbp translation. PMID:26379499

  12. Active debris removal of multiple priority targets

    NASA Astrophysics Data System (ADS)

    Braun, Vitali; Lüpken, A.; Flegel, S.; Gelhaus, J.; Möckel, M.; Kebschull, C.; Wiedemann, C.; Vörsmann, P.

    2013-05-01

    Today's space debris environment shows major concentrations of objects within distinct orbital regions for nearly all size regimes. The most critical region is found at orbital altitudes near 800 km with high declinations. Within this region many satellites are operated in so called sun-synchronous orbits (SSO). Among those, there are Earth observation, communication and weather satellites. Due to the orbital geometry in SSO, head-on encounters with relative velocities of about 15 km/s are most probable and would thus result in highly energetic collisions, which are often referred to as catastrophic collisions, leading to the complete fragmentation of the participating objects. So called feedback collisions can then be triggered by the newly generated fragments, thus leading to a further population increase in the affected orbital region. This effect is known as the Kessler syndrome.Current studies show that catastrophic collisions are not a major problem today, but will become the main process for debris generation within the SSO region in the near future, even without any further launches. In order to avoid this effect, objects with a major impact on collisional cascading have to be actively removed from the critical region after their end of life. Not having the capability to perform an end-of-life maneuver in order to transfer to a graveyard orbit or to de-orbit, many satellites and rocket bodies would have to be de-orbited within a dedicated mission. In such a mission, a service satellite would perform a de-orbit maneuver, after having docked to a specific target.In this paper, chemical and electric propulsion systems were analysed with the main focus on removing multiple targets within one single mission. The targets were chosen from a previously defined priority list in order to enhance the mission efficiency. Total mission time, ΔV and system mass were identified as key parameters to allow for an evaluation of the different concepts. It was shown that it

  13. Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities.

    PubMed

    Alqarni, Mohammed; Myint, Kyaw Zeyar; Tong, Qin; Yang, Peng; Bartlow, Patrick; Wang, Lirong; Feng, Rentian; Xie, Xiang-Qun

    2014-09-26

    We performed molecular modeling and docking to predict a putative binding pocket and associated ligand-receptor interactions for human cannabinoid receptor 2 (CB2). Our data showed that two hydrophobic residues came in close contact with three structurally distinct CB2 ligands: CP-55,940, SR144528 and XIE95-26. Site-directed mutagenesis experiments and subsequent functional assays implicated the roles of Valine residue at position 3.32 (V113) and Leucine residue at position 5.41 (L192) in the ligand binding function and downstream signaling activities of the CB2 receptor. Four different point mutations were introduced to the wild type CB2 receptor: V113E, V113L, L192S and L192A. Our results showed that mutation of Val113 with a Glutamic acid and Leu192 with a Serine led to the complete loss of CB2 ligand binding as well as downstream signaling activities. Substitution of these residues with those that have similar hydrophobic side chains such as Leucine (V113L) and Alanine (L192A), however, allowed CB2 to retain both its ligand binding and signaling functions. Our modeling results validated by competition binding and site-directed mutagenesis experiments suggest that residues V113 and L192 play important roles in ligand binding and downstream signaling transduction of the CB2 receptor. PMID:25148941

  14. Active Debris Removal of Multiple Priority Targets

    NASA Astrophysics Data System (ADS)

    Braun, Vitali; Flegel, Sven Kevin; Vörsmann, Peter; Wiedemann, Carsten; Gelhaus, Johannes; Moeckel, Marek; Kebschull, Christopher

    2012-07-01

    Today's space debris environment shows major concentrations of objects within distinct orbital regions for nearly all size regimes. The most critical region is found at orbital altitudes near 800 kilometers with high declinations. Within this region many satellites are operated in so called sun-synchronous orbits (SSO). Among those, there are Earth observation, communication and weather satellites. Due to the orbital geometry, head-on encounters with relative velocities of about 15 km/s are most probable and would thus result in highly energetic collisions, which are often referred to as catastrophic collisions, leading to the complete fragmentation of the participating objects. So called feedback collisions can then be triggered by the newly generated fragments, thus leading to a further population increase in the affected orbital region. This effect is known as the Kessler syndrome. Current studies show that catastrophic collisions are not a major problem today, but will become the main process for debris generation within the SSO region in the near future, even without any future launches. In order to avoid this effect, objects with a major impact on collisional cascading have to be actively removed from the critical region after their end of life. Not having the capability to perform an end-of-life maneuver in order to transfer to a graveyard orbit or to de-orbit, many satellites and rocket bodies would have to be de-orbited within a dedicated mission. In such a mission, a service satellite would perform a de-orbit maneuver, after having docked to a specific target. In this paper several systems, e.g. chemical and electrical engines are analysed with the main focus on removing multiple targets within one single mission. The service satellite has to undock from the previously de-orbited target in order to start the rendezvous and docking phase for a subsequent target. The targets are chosen from a previously defined priority list in order to enhance the mission

  15. Hep88 mAb-mediated paraptosis-like apoptosis in HepG2 cells via downstream upregulation and activation of caspase-3, caspase-8 and caspase-9.

    PubMed

    Mitupatum, Thantip; Aree, Kalaya; Kittisenachai, Suthathip; Roytrakul, Sittiruk; Puthong, Songchan; Kangsadalampai, Sasichai; Rojpibulstit, Panadda

    2015-01-01

    Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Presently, targeted therapy via monoclonal antibodies to specific tumor-associated antigens is being continuously developed. Hep88 mAb has proven to exert tumoricidal effects on the HepG2 cell via a paraptosis-like morphology. To verify the pathway, we then demonstrated downstream up-regulation of caspase-3, caspase-8 and caspase-9, assessingmRNA expression by real-time PCR and associated enzyme activity by colorimetric assay. Active caspase-3 determination was also accomplished by flow cytometry. Active caspase-3 expression was increased by Hep88 mAb treatment in a dose-and time-dependent manner. All of the results indicated that Hep88 mAb induced programmed cell death in the HepG2 cell line from paraptosis-like to apoptosis by downstream induction of caspases. These conclusions imply that Hep88mAb might be a promising tool for the effective treatment of HCC in the future. PMID:25773824

  16. Assembly-driven activation of the AIM2 foreign-dsDNA sensor provides a polymerization template for downstream ASC

    NASA Astrophysics Data System (ADS)

    Morrone, Seamus R.; Matyszewski, Mariusz; Yu, Xiong; Delannoy, Michael; Egelman, Edward H.; Sohn, Jungsan

    2015-07-01

    AIM2 recognizes foreign dsDNA and assembles into the inflammasome, a filamentous supramolecular signalling platform required to launch innate immune responses. We show here that the pyrin domain of AIM2 (AIM2PYD) drives both filament formation and dsDNA binding. In addition, the dsDNA-binding domain of AIM2 also oligomerizes and assists in filament formation. The ability to oligomerize is critical for binding dsDNA, and in turn permits the size of dsDNA to regulate the assembly of the AIM2 polymers. The AIM2PYD oligomers define the filamentous structure, and the helical symmetry of the AIM2PYD filament is consistent with the filament assembled by the PYD of the downstream adaptor ASC. Our results suggest that the role of AIM2PYD is not autoinhibitory, but generating a structural template by coupling ligand binding and oligomerization is a key signal transduction mechanism in the AIM2 inflammasome.

  17. 4-Aminoethylamino-emodin – a novel potent inhibitor of GSK-3β– acts as an insulin-sensitizer avoiding downstream effects of activated β-catenin

    PubMed Central

    Gebhardt, Rolf; Lerche, Katja S; Götschel, Frank; Günther, Robert; Kolander, Jens; Teich, Lars; Zellmer, Sebastian; Hofmann, Hans-Jörg; Eger, Kurt; Hecht, Andreas; Gaunitz, Frank

    2010-01-01

    Abstract Glycogen synthase kinase-3β (GSK-3β) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3β with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3β inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular ATP levels. Physiologically, L4 acts as an insulin-sensitizing agent that is able to enhance hepatocellular glycogen and fatty acid biosynthesis. These functions are particularly stimulated in the presence of elevated concentrations of glucose and in synergy with the hormone action at moderate but not high insulin levels. In contrast to other low molecular weight GSK-3β inhibitors (SB216763 and LiCl) or Wnt-3α-conditioned medium, however, L4 does not induce reporter and target genes of activated β-catenin such as TOPflash, Axin2 and glutamine synthetase. Moreover, when present together with SB216763 or LiCl, L4 counteracts expression of TOPflash or induction of glutamine synthetase by these inhibitors. Because L4 slightly activates β-catenin on its own, these results suggest that a downstream molecular step essential for activation of gene transcription by β-catenin is also inhibited by L4. It is concluded that L4 represents a potent insulin-sensitizing agent favouring physiological effects of insulin mediated by GSK-3β inhibition but avoiding hazardous effects such as activation of β-catenin-dependent gene expression which may lead to aberrant induction of cell proliferation and cancer. PMID:19228266

  18. Transcriptomic-based effects monitoring for endocrine active chemicals: Assessing relative contribution of treated wastewater to downstream pollution

    EPA Science Inventory

    The present study investigated whether combining of targeted analytical chemistry methods with unsupervised, data-rich methodologies (i.e. transcriptomics) can be utilized to evaluate relative contributions of wastewater treatment plant (WWTP) effluents to biological effects. The...

  19. Upstream to downstream: a multiple-assessment-point approach for targeting non-point-source priority management areas at large watershed scale

    NASA Astrophysics Data System (ADS)

    Chen, L.; Zhong, Y.; Wei, G.; Shen, Z.

    2014-04-01

    The identification of priority management areas (PMAs) is essential for the control of non-point-source (NPS) pollution, especially for a large-scale watershed. However, previous studies have typically focused on small-scale catchments adjacent to specific assessment points; thus, the interactions between multiple river points remain poorly understood. In this study, a multiple-assessment-point PMA (MAP-PMA) framework was proposed by integrating the upstream sources and the downstream transport aspects of NPS pollution. Daning River watershed was taken as a case study in this paper, which has demonstrated that the integration of the upstream input changes was vital for the final PMAs map, especially for downstream areas. Contrary to conventional wisdom, this research recommended that the NPS pollutants could be best controlled among the upstream high-level PMAs when protecting the water quality of the entire watershed. The MAP-PMA framework provided a more cost-effective tool for the establishment of conservation practices, especially for a large-scale watershed.

  20. Triptolide Induces Cell Apoptosis by Targeting H3K4me3 and Downstream Effector Proteins in KM3 Multiple Myeloma Cells.

    PubMed

    Wen, Lu; Chen, Yan; Zeng, Ling L; Zhao, Fei; Yi, Sha; Yang, Li J; Zhang, Ben P; Zhao, Jie; Zhao, Zi C; Zhang, Chun

    2015-01-01

    As the principal active ingredient in the Chinese herb Tripterygium wilfordii Hook.F (TwHF), triptolide has been shown to have very strong antitumor properties. The trimethylation of lysine 4 on histone H3 (H3K4me3) has been proposed to promote gene expression, and the accumulation of H3K4me3 at the transcriptional start sites of oncogenes is involved in carcinogenesis. To identify the association between the reduction of H3K4me3 and the apoptosis of MM cells induced by triptolide, we investigated the global patterns of H3K4me3 occupancy in the MM cell genome. Combined analyses using ChIP-on-chip and western blotting showed that H3K4me3 were highly enriched on the gene promoters of c-Myc and VEGFA and were associated with the up-regulation of both genes. Treatment of KM3 cells with triptolide and siRNA targeting ASH2L reduced the expression of c-Myc and VEGFA. These results suggest that triptolide can down-regulate c-Myc and VEGFA expression by blocking the accumulation of H3K4me3 on their promoters,and thus play an important role in anti-MM mechanism. PMID:26420049

  1. Constitutive activation of Pim1 kinase is a therapeutic target for adult T-cell leukemia.

    PubMed

    Bellon, Marcia; Lu, Ling; Nicot, Christophe

    2016-05-19

    Human T-cell leukemia virus type 1 (HTLV-1)-associated adult T-cell leukemia and T-cell lymphoma (ATL) are aggressive diseases with poor prognoses, limited therapeutic options, and no curative treatment. In this study, we used a mouse model of ATL and restored expression of the microRNA, miR-124a, to identify in vivo downstream effectors responsible for its tumor-suppressive functions in ATL cells. Our results revealed that STAT3, a direct target of miR-124a, is constitutively activated in HTLV-I-transformed cells and ATL cells, and activating STAT3 mutations were detected in 25.5% of primary ATL patients. Interestingly, we found that the STAT3 downstream kinase effector, Pim1, is constitutively activated in ATL cells. The dependence of ATL cells to Pim1 activity was demonstrated using 2 Pim1 small inhibitors, SMI-4a and AZD1208. These studies indicated that HTLV-I-transformed and ATL cells, but not normal peripheral blood mononuclear cells, are highly sensitive to AZD1208, and the inhibition of Pim1 signaling triggers an apoptotic signal in leukemic cells. Finally, preclinical testing of AZD1208 in a mouse model of ATL resulted in significant prevention of tumor growth in vivo. In conclusion, our studies suggest that constitutive activation of the STAT3-Pim1 pathway represents a novel therapeutic target for the treatment of ATL. PMID:26813676

  2. p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation.

    PubMed

    Sohn, Dennis; Essmann, Frank; Schulze-Osthoff, Klaus; Jänicke, Reiner U

    2006-12-01

    The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G(2)-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria. PMID:17145870

  3. Diversity of Stability, Localization, Interaction and Control of Downstream Gene Activity in the Maize Aux/IAA Protein Family

    PubMed Central

    Ludwig, Yvonne; Berendzen, Kenneth W.; Xu, Changzheng; Piepho, Hans-Peter; Hochholdinger, Frank

    2014-01-01

    AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are central regulators of auxin signal transduction. They control many aspects of plant development, share a conserved domain structure and are localized in the nucleus. In the present study, five maize Aux/IAA proteins (ZmIAA2, ZmIAA11, ZmIAA15, ZmIAA20 and ZmIAA33) representing the evolutionary, phylogenetic and expression diversity of this gene family were characterized. Subcellular localization studies revealed that ZmIAA2, ZmIAA11 and ZmIAA15 are confined to the nucleus while ZmIAA20 and ZmIAA33 are localized in both the nucleus and the cytoplasm. Introduction of specific point mutations in the degron sequence (VGWPPV) of domain II by substituting the first proline by serine or the second proline by leucine stabilized the Aux/IAA proteins. While protein half-life times between ∼11 min (ZmIAA2) to ∼120 min (ZmIAA15) were observed in wild-type proteins, the mutated forms of all five proteins were almost as stable as GFP control proteins. Moreover, all five maize Aux/IAA proteins repressed downstream gene expression in luciferase assays to different degrees. In addition, bimolecular fluorescence complementation (BiFC) analyses demonstrated interaction of all five Aux/IAA proteins with RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1, ZmIAA10) while only ZmIAA15 and ZmIAA33 interacted with the RUM1 paralog RUL1 (RUM-LIKE 1, ZmIAA29). Moreover, ZmIAA11, ZmIAA15 ZmIAA33 displayed homotypic interaction. Hence, despite their conserved domain structure, maize Aux/IAA proteins display a significant variability in their molecular characteristics which is likely associated with the wide spectrum of their developmental functions. PMID:25203637

  4. Survey of activated kinase proteins reveals potential targets for cholangiocarcinoma treatment.

    PubMed

    Dokduang, Hasaya; Juntana, Sirinun; Techasen, Anchalee; Namwat, Nisana; Yongvanit, Puangrat; Khuntikeo, Narong; Riggins, Gregory J; Loilome, Watcharin

    2013-12-01

    Improving therapy for patients with cholangiocarcinoma (CCA) presents a significant challenge. This is made more difficult by a lack of a clear understanding of potential molecular targets, such as deregulated kinases. In this work, we profiled the activated kinases in CCA in order to apply them as the targets for CCA therapy. Human phospho-receptor tyrosine kinases (RTKs) and phospho-kinase array analyses revealed that multiple kinases are activated in both CCA cell lines and human CCA tissues that included cell growth, apoptosis, cell to cell interaction, movement, and angiogenesis RTKs. Predominately, the kinases activated downstream were those in the PI3K/Akt, Ras/MAPK, JAK/STAT, and Wnt/β-catenin signaling pathways. Western blot analysis confirms that Erk1/2 and Akt activation were increased in CCA tissues when compared with their normal adjacent tissue. The inhibition of kinase activation using multi-targeted kinase inhibitors, sorafenib and sunitinib led to significant cell growth inhibition and apoptosis induction via suppression of Erk1/2 and Akt activation, whereas drugs with specificity to a single kinase showed less potency. In conclusion, our study reveals the involvement of multiple kinase proteins in CCA growth that might serve as therapeutic targets for combined kinase inhibition. PMID:23812726

  5. Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling.

    PubMed

    Gerber, Simon D; Amann, Ruth; Wyder, Stefan; Trueb, Beat

    2012-01-01

    Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron. PMID:22432025

  6. Comparison of the Gene Expression Profiles from Normal and Fgfrl1 Deficient Mouse Kidneys Reveals Downstream Targets of Fgfrl1 Signaling

    PubMed Central

    Gerber, Simon D.; Amann, Ruth; Wyder, Stefan; Trueb, Beat

    2012-01-01

    Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron. PMID:22432025

  7. How targets select activation or repression in response to Wnt.

    PubMed

    Murgan, Sabrina; Bertrand, Vincent

    2015-01-01

    In metazoans, the Wnt signaling pathway plays a key role in the regulation of binary decisions during development. During this process different sets of target genes are activated in cells where the Wnt pathway is active (classic target genes) versus cells where the pathway is inactive (opposite target genes). While the mechanism of transcriptional activation is well understood for classic target genes, how opposite target genes are activated in the absence of Wnt remains poorly characterized. Here we discuss how the key transcriptional mediator of the Wnt pathway, the TCF family member POP-1, regulates opposite target genes during C. elegans development. We examine recent findings suggesting that the direction of the transcriptional output (activation or repression) can be determined by the way TCF is recruited and physically interacts with its target gene. PMID:27123368

  8. An active target concept for the electronuclear reactor

    SciTech Connect

    Grebyonkin, K.F.; Shzerebzov, A.L.; Kandiev, Ya.Z.; Maloyaroslavtsev, A.N.; Modin, V.N.; Orlov, A.I.; Peschkov, I.A.; Scherbakov, A.P.

    1995-12-31

    Preliminary identification of the components and efficiency estimations for the proposed (by Chelyabinsk-70) concept of active target for electronuclear reactor are goals of this work. (The electronuclear reactor comprises a high-energy proton acclerator, a high-atomic-number target (lead, tungsten) which produces neutrons from the protons, and a subcritical blanket.) Results of preliminary neutron and thermal-hydraulic simulations of the target are represented in the paper and preliminary detailing of the active target components is performed. It is shown that the use of active target can lead to an essential reduction of the requirements to the accelerator power without deterioration of the safety of the system.

  9. The down regulation of target genes by photo activated DNA nanoscissors.

    PubMed

    Tsai, Tsung-Lin; Shieh, Dar-Bin; Yeh, Chen-Sheng; Tzeng, Yonhua; Htet, Khant; Chuang, Kao-Shu; Hwu, Jih Ru; Su, Wu-Chou

    2010-09-01

    An artificial, targeted, light-activated nanoscissor (ATLANS) was developed for precision photonic cleavage of DNA at selectable target sequences. The ATLANS is comprised of nanoparticle core and a monolayer of hydrazone-modified triplex-forming oligonucleotides (TFOs), which recognize and capture the targeted DNA duplex. Upon photo-illumination (lambda = 460 nm), the attached hydrazone scissor specifically cleaves the targeted DNA at a pre-designed nucleotide pair. Electrophoretic mobility shift and co-precipitation assays revealed sequence-specific binding with the short-fragment and long-form plasmid DNA of both TFO and TFO-nanoparticle probes. Upon photo-illumination, ATLANS introduced a precise double-stranded break 12bp downstream the TFO binding sequence and down-regulated the target gene in HeLa cell system. Gold nanoparticles multiplexed the cutting efficiency and potential for simultaneous manipulation of multiple targets, as well as protected DNA from non-specific photo-damage. This photon-mediated DNA manipulation technology will facilitate high spatial and temporal precision in simultaneous silencing at the genome level, and advanced simultaneous manipulation of multiple targeted genes. PMID:20605206

  10. Khellin and Visnagin Differentially Modulate AHR Signaling and Downstream CYP1A Activity in Human Liver Cells

    PubMed Central

    Proksch, Peter; Abel, Josef; Dvorak, Zdenek; Haarmann-Stemmann, Thomas

    2013-01-01

    Khellin and visnagin are two furanochromones that can be frequently found in ethnomedical formulations in Asia and the Middle East. Both compounds possess anti-inflammatory and analgesic properties, therefore modern medicine uses these compounds or structurally related derivatives for treatment of vitiligo, bronchial asthma and renal colics. Despite their frequent usage, the potential toxic properties of visnagin and khellin are not well characterized up-to-now. Many natural compounds modulate the expression and activity of cytochrome P450 1A1 (CYP1A1), which is well-known to bioactivate pro-carcinogens. The expression of this enzyme is controlled by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor and regulator of drug metabolism. Here, we investigated the influence of both furanochromones on AHR signaling in human HepG2 hepatocarcinoma cells and primary human hepatocytes. Both compounds transactivated xenobiotic response element (XRE)-driven reporter gene activity in a dose-dependent manner and induced CYP1A1 transcription in HepG2 cells and primary hepatocytes. The latter was abolished in presence of a specific AHR antagonist. CYP1A enzyme activity assays done in HepG2 cells and primary hepatocytes revealed an inhibition of enzyme activity by both furanochromones, which may become relevant regarding the metabolism of xenobiotics and co-administered therapeutic drugs. The observed induction of several other members of the AHR gene battery, whose gene products are involved in regulation of cell growth, differentiation and migration, indicates that a further toxicological characterization of visnagin and khelllin is urgently required in order to minimize potential drug-drug interactions and other toxic side-effects that may occur during therapeutic usage of these furanochromones. PMID:24069365

  11. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus.

    PubMed

    Wang, Xi; Pan, Ya-Jie; Chang, Bo-Wen; Hu, Yan-Bo; Guo, Xiao-Rui; Tang, Zhong-Hua

    2016-01-01

    We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. PMID:27314017

  12. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus

    PubMed Central

    Wang, Xi; Pan, Ya-Jie; Chang, Bo-Wen; Hu, Yan-Bo; Guo, Xiao-Rui; Tang, Zhong-Hua

    2016-01-01

    We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. PMID:27314017

  13. GPR30 Promotes Prostate Stromal Cell Activation via Suppression of ERα Expression and Its Downstream Signaling Pathway.

    PubMed

    Jia, Bona; Gao, Yu; Li, Mingming; Shi, Jiandang; Peng, Yanfei; Du, Xiaoling; Klocker, Helmut; Sampson, Natalie; Shen, Yongmei; Liu, Mengyang; Zhang, Ju

    2016-08-01

    Cancer-associated fibroblasts (CAFs) play a vital role in malignant transformation and progression of prostate cancer (PCa), and accumulating evidence suggests an enhancing effect of estrogens on PCa. The present study aimed to investigate the possible origin of prostate CAFs and the effects of estrogen receptors, G protein-coupled receptor 30 (GPR30) and estrogen receptor (ER)-α, on stromal cell activation. High expression of fibroblast activation protein (FAP), CD44, and nonmuscle myosin heavy chain B (SMemb) accompanied by low expression of smooth muscle differentiation markers was found in the stromal cells of PCa tissues and in cultured human prostate CAFs. Additionally, SMemb expression, which is coupled to cell phenotype switching and proliferation, was coexpressed with FAP, a marker of activated stromal cells, and with the stem cell marker CD44 in the stromal cells of PCa tissue. Prostate CAFs showed high GPR30 and low ERα expression. Moreover, GPR30 was coexpressed with FAP, CD44, and SMemb. Furthermore, the study demonstrated that the overexpression of GPR30 or the knockdown of ERα in prostate stromal cells induced the up-regulation of FAP, CD44, Smemb, and the down-regulation of smooth muscle markers. The conditioned medium from these cells promoted the proliferation and migration of LNCaP and PC3 PCa cells. GPR30 knockdown or ERα overexpression showed opposite effects. Finally, we present a novel mechanism whereby GPR30 limits ERα expression via inhibition of the cAMP/protein kinase A signaling pathway. These results suggest that stem-like cells within the stroma are a possible source of prostate CAFs and that the negative regulation of ERα expression by GPR30 is centrally involved in prostate stromal cell activation. PMID:27163843

  14. Chemical downstream etching of tungsten

    SciTech Connect

    Blain, M.G.; Jarecki, R.L.; Simonson, R.J.

    1998-07-01

    The downstream etching of tungsten and tungsten oxide has been investigated. Etching of chemical vapor deposited tungsten and e-beam deposited tungsten oxide samples was performed using atomic fluorine generated by a microwave discharge of argon and NF{sub 3}. Etching was found to be highly activated with activation energies approximated to be 6.0{plus_minus}0.5thinspkcal/mol and 5.4{plus_minus}0.4thinspkcal/mol for W and WO{sub 3}, respectively. In the case of F etching of tungsten, the addition of undischarged nitric oxide (NO) directly into the reaction chamber results in the competing effects of catalytic etch rate enhancement and the formation of a nearly stoichiometric WO{sub 3} passivating tungsten oxide film, which ultimately stops the etching process. For F etching of tungsten oxide, the introduction of downstream NO reduces the etch rate. {copyright} {ital 1998 American Vacuum Society.}

  15. Increased Cortical Synaptic Activation of TrkB and Downstream Signaling Markers in a Mouse Model of Down Syndrome

    PubMed Central

    Nosheny, RL; Belichenko, PV; Busse, BL; Weissmiller, AM; Dang, V; Das, D; Fahimi, A; Salehi, A; Smith, SJ; Mobley, WC

    2015-01-01

    Down Syndrome (DS), trisomy 21, is characterized by synaptic abnormalities and cognitive deficits throughout the lifespan and with development of Alzheimer’s disease (AD) neuropathology and progressive cognitive decline in adults. Synaptic abnormalities are also present in the Ts65Dn mouse model of DS, but which synapses are affected and the mechanisms underlying synaptic dysfunction are unknown. Here we show marked increases in the levels and activation status of TrkB and associated signaling proteins in cortical synapses in Ts65Dn mice. Proteomic analysis at the single synapse level of resolution using array tomography (AT) uncovered increased colocalization of activated TrkB with signaling endosome related proteins, and demonstrated increased TrkB signaling. The extent of increases in TrkB signaling differed in each of the cortical layers examined and with respect to the type of synapse, with the most marked increases seen in inhibitory synapses. These findings are evidence of markedly abnormal TrkB-mediated signaling in synapses. They raise the possibility that dysregulated TrkB signaling contributes to synaptic dysfunction and cognitive deficits in DS. PMID:25753471

  16. Exposure histories derived from selenium in otoliths of three cold-water fish species captured downstream from coal mining activity.

    PubMed

    Friedrich, Lisa A; Orr, Patricia L; Halden, Norman M; Yang, Panseok; Palace, Vince P

    2011-10-01

    Establishing exposure to contaminants within a given environment is often difficult for fish species with large home ranges. Chemical analyses of muscle or visceral tissue are useful indicators of recent exposure, but depuration, metabolic transformation, and tissue redistributions can alter temporal resolution. Otoliths are metabolically stable and thus provide complete chemical records within their calcified tissues that, when coupled to the annular structure, can provide temporal resolution for exposure to trace metals. Otoliths from bull trout, cutthroat trout, and mountain whitefish from an area rich in seleniferous soils and with active coal mining activity were analyzed for selenium to determine any history of exposure to elevated levels of selenium. Selenium concentrations in otolith primordia tended to be low, indicating that these fish emerged in low selenium areas. Later life stages showed peaks of high Se concentrations, suggesting that individuals moved into areas of increased selenium later in life. Individuals captured from the same area had a wide variety of selenium exposure profiles, indicating that these fish do not move en masse into and out of high-selenium areas. Year-to-year variability of selenium exposure patterns within an otolith suggests inconsistent utilization of high- and low-selenium areas by the individual. The inconsistent exposure profiles for these fish, in addition to their home range of tens of kilometres, indicate that soft tissue concentrations, while useful indicators of recent exposure, cannot be relied upon to provide a life history recording of exposure. PMID:21899824

  17. Targeting and valuing conservation investments in support of a water fund: linking upstream land management with downstream services in the Upper Tana catchment, Kenya

    NASA Astrophysics Data System (ADS)

    Bryant, B. P.; Droogers, P.; Hunink, J.; Vogl, A.; Wolny, S.

    2014-12-01

    We apply an integrated modeling framework to both target and value watershed management interventions in the Upper Tana watershed, which provides municipal water, irrigation water, and hydropower services to Nairobi and surrounding areas. The analysis begins by applying an index model approach that incorporates existing land use and land surface characteristics to prioritize the type and location of conservation investments in different subbasins, subject to budget constraints and stakeholder concerns (Resource Investment Optimization System -- RIOS). We then run the Soil and Water Assessment Tool (SWAT) using the RIOS-identified investment scenarios to produce spatially explicit scenarios that simulate changes in water yield and suspended sediment. Finally, we link those biophysical outputs to monetary and non-monetary human well-being metrics for multiple benefit streams, including: Reduced water treatment costs, increased hydropower production, and crop yield benefits for upstream farmers in the conservation area. The viability of a payment for watershed services scheme is discussed, with attention to the various components of value assessed and to dependencies on water management approaches. While other studies have examined links between land use and the provision of hydrologic services, this study is novel in that it presents an integrated analysis that targets interventions in a decision context and then relies on calibrated, process-based, biophysical models to demonstrate the return on those investments considering multiple (and sometimes competing) hydrological services, doing so at a sub-annual time-scale.

  18. Atypical nuclear apoptosis downstream to caspase-3 activation in ara-C treated CCRF-CEM cells.

    PubMed

    Badran, Adel; Iwasaki, Hiromichi; Inoue, Hitoshi; Ueda, Takanori

    2003-03-01

    The necessity of internucleosomal DNA fragmentation for the complete execution of apoptosis is still controversial. While investigating the apoptotic pathway induced by 1-beta-D-arabinofuranosylcytosine (ara-C) in the human T-lymphoblastic leukemia CCRF-CEM (CEM) cells, we could easily retrieve high molecular weight (HMW) DNA fragments with a predominant size of 50 kb. However, under the same circumstances, internucleosomal DNA fragmentation characteristic of apoptosis was undetectable despite estimated heightened caspase-3 activity. Paradoxically, generation of low molecular weight DNA fragments was readily demonstrable by flow cytometric and immunohistochemical evidence in the absence of any detectable DNA ladder formation. These findings present a proof that, within certain contexts, small-sized DNA fragmentation occurring in apoptosis may not necessarily be of the ladder yielding internucleosomal integer multiples' pattern. Our data also add to the evidence that the machinery underlying HMW DNA fragmentation is distinct from that responsible for the internucleosomal one. PMID:12579303

  19. A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

    PubMed

    García-Rodríguez, Luis J; De Piccoli, Giacomo; Marchesi, Vanessa; Jones, Richard C; Edmondson, Ricky D; Labib, Karim

    2015-10-15

    Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint. PMID:26250113

  20. Effects of urine composition on epithelial Na+ channel-targeted protease activity.

    PubMed

    Berman, Jonathan M; Awayda, Ryan G; Awayda, Mouhamed S

    2015-11-01

    We examined human urinary proteolytic activity toward the Epithelial Sodium Channel (ENaC). We focused on two sites in each of alpha and gamma ENaC that are targets of endogenous and exogenous proteases. We examined the effects of ionic strength, pH and urinary H(+)-buffers, metabolic intermediates, redox molecules, and large urinary proteins. Monoatomic cations caused the largest effect, with sodium inhibiting activity in the 15-515 mEq range. Multivalent cations zinc and copper inhibited urinary proteolytic activity at concentrations below 100 μmol/L. Similar to sodium, urea caused a 30% inhibition in the 0-500 mmol/L range. This was not observed with acetone and ethanol. Modulating urinary redox status modified activity with H2O2 stimulated and ascorbate inhibited activity. Minimal effects (<10%) were observed with caffeine, glucose, several TCA cycle intermediates, salicylic acid, inorganic phosphate, albumin, creatinine, and Tamm-Horsfall protein. The cumulative activity of ENaC-cleaving proteases was highest at neutral pH, however, alpha and gamma proteases exhibited an inverse dependence with alpha stimulated at acidic and gamma stimulated at alkaline pH. These data indicate that ENaC-targeting urinary proteolytic activity is sensitive to sodium, urea and pH and changes in these components can modify channel cleavage and activation status, and likely downstream sodium absorption unrelated to changes in protein or channel density. PMID:26564065

  1. Camera calibration approach based on adaptive active target

    NASA Astrophysics Data System (ADS)

    Zhang, Yalin; Zhou, Fuqiang; Deng, Peng

    2011-12-01

    Aiming at calibrating camera on site, where the lighting condition is hardly controlled and the quality of target images would be declined when the angle between camera and target changes, an adaptive active target is designed and the camera calibration approach based on the target is proposed. The active adaptive target in which LEDs are embedded is flat, providing active feature point. Therefore the brightness of the feature point can be modified via adjusting the electricity, judging from the threshold of image feature criteria. In order to extract features of the image accurately, the concept of subpixel-precise thresholding is also proposed. It converts the discrete representation of the digital image to continuous function by bilinear interpolation, and the sub-pixel contours are acquired by the intersection of the continuous function and the appropriate selection of threshold. According to analysis of the relationship between the features of the image and the brightness of the target, the area ratio of convex hulls and the grey value variance are adopted as the criteria. Result of experiments revealed that the adaptive active target accommodates well to the changing of the illumination in the environment, the camera calibration approach based on adaptive active target can obtain high level of accuracy and fit perfectly for image targeting in various industrial sites.

  2. Identification of putative pathogenic microRNA and its downstream targets in anaplastic lymphoma kinase-negative anaplastic large cell lymphoma.

    PubMed

    Mehrotra, Meenakshi; Medeiros, L Jeffrey; Luthra, Rajyalakshmi; Sargent, Rachel L; Yao, Hui; Barkoh, Bedia A; Singh, Rajesh; Patel, Keyur P

    2014-10-01

    Anaplastic large cell lymphomas (ALCL) are tumors of T/null-cell lineage characterized by uniform CD30 expression. The 2008 World Health Organization classification subdivided ALCLs into 2 groups: anaplastic lymphoma kinase (ALK)-positive (established entity) and ALK-negative (proposed new entity) ALCL. The genetic basis for the pathogenesis of newly categorized ALK- ALCL is poorly understood. In this study, we used microRNA microarray analysis to identify differentially expressed microRNAs in ALK+ and ALK- ALCL. ALK- ALCL showed significantly higher expression of miR-155 (0.888 ± 0.228) compared with ALK+ ALCL (0.0565 ± 0.009) on microarray and by quantitative real-time polymerase chain reaction in ALK- ALCL compared with ALK+ ALCL (P < .05) with a strong correlation between the 2 platforms (R = 0.9, P < .0003). A novel in situ hybridization method allows direct visualization of expression patterns and relative quantitation of miR-155 (mean score, 2.3 versus 1.3; P = .01) for the first time in tissue sections of ALCL. Among computationally predicted targets of miR-155, we identified ZNF652 (r = -0.57, P = .05), BACH1 (r = 0.88, P = .02), RBAK (r = 0.81, P = .05), TRIM32 (r = 0.92, P = .01), E2F2 (r = 0.81, P = .05), and TP53INP1 (r = -0.31, P = .03) as genes whose expression by quantitative real-time polymerase chain reaction correlated significantly with the level of miR-155 in ALCL tumor tissue. PMID:25128227

  3. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-03-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  4. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-01-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  5. Targeted, noninvasive blockade of cortical neuronal activity

    NASA Astrophysics Data System (ADS)

    McDannold, Nathan; Zhang, Yongzhi; Power, Chanikarn; Arvanitis, Costas D.; Vykhodtseva, Natalia; Livingstone, Margaret

    2015-11-01

    Here we describe a novel method to noninvasively modulate targeted brain areas through the temporary disruption of the blood-brain barrier (BBB) via focused ultrasound, enabling focal delivery of a neuroactive substance. Ultrasound was used to locally disrupt the BBB in rat somatosensory cortex, and intravenous administration of GABA then produced a dose-dependent suppression of somatosensory-evoked potentials in response to electrical stimulation of the sciatic nerve. No suppression was observed 1-5 days afterwards or in control animals where the BBB was not disrupted. This method has several advantages over existing techniques: it is noninvasive; it is repeatable via additional GABA injections; multiple brain regions can be affected simultaneously; suppression magnitude can be titrated by GABA dose; and the method can be used with freely behaving subjects. We anticipate that the application of neuroactive substances in this way will be a useful tool for noninvasively mapping brain function, and potentially for surgical planning or novel therapies.

  6. The Epithelial-Mesenchymal Transition (EMT) Regulatory Factor SLUG (SNAI2) Is a Downstream Target of SPARC and AKT in Promoting Melanoma Cell Invasion

    PubMed Central

    Fenouille, Nina; Tichet, Mélanie; Dufies, Maeva; Pottier, Anaïs; Mogha, Ariane; Soo, Julia K.; Rocchi, Stéphane; Mallavialle, Aude; Galibert, Marie-Dominique; Khammari, Amir; Lacour, Jean-Philippe; Ballotti, Robert; Deckert, Marcel; Tartare-Deckert, Sophie

    2012-01-01

    During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAFV600E using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAFV600E. Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG

  7. EWS/FLI and its Downstream Target NR0B1 Interact Directly to Modulate Transcription and Oncogenesis in Ewing's Sarcoma

    PubMed Central

    Kinsey, Michelle; Smith, Richard; Iyer, Anita K.; McCabe, Edward R.B.; Lessnick, Stephen L.

    2009-01-01

    Most Ewing's sarcomas harbor chromosomal translocations that encode fusions between EWS and ETS family members. The most common fusion, EWS/FLI, consists of an EWSR1-derived strong transcriptional activation domain fused, in frame, to the DNA binding domain-containing portion of FLI1. EWS/FLI functions as an aberrant transcription factor to regulate genes that mediate the oncogenic phenotype of Ewing's sarcoma. One of these regulated genes, NR0B1, encodes a co-repressor protein, and likely plays a transcriptional role in tumorigenesis. However, the genes that NR0B1 regulates and the transcription factors it interacts with in Ewing's sarcoma are largely unknown. We used transcriptional profiling and chromatin immunoprecipitation to identify genes that are regulated by NR0B1, and compared these data to similar data for EWS/FLI. While the transcriptional profile overlapped as expected, we also found that the genome-wide localization of NR0B1and EWS/FLI overlapped as well, suggesting that they regulate some genes coordinately. Further analysis revealed that NR0B1 and EWS/FLI physically interact. This protein-protein interaction is likely to be relevant for Ewing's sarcoma development because mutations in NR0B1 that disrupt the interaction have transcriptional consequences and also abrogate oncogenic transformation. Taken together, these data suggest that EWS/FLI and NR0B1 physically interact, coordinately modulate gene expression, and mediate the transformed phenotype of Ewing's sarcoma. PMID:19920188

  8. Identification of Phosphotyrosine Binding Domain-Containing Proteins as Novel Downstream Targets of the EphA8 Signaling Function▿ †

    PubMed Central

    Shin, Jongdae; Gu, Changkyu; Park, Eunjeong; Park, Soochul

    2007-01-01

    Eph receptors and ephrins have been implicated in a variety of cellular processes, including morphology and motility, because of their ability to modulate intricate signaling networks. Here we show that the phosphotyrosine binding (PTB) domain-containing proteins AIDA-1b and Odin are tightly associated with the EphA8 receptor in response to ligand stimulation. Both AIDA-1b and Odin belong to the ankyrin repeat and sterile alpha motif domain-containing (Anks) protein family. The PTB domain of Anks family proteins is crucial for their association with the juxtamembrane domain of EphA8, whereas EphA8 tyrosine kinase activity is not required for this protein-protein interaction. In addition, we found that Odin is a more physiologically relevant partner of EphA8 in mammalian cells. Interestingly, overexpression of the Odin PTB domain alone attenuated EphA8-mediated inhibition of cell migration in HEK293 cells, suggesting that it acts as a dominant-negative mutant of the endogenous Odin protein. More importantly, small interfering RNA-mediated Odin silencing significantly diminished ephrinA5-induced EphA8 signaling effects, which inhibit cell migration in HEK293 cells and retract growing neurites of Neuro2a cells. Taken together, our findings support a possible function for Anks family proteins as scaffolding proteins of the EphA8 signaling pathway. PMID:17875921

  9. Active calibration target for bistatic radar cross-section measurements

    NASA Astrophysics Data System (ADS)

    Pienaar, M.; Odendaal, J. W.; Joubert, J.; Cilliers, J. E.; Smit, J. C.

    2016-05-01

    Either passive calibration targets are expensive and complex to manufacture or their bistatic radar cross section (RCS) levels are significantly lower than the monostatic RCS levels of targets such as spheres, dihedral, and trihedral corner reflectors. In this paper the performance of an active calibration target with relative high bistatic RCS values is illustrated as a reference target for bistatic RCS measurements. The reference target is simple to manufacture, operates over a wide frequency range, and can be configured to calibrate all four polarizations (VV, HH, HV, and VH). Bistatic RCS measurements of canonical targets, performed in a controlled environment, are calibrated with the reference target and the results are compared to simulated results using FEKO.

  10. N-myc downstream regulated gene 2 overexpression reduces matrix metalloproteinase-2 and -9 activities and cell invasion of A549 lung cancer cell line in vitro

    PubMed Central

    Faraji, Seyed Nooredin; Mojtahedi, Zahra; Ghalamfarsa, Ghasem; Takhshid, Mohammad Ali

    2015-01-01

    Objective(s): N-myc downstream regulated gene 2 (NDRG2) is a candidate gene for tumor suppression. The expression of NDRG2 is down-regulated in several tumors including lung cancer. The aim of this study was to explore the effect of NDRG2 overexpression on invasion, migration, and enzymatic activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in human lung adenocarcinoma A549 cells. Materials and Methods: A recombinant plasmid encoding green fluorescent protein (GFP)-tagged NDRG2 (pCMV6-AC-NDRG2-GFP) was used to overexpress GFP-tagged NDRG2 in A549 cells. The cells in the experimental group and those in the control group were transfected with pCMV6-AC-NDRG2-GFP and a control plasmid without NDRG2 (pCMV6-AC-GFP), respectively. Fluorescent microscopy and flowcytometry analysis of GFP expression were used to evaluate the cellular expression of GFP-tagged NDRG2 and the efficiency of transfection. The effects of NDRG2 expression on cell invasion and migration were evaluated using transwell filter migration assay. The gelatinase activity of secreted MMP-2 and MMP-9 was measured by gelatin zymography. Results: Our results demonstrated the expression of GFP-tagged NDRG2 in the cytoplasm and nucleus of A549 cells. The findings of transwell assay showed that NDRG2 overexpression reduced migration and invasion of A549 cells compared to control cells. Gelatin zymography analyses revealed that NDRG2 overexpression decreased the gelatinase activity of secreted MMP-2 and MMP-9. Conclusion: These findings suggest that NDRG2 may be a new anti-invasion factor in lung cancer that inhibits MMPs activities. PMID:26557966

  11. Cancer active targeting by nanoparticles: a comprehensive review of literature

    PubMed Central

    Bazak, Remon; Houri, Mohamad; Achy, Samar El; Kamel, Serag

    2016-01-01

    Purpose Cancer is one of the leading causes of death, and thus, the scientific community has but great efforts to improve cancer management. Among the major challenges in cancer management is development of agents that can be used for early diagnosis and effective therapy. Conventional cancer management frequently lacks accurate tools for detection of early tumors and has an associated risk of serious side effects of chemotherapeutics. The need to optimize therapeutic ratio as the difference with which a treatment affects cancer cells versus healthy tissues lead to idea that it is needful to have a treatment that could act a the “magic bullet”—recognize cancer cells only. Nanoparticle platforms offer a variety of potentially efficient solutions for development of targeted agents that can be exploited for cancer diagnosis and treatment. There are two ways by which targeting of nanoparticles can be achieved, namely passive and active targeting. Passive targeting allows for the efficient localization of nanoparticles within the tumor microenvironment. Active targeting facilitates the active uptake of nanoparticles by the tumor cells themselves. Methods Relevant English electronic databases and scientifically published original articles and reviews were systematically searched for the purpose of this review. Results In this report, we present a comprehensive review of literatures focusing on the active targeting of nanoparticles to cancer cells, including antibody and antibody fragment-based targeting, antigen-based targeting, aptamer-based targeting, as well as ligand-based targeting. Conclusion To date, the optimum targeting strategy has not yet been announced, each has its own advantages and disadvantages even though a number of them have found their way for clinical application. Perhaps, a combination of strategies can be employed to improve the precision of drug delivery, paving the way for a more effective personalized therapy. PMID:25005786

  12. FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

    PubMed

    Kamei, Yasutomi; Hattori, Maki; Hatazawa, Yukino; Kasahara, Tomomi; Kanou, Masanobu; Kanai, Sayaka; Yuan, Xunmei; Suganami, Takayoshi; Lamers, Wouter H; Kitamura, Tadahiro; Ogawa, Yoshihiro

    2014-09-15

    Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle. PMID:25074987

  13. DNA G-quadruplex formation in response to remote downstream transcription activity: long-range sensing and signal transducing in DNA double helix.

    PubMed

    Zhang, Chao; Liu, Hong-He; Zheng, Ke-Wei; Hao, Yu-Hua; Tan, Zheng

    2013-08-01

    G-quadruplexes, four-stranded structures formed by Guanine-rich nucleic acids, are implicated in many physiological and pathological processes. G-quadruplex-forming sequences are abundant in genomic DNA, and G-quadruplexes have recently been shown to exist in the genome of mammalian cells. However, how G-quadruplexes are formed in the genomes remains largely unclear. Here, we show that G-quadruplex formation can be remotely induced by downstream transcription events that are thousands of base pairs away. The induced G-quadruplexes alter protein recognition and cause transcription termination at the local region. These results suggest that a G-quadruplex-forming sequence can serve as a sensor or receiver to sense remote DNA tracking activity in response to the propagation of mechanical torsion in a DNA double helix. We propose that the G-quadruplex formation may provide a mean for long-range sensing and communication between distal genomic locations to coordinate regulatory transactions in genomic DNA. PMID:23716646

  14. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis1[OPEN

    PubMed Central

    Kong, Qing; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-01-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  15. View downstream of timber guide wall downstream from SE corner ...

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

    View downstream of timber guide wall downstream from SE corner of lock, view towards east - St. Lucie Canal, St. Lucie Lock No. 1, St. Lucie, Cross State Canal, Okeechobee Intracoastal Waterway, Stuart, Martin County, FL

  16. Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    PubMed Central

    Akhtar, Saghir; Yousif, Mariam H. M.; Dhaunsi, Gursev S.; Sarkhouh, Fatma; Chandrasekhar, Bindu; Attur, Sreeja; Benter, Ibrahim F.

    2013-01-01

    Diabetes mellitus leads to vascular complications but the underlying signalling mechanisms are not fully understood. Here, we examined the role of ErbB2 (HER2/Neu), a transmembrane receptor tyrosine kinase of the ErbB/EGFR (epidermal growth factor receptor) family, in mediating diabetes-induced vascular dysfunction in an experimental model of type 1 diabetes. Chronic treatment of streptozotocin-induced diabetic rats (1 mg/kg/alt diem) or acute, ex-vivo (10−6, 10−5 M) administration of AG825, a specific inhibitor of ErbB2, significantly corrected the diabetes-induced hyper-reactivity of the perfused mesenteric vascular bed (MVB) to the vasoconstrictor, norephinephrine (NE) and the attenuated responsiveness to the vasodilator, carbachol. Diabetes led to enhanced phosphorylation of ErbB2 at multiple tyrosine (Y) residues (Y1221/1222, Y1248 and Y877) in the MVB that could be attenuated by chronic AG825 treatment. Diabetes- or high glucose-mediated upregulation of ErbB2 phosphorylation was coupled with activation of Rho kinases (ROCKs) and ERK1/2 in MVB and in cultured vascular smooth muscle cells (VSMC) that were attenuated upon treatment with either chronic or acute AG825 or with anti-ErbB2 siRNA. ErbB2 likley heterodimerizes with EGFR, as evidenced by increased co-association in diabetic MVB, and further supported by our finding that ERK1/2 and ROCKs are common downstream effectors since their activation could also be blocked by AG1478. Our results show for the first time that ErbB2 is an upstream effector of ROCKs and ERK1/2 in mediating diabetes-induced vascular dysfunction. Thus, potential strategies aimed at modifying actions of signal transduction pathways involving ErbB2 pathway may prove to be beneficial in treatment of diabetes-induced vascular complications. PMID:23826343

  17. High efficiency cell-specific targeting of cytokine activity

    NASA Astrophysics Data System (ADS)

    Garcin, Geneviève; Paul, Franciane; Staufenbiel, Markus; Bordat, Yann; van der Heyden, José; Wilmes, Stephan; Cartron, Guillaume; Apparailly, Florence; de Koker, Stefaan; Piehler, Jacob; Tavernier, Jan; Uzé, Gilles

    2014-01-01

    Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-by-targeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

  18. An active target for the accelerator-based transmutation system

    SciTech Connect

    Grebyonkin, K. F.

    1995-09-15

    Consideration is given to the possibility of radical reduction in power requirements to the proton accelerator of the electronuclear reactor due to neutron multiplication both in the blanket and the target of an active material. The target is supposed to have the fast-neutron spectrum, and the blanket--the thermal one. The blanket and the target are separated by the thermal neutrons absorber, which is responsible for the neutron decoupling of the active target and blanket. Also made are preliminary estimations which illustrate that the realization of the idea under consideration can lead to significant reduction in power requirements to the proton beam and, hence considerably improve economic characteristics of the electronuclear reactor.

  19. A review of the ligands and related targeting strategies for active targeting of paclitaxel to tumours.

    PubMed

    Li, Juan; Wang, Fengshan; Sun, Deqing; Wang, Rongmei

    2016-08-01

    It has been 30 years since the discovery of the anti-tumour property of paclitaxel (PTX), which has been successfully applied in clinic for the treatment of carcinomas of the lungs, breast and ovarian. However, PTX is poorly soluble in water and has no targeting and selectivity to tumour tissue. Recent advances in active tumour targeting of PTX delivery vehicles have addressed some of the issues related to lack of solubility in water and non-specific toxicities associated with PTX. These PTX delivery vehicles are designed for active targeting to specific cancer cells by the addition of ligands for recognition by specific receptors/antigens on cancer cells. This article will focus on various ligands and related targeting strategies serving as potential tools for active targeting of PTX to tumour tissues, illustrating their use in different tumour models. This review also highlights the need of further studies on the discovery of receptors in different cells of specific organ and ligands with binding efficiency to these specific receptors. PMID:26878228

  20. IKK is a therapeutic target in KRAS-Induced lung cancer with disrupted p53 activity

    PubMed Central

    Bassères, Daniela S.; Ebbs, Aaron; Cogswell, Patricia C.; Baldwin, Albert S.

    2014-01-01

    Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic deletion of IKKβ or the RELA/p65 subunit of NF-κB. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKKβ inhibitor Compound A (CmpdA) led to NF-κB inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKKα or IKKβ reduced activity of the NF-κB canonical pathway. Next, we determined that both IKKα and IKKβ contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-KrasG12D) combined with loss of p53 (LSL-KrasG12D/p53fl/fl). CmpdA treatment was well tolerated and mice treated with this IKKβ inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKKβ inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity. PMID:24955217

  1. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

    PubMed

    Mulder, Monique P C; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C O; Schulman, Brenda A; Komander, David; Neefjes, Jacques; El Oualid, Farid; Ovaa, Huib

    2016-07-01

    Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades. PMID:27182664

  2. Targeting the Metastasis Suppressor, N-Myc Downstream Regulated Gene-1, with Novel Di-2-Pyridylketone Thiosemicarbazones: Suppression of Tumor Cell Migration and Cell-Collagen Adhesion by Inhibiting Focal Adhesion Kinase/Paxillin Signaling.

    PubMed

    Wangpu, Xiongzhi; Lu, Jiaoyang; Xi, Ruxing; Yue, Fei; Sahni, Sumit; Park, Kyung Chan; Menezes, Sharleen; Huang, Michael L H; Zheng, Minhua; Kovacevic, Zaklina; Richardson, Des R

    2016-05-01

    Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway. PMID:26895766

  3. A helium gas scintillator active target for photoreaction measurements

    NASA Astrophysics Data System (ADS)

    Al Jebali, Ramsey; Annand, John R. M.; Adler, Jan-Olof; Akkurt, Iskender; Buchanan, Emma; Brudvik, Jason; Fissum, Kevin; Gardner, Simon; Hamilton, David J.; Hansen, Kurt; Isaksson, Lennart; Livingston, Kenneth; Lundin, Magnus; McGeorge, John C.; MacGregor, Ian J. D.; MacRae, Roderick; Middleton, Duncan G.; Reiter, Andreas J. H.; Rosner, Günther; Schröder, Bent; Sjögren, Johan; Sokhan, Daria; Strandberg, Bruno

    2015-10-01

    A multi-cell He gas scintillator active target, designed for the measurement of photoreaction cross sections, is described. The target has four main chambers, giving an overall thickness of 0.103 g/cm3 at an operating pressure of 2 MPa. Scintillations are read out by photomultiplier tubes and the addition of small amounts of N2 to the He, to shift the scintillation emission from UV to visible, is discussed. First results of measurements at the MAX IV Laboratory tagged-photon facility show that the target has a timing resolution of around 1 ns and can cope well with a high-flux photon beam. The determination of reaction cross sections from target yields relies on a Monte Carlo simulation, which considers scintillation light transport, photodisintegration processes in 4He, background photon interactions in target windows and interactions of the reaction-product particles in the gas and target container. The predictions of this simulation are compared to the measured target response.

  4. A CONSTANS-like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1 in rice.

    PubMed

    Sheng, Peike; Wu, Fuqing; Tan, Junjie; Zhang, Huan; Ma, Weiwei; Chen, Liping; Wang, Jiachang; Wang, Jie; Zhu, Shanshan; Guo, Xiuping; Wang, Jiulin; Zhang, Xin; Cheng, Zhijun; Bao, Yiqun; Wu, Chuanyin; Liu, Xuanming; Wan, Jianmin

    2016-09-01

    Flowering time determines the adaptability of crop plants to different local environments, thus being one of the most important agronomic traits targeted in breeding programs. Photoperiod is one of the key factors that control flowering in plant. A number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis, as well as in short-day plants such as Oryza sativa. Of those, CONSTANS (CO) as a floral integrator promotes flowering in Arabidopsis under long day conditions. In rice, Heading date1 (Hd1), a homologue of CO, functions in an opposite way, which inhibits flowering under long day conditions and induces flowering under short day conditions. Here, we show that another CONSTANS-like (COL) gene, OsCOL13, negatively regulates flowering in rice under both long and short day conditions. Overexpression of OsCOL13 delays flowering regardless of day length. We also demonstrated that OsCOL13 has a constitutive and rhythmic expression pattern, and that OsCOL13 is localized to the nucleus. OsCOL13 displays transcriptional activation activity in the yeast assays and likely forms homodimers in vivo. OsCOL13 suppresses the florigen genes Hd3a and RFT1 by repressing Ehd1, but has no relationship with other known Ehd1 regulators as determined by using mutants or near isogenic lines. In addition, the transcriptional level of OsCOL13 significantly decreased in the osphyb mutant, but remained unchanged in the osphya and osphyc mutants. Thus, we conclude that OsCOL13 functions as a negative regulator downstream of OsphyB and upstream of Ehd1 in the photoperiodic flowering in rice. PMID:27405463

  5. Active Targeted Drug Delivery for Microbes Using Nano-Carriers

    PubMed Central

    Lin, Yung-Sheng; Lee, Ming-Yuan; Yang, Chih-Hui; Huang, Keng-Shiang

    2015-01-01

    Although vaccines and antibiotics could kill or inhibit microbes, many infectious diseases remain difficult to treat because of acquired resistance and adverse side effects. Nano-carriers-based technology has made significant progress for a long time and is introducing a new paradigm in drug delivery. However, it still has some challenges like lack of specificity toward targeting the infectious site. Nano-carriers utilized targeting ligands on their surface called ‘active target’ provide the promising way to solve the problems like accelerating drug delivery to infectious areas and preventing toxicity or side-effects. In this mini review, we demonstrate the recent studies using the active targeted strategy to kill or inhibit microbes. The four common nano-carriers (e.g. liposomes, nanoparticles, dendrimers and carbon nanotubes) delivering encapsulated drugs are introduced. PMID:25877093

  6. Acoustic gaze adjustments during active target selection in echolocating porpoises.

    PubMed

    Wisniewska, Danuta Maria; Johnson, Mark; Beedholm, Kristian; Wahlberg, Magnus; Madsen, Peter Teglberg

    2012-12-15

    Visually dominant animals use gaze adjustments to organize perceptual inputs for cognitive processing. Thereby they manage the massive sensory load from complex and noisy scenes. Echolocation, as an active sensory system, may provide more opportunities to control such information flow by adjusting the properties of the sound source. However, most studies of toothed whale echolocation have involved stationed animals in static auditory scenes for which dynamic information control is unnecessary. To mimic conditions in the wild, we designed an experiment with captive, free-swimming harbor porpoises tasked with discriminating between two hydrophone-equipped targets and closing in on the selected target; this allowed us to gain insight into how porpoises adjust their acoustic gaze in a multi-target dynamic scene. By means of synchronized cameras, an acoustic tag and on-target hydrophone recordings we demonstrate that porpoises employ both beam direction control and range-dependent changes in output levels and pulse intervals to accommodate their changing spatial relationship with objects of immediate interest. We further show that, when switching attention to another target, porpoises can set their depth of gaze accurately for the new target location. In combination, these observations imply that porpoises exert precise vocal-motor control that is tied to spatial perception akin to visual accommodation. Finally, we demonstrate that at short target ranges porpoises narrow their depth of gaze dramatically by adjusting their output so as to focus on a single target. This suggests that echolocating porpoises switch from a deliberative mode of sensorimotor operation to a reactive mode when they are close to a target. PMID:23175527

  7. Effect of target probability on pre-stimulus brain activity.

    PubMed

    Lucci, G; Berchicci, M; Perri, R L; Spinelli, D; Di Russo, F

    2016-05-13

    Studies on perceptual decision-making showed that manipulating the proportion of target and non-target stimuli affects the behavioral performance. Tasks with high frequency of targets are associated to faster response times (RTs) conjunctively to higher number of errors (reflecting a response bias characterized by speed/accuracy trade-off) when compared to conditions with low frequency of targets. Electroencephalographic studies well described modulations of post-stimulus event-related potentials as effect of the stimulus probability; in contrast, in the present study we focused on the pre-stimulus preparatory activities subtending the response bias. Two versions of a Go/No-go task characterized by different proportion of Go stimuli (88% vs. 12%) were adopted. In the task with frequent go trials, we observed a strong enhancement in the motor preparation as indexed by the Bereitschaftspotential (BP, previously associated with activity within the supplementary motor area), faster RTs, and larger commission error rate than in the task with rare go trials. Contemporarily with the BP, a right lateralized prefrontal negativity (lateral pN, previously associated with activity within the dorsolateral prefrontal cortex) was larger in the task with rare go trial. In the post-stimulus processing stage, we confirmed that the N2 and the P3 components were larger for rare trials, irrespective of the Go/No-go stimulus category. The increase of activities recorded in the preparatory phase related to frequency of targets is consistent with the view proposed in accumulation models of perceptual decision for which target frequency affects the subjective baseline, reducing the distance between the starting-point and the response boundary, which determines the response speed. PMID:26912279

  8. Cilostazol Induces PGI2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

    PubMed Central

    Hashimoto, Ayako

    2015-01-01

    Background Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is known as an anti-platelet drug and acts directly on platelets. Cilostazol has been shown to exhibit vascular protection in ischemic diseases. Although vascular endothelium-derived prostaglandin I2 (PGI2) plays an important role in vascular protection, it is unknown whether cilostazol directly stimulates PGI2 synthesis in endothelial cells. Here, we elucidate the mechanism of cilostazol-induced PGI2 stimulation in endothelial cells. Methods and Results Human aortic endothelial cells (HAECs) were stimulated with cilostazol and PGI2 accumulation in the culture media was measured. Cilostazol increased PGI2 synthesis via the arachidonic acid pathway. Cilostazol-induced intracellular calcium also promoted PGI2 synthesis via the inositol 1,4,5-trisphosphate receptor. Using RNAi, silencing of PDE3B abolished the induction effect of cilostazol on PGI2 synthesis and intracellular cAMP accumulation. Inhibition of the exchange protein, which was directly activated by cyclic AMP 1 (Epac-1) and its downstream signal the Ras-like small GTPase (Rap-1), abolished cilostazol-induced PGI2 synthesis, but this did not take place via protein kinase A (PKA). Inhibition of downstream signaling, such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) γ, and phospholipase C (PLC) ε, suppressed cilostazol-induced PGI2 synthesis. Conclusions The PDE3/Epac-1/Rap-1 signaling pathway plays an important role in cilostazol-induced PGI2 synthesis. Namely, stimulation of HAECs with cilostazol induces intracellular calcium elevation via the Rap-1/PLCε/IP3 pathway, along with MAPK activation via direct activation by Epac-1/Rap-1 and indirect activation by Epac-1/Rap-1/PI3Kγ, resulting in synergistically induced PGI2 synthesis. PMID:26181635

  9. Eliciting Production of L2 Target Structures through Priming Activities

    ERIC Educational Resources Information Center

    McDonough, Kim; Trofimovich, Pavel; Neumann, Heike

    2015-01-01

    This study focuses on the pedagogical applications of structural priming research in an English for academic purposes (EAP) context, investigating whether priming activities are an effective tool for eliciting production of target grammatical structures. University students across four EAP classes carried out a total of 6 information-exchange…

  10. The unfolded protein response selectively targets active smoothened mutants.

    PubMed

    Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

    2013-06-01

    The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

  11. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  12. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    PubMed

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  13. Active helium target: Neutron scalar polarizability extraction via Compton scattering

    SciTech Connect

    Morris, Meg Hornidge, David; Annand, John; Strandberg, Bruno

    2015-12-31

    Precise measurement of the neutron scalar polarizabilities has been a lasting challenge because of the lack of a free-neutron target. Led by the University of Glasgow and the Mount Allison University groups of the A2 collaboration in Mainz, Germany, preparations have begun to test a recent theoretical model with an active helium target with the hope of determining these elusive quantities with small statistical, systematic, and model-dependent errors. Apparatus testing and background-event simulations have been carried out, with the full experiment projected to run in 2015. Once determined, these values can be applied to help understand quantum chromodynamics in the nonperturbative region.

  14. Active helium target: Neutron scalar polarizability extraction via Compton scattering

    NASA Astrophysics Data System (ADS)

    Morris, Meg; Annand, John; Hornidge, David; Strandberg, Bruno

    2015-12-01

    Precise measurement of the neutron scalar polarizabilities has been a lasting challenge because of the lack of a free-neutron target. Led by the University of Glasgow and the Mount Allison University groups of the A2 collaboration in Mainz, Germany, preparations have begun to test a recent theoretical model with an active helium target with the hope of determining these elusive quantities with small statistical, systematic, and model-dependent errors. Apparatus testing and background-event simulations have been carried out, with the full experiment projected to run in 2015. Once determined, these values can be applied to help understand quantum chromodynamics in the nonperturbative region.

  15. Reversible, activity-dependent targeting of profilin to neuronal nuclei

    SciTech Connect

    Birbach, Andreas . E-mail: andreas.birbach@lbicr.lbg.ac.at; Verkuyl, J. Martin; Matus, Andrew . E-mail: aim@fmi.ch

    2006-07-15

    The actin cytoskeleton in pyramidal neurons plays a major role in activity-dependent processes underlying neuronal plasticity. The small actin-binding protein profilin shows NMDA receptor-dependent accumulation in dendritic spines, which is correlated with suppression of actin dynamics and long-term stabilization of synaptic morphology. Here we show that following NMDA receptor activation profilin also accumulates in the nucleus of hippocampal neurons via a process involving rearrangement of the actin cytoskeleton. This simultaneous targeting to dendritic spines and the cell nucleus suggests a novel mechanism of neuronal plasticity in which profilin both tags activated synapses and influences nuclear events.

  16. p53 activated by AND gate genetic circuit under radiation and hypoxia for targeted cancer gene therapy

    PubMed Central

    Ding, Miao; Li, Rong; He, Rong; Wang, Xingyong; Yi, Qijian; Wang, Weidong

    2015-01-01

    Radio-activated gene therapy has been developed as a novel therapeutic strategy against cancer; however, expression of therapeutic gene in peritumoral tissues will result in unacceptable toxicity to normal cells. To restrict gene expression in targeted tumor mass, we used hypoxia and radiation tolerance features of tumor cells to develop a synthetic AND gate genetic circuit through connecting radiation sensitivity promoter cArG6, heat shock response elements SNF1, HSF1 and HSE4 with retroviral vector plxsn. Their construction and dynamic activity process were identified through downstream enhanced green fluorescent protein and wtp53 expression in non-small cell lung cancer A549 cells and in a nude mice model. The result showed that AND gate genetic circuit could be activated by lower required radiation dose (6 Gy) and after activated, AND gate could induce significant apoptosis effects and growth inhibition of cancer cells in vitro and in vivo. The radiation- and hypoxia-activated AND gate genetic circuit, which could lead to more powerful target tumoricidal activity represented a promising strategy for both targeted and effective gene therapy of human lung adenocarcinoma and low dose activation character of the AND gate genetic circuit implied that this model could be further exploited to decrease side-effects of clinical radiation therapy. PMID:26177264

  17. DARHT-II Downstream Beam Transport Beamline

    SciTech Connect

    Westenskow, G A; Bertolini, L R; Duffy, P T; Paul, A C

    2000-08-01

    This paper describes the mechanical design of the downstream beam transport line for the second axis of the Dual Axis Radiographic Hydrodynamic Test (DARHT II) Facility. The DARHT-II project is a collaboration between LANL, LBNL and LLNL. DARHT II is a 20-MeV, 2000-Amperes, 2-{micro}sec linear induction accelerator designed to generate short bursts of x-rays for the purpose of radiographing dense objects. The downstream beam transport line is approximately 20-meter long region extending from the end of the accelerator to the bremsstrahlung target. Within this proposed transport line there are 15 conventional solenoid, quadrupole and dipole magnets; as well as several specialty magnets, which transport and focus the beam to the target and to the beam dumps. There are two high power beam dumps, which are designed to absorb 80-kJ per pulse during accelerator start-up and operation. Aspects of the mechanical design of these elements are presented.

  18. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    PubMed

    Liou, Yu-Ren; Wang, Yu-Hsin; Lee, Chia-Ying; Li, Pai-Chi

    2015-01-01

    Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+)) and MDA-MB-453 cells (CD44-), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+) is a commonly used cancer-stem-cell biomarker, our

  19. Buoyancy-Activated Cell Sorting Using Targeted Biotinylated Albumin Microbubbles

    PubMed Central

    Liou, Yu-Ren; Wang, Yu-Hsin; Lee, Chia-Ying; Li, Pai-Chi

    2015-01-01

    Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including florescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10g for 1 min, and then allowed 1 hour at 4°C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44+) and MDA-MB-453 cells (CD44–), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44+ is a commonly used cancer-stem-cell biomarker, our targeted

  20. Downstream in Mawrth Valles

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site]

    The THEMIS VIS camera is capable of capturing color images of the Martian surface using five different color filters. In this mode of operation, the spatial resolution and coverage of the image must be reduced to accommodate the additional data volume produced from using multiple filters. To make a color image, three of the five filter images (each in grayscale) are selected. Each is contrast enhanced and then converted to a red, green, or blue intensity image. These three images are then combined to produce a full color, single image. Because the THEMIS color filters don't span the full range of colors seen by the human eye, a color THEMIS image does not represent true color. Also, because each single-filter image is contrast enhanced before inclusion in the three-color image, the apparent color variation of the scene is exaggerated. Nevertheless, the color variation that does appear is representative of some change in color, however subtle, in the actual scene. Note that the long edges of THEMIS color images typically contain color artifacts that do not represent surface variation.

    This false color image is from further downstream in Mawrth Valles than yesterday's image. The channel here is at the end of the vallis. This image was collected during the Northern Spring season.

    Image information: VIS instrument. Latitude 26.7, Longitude 340.2 East (19.8 West). 37 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages

  1. Pyrvinium attenuates Hedgehog signaling downstream of smoothened.

    PubMed

    Li, Bin; Fei, Dennis Liang; Flaveny, Colin A; Dahmane, Nadia; Baubet, Valérie; Wang, Zhiqiang; Bai, Feng; Pei, Xin-Hai; Rodriguez-Blanco, Jezabel; Hang, Brian; Orton, Darren; Han, Lu; Wang, Baolin; Capobianco, Anthony J; Lee, Ethan; Robbins, David J

    2014-09-01

    The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds, vismodegib, is now FDA-approved for patients with advanced basal cell carcinoma, acquired mutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independent activation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trials against other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms of action, particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinworm compound pyrvinium as a novel, potent (IC50, 10 nmol/L) casein kinase-1α (CK1α) agonist. We show here that pyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family of transcription factors. Consistent with CK1α agonists acting on these most distal components of the HH signaling pathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib -resistant Smo mutant, as well as the Gli activity resulting from loss of the negative regulator suppressor of fused. We go on to demonstrate the utility of this small molecule in vivo, against the HH-dependent cancer medulloblastoma, attenuating its growth and reducing the expression of HH biomarkers. PMID:24994715

  2. AMPK activation: a therapeutic target for type 2 diabetes?

    PubMed Central

    Coughlan, Kimberly A; Valentine, Rudy J; Ruderman, Neil B; Saha, Asish K

    2014-01-01

    Type 2 diabetes (T2D) is a metabolic disease characterized by insulin resistance, β-cell dysfunction, and elevated hepatic glucose output. Over 350 million people worldwide have T2D, and the International Diabetes Federation projects that this number will increase to nearly 600 million by 2035. There is a great need for more effective treatments for maintaining glucose homeostasis and improving insulin sensitivity. AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase whose activation elicits insulin-sensitizing effects, making it an ideal therapeutic target for T2D. AMPK is an energy-sensing enzyme that is activated when cellular energy levels are low, and it signals to stimulate glucose uptake in skeletal muscles, fatty acid oxidation in adipose (and other) tissues, and reduces hepatic glucose production. There is substantial evidence suggesting that AMPK is dysregulated in animals and humans with metabolic syndrome or T2D, and that AMPK activation (physiological or pharmacological) can improve insulin sensitivity and metabolic health. Numerous pharmacological agents, natural compounds, and hormones are known to activate AMPK, either directly or indirectly – some of which (for example, metformin and thiazolidinediones) are currently used to treat T2D. This paper will review the regulation of the AMPK pathway and its role in T2D, some of the known AMPK activators and their mechanisms of action, and the potential for future improvements in targeting AMPK for the treatment of T2D. PMID:25018645

  3. The Platin-X series: activation, targeting, and delivery.

    PubMed

    Basu, Uttara; Banik, Bhabatosh; Wen, Ru; Pathak, Rakesh K; Dhar, Shanta

    2016-08-16

    Anticancer platinum (Pt) complexes have long been considered to be one of the biggest success stories in the history of medicinal inorganic chemistry. Yet there remains the hunt for the "magic bullet" which can satisfy the requirements of an effective chemotherapeutic drug formulation. Pt(iv) complexes are kinetically more inert than the Pt(ii) congeners and offer the opportunity to append additional functional groups/ligands for prodrug activation, tumor targeting, or drug delivery. The ultimate aim of functionalization is to enhance the tumor selective action and attenuate systemic toxicity of the drugs. Moreover, an increase in cellular accumulation to surmount the resistance of the tumor against the drugs is also of paramount importance in drug development and discovery. In this review, we will address the attempts made in our lab to develop Pt(iv) prodrugs that can be activated and delivered using targeted nanotechnology-based delivery platforms. PMID:27493131

  4. Feasibility study of an active target for the MEG experiment

    NASA Astrophysics Data System (ADS)

    Papa, A.; Cavoto, G.; Ripiccini, E.

    2014-03-01

    We consider the possibility to have an active target for the upgrade of the MEG experiment (MEG II). The active target should work as (1) a beam monitoring, to continuously measure the muon stopping rate and therefore provide a direct evaluation of the detector acceptance (or an absolute normalization of the stopped muon); and as (2) an auxiliary device for the spectrometer, to improve the determination of the muon decay vertex and consequently to achieve a better positron momentum and angular resolutions, detecting the positron from the muon decay. In this work we studied the feasibility of detecting minimum ionizing particle with a single layer of 250 μm fiber and the capability to discriminate between the signal induced by either a muon or a positron.

  5. Expression of nuclear factor of activated T cells (NFAT) and downstream muscle-specific proteins in ground squirrel skeletal and heart muscle during hibernation.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-01-01

    The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) undergoes remarkable adaptive changes during hibernation. Interestingly, skeletal muscle remodelling occurs during the torpor-arousal cycle of hibernation to prevent net muscle loss despite inactivity. Reversible cardiomyocyte hypertrophy occurs in cardiac muscle, allowing the heart to preserve cardiac output during hibernation, while avoiding chronic maladaptive hypertrophy post-hibernation. We propose that calcium signalling proteins [calcineurin (Cn), calmodulin (CaM), and calpain], the nuclear factor of activated T cell (NFAT) family of transcription factors, and the NFAT targets myoferlin and myomaker contribute significantly to adaptations taking place in skeletal and cardiac muscle during hibernation. Protein-level analyses were performed over several conditions: euthermic room temperature (ER), euthermic cold room (EC), entrance into (EN), early (ET), and late torpor (LT) time points, in addition to early (EA), interbout (IA), and late arousal (LA) time points using immunoblotting and DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISAs). In skeletal and cardiac muscle, NFATc2 protein levels were elevated during torpor. NFATc4 increased throughout the torpor-arousal cycle in both tissues, and NFATc1 showed this trend in cardiac muscle only. NFATc3 showed an elevation in DNA-binding activity but not expression during torpor. Myoferlin protein levels dramatically increased during torpor in both skeletal and cardiac muscle. Myomaker levels also increased significantly in cardiac muscle during torpor. Cardiac Cn levels remained stable, whereas CaM and calpain decreased throughout the torpor-arousal cycle. Activation and/or upregulation of NFATc2, c3, myoferlin, and myomaker at torpor could be part of a stress-response mechanism to preserve skeletal muscle mass, whereas CaM and calpain appear to initiate the rapid reversal of cardiac hypertrophy during arousal through

  6. Active multispectral near-IR detection of small surface targets

    NASA Astrophysics Data System (ADS)

    de Jong, Arie N.; Winkel, Hans; Roos, Marco J. J.

    2001-10-01

    The detection and identification of small surface targets with Electro-Optical sensors is seriously hampered by ground clutter, leading to false alarms and reduced detection probabilities. Active ground illumination can improve the detection performance of EO sensors compared to passive skylight illumination because of the knowledge of the illumination level and of its temporal stability. Sun and sky cannot provide this due to the weather variability. In addition multispectral sensors with carefully chosen spectral bands ranging from the visual into the near IR from 400-2500 nm wavelength can take benefit of a variety of cheap active light sources, ranging from lasers to Xenon or halogen lamps. Results are presented, obtained with a two- color laser scanner with one wavelength in the chlorophyll absorption dip. Another active scanner is described operating at 4 wavebands between 1400 and 2300 nm, using tungsten halogen lamps. Finally a simple TV camera was used with either a ste of narrow band spectral filters or polarization filters in front of the lamps. The targets consisted of an array of mixed objects, most of them real mines. The results how great promise in enhancing the detection and identification probabilities of EO sensors against small surface targets.

  7. Target system of IFMIF/EVEDA in Japanese activities

    NASA Astrophysics Data System (ADS)

    Ida, M.; Fukada, S.; Furukawa, T.; Hirakawa, Y.; Horiike, H.; Kanemura, T.; Kondo, H.; Miyashita, M.; Nakamura, H.; Sigiura, H.; Suzuki, A.; Terai, T.; Tsuji, Y.; Ushimaru, H.; Watanabe, K.; Yagi, J.

    2011-10-01

    The Engineering Validation and Engineering Design Activities (EVEDA) of the International Fusion Materials Irradiation Facility (IFMIF) have been started. As Japanese activities for the target system, the EVEDA Lithium (Li) Test Loop to simulate hydraulic and impurity conditions of the IFMIF Li loop is under design. The feasibility of the thermo-mechanical structure of the target assembly and the replaceable back-plate made of F82H and 316L stainless steel is a key research subject. Toward final validation at the EVEDA loop, diagnostics systems applicable to the high-speed free-surface Li flow and hot traps to control nitrogen and hydrogen in Li loop have been investigated. In the remote handling subject of target assemblies and the replaceable back-plates activated by irradiation up to 50 dpa/y, lip welds on 316L-316L by laser and dissimilar metal welds on F82H-316L are necessary. Water experiments and hydraulic/thermo-mechanical analyses of the back-plate are underway.

  8. Brain activation underlying threat detection to targets of different races.

    PubMed

    Senholzi, Keith B; Depue, Brendan E; Correll, Joshua; Banich, Marie T; Ito, Tiffany A

    2015-01-01

    The current study examined blood oxygen level-dependent signal underlying racial differences in threat detection. During functional magnetic resonance imaging, participants determined whether pictures of Black or White individuals held weapons. They were instructed to make shoot responses when the picture showed armed individuals but don't shoot responses to unarmed individuals, with the cost of not shooting armed individuals being greater than that of shooting unarmed individuals. Participants were faster to shoot armed Blacks than Whites, but faster in making don't shoot responses to unarmed Whites than Blacks. Brain activity differed to armed versus unarmed targets depending on target race, suggesting different mechanisms underlying threat versus safety decisions. Anterior cingulate cortex was preferentially engaged for unarmed Whites than Blacks. Parietal and visual cortical regions exhibited greater activity for armed Blacks than Whites. Seed-based functional connectivity of the amygdala revealed greater coherence with parietal and visual cortices for armed Blacks than Whites. Furthermore, greater implicit Black-danger associations were associated with increased amygdala activation to armed Blacks, compared to armed Whites. Our results suggest that different neural mechanisms may underlie racial differences in responses to armed versus unarmed targets. PMID:26357911

  9. Limits of downstream hydraulic geometry

    NASA Astrophysics Data System (ADS)

    Wohl, Ellen

    2004-10-01

    Adjustments to flow width, depth, and velocity in response to changes in discharge are commonly characterized by using downstream hydraulic geometry relationships. The spatial limits of these relationships within a drainage basin have not been systematically quantified. Where the erosional resistance of the channel substrate is sufficiently large, hydraulic driving forces presumably will be unable to adjust channel form. Data sets from 10 mountain rivers in the United States, Panama, Nepal, and New Zealand are used in this study to explore the limits of downstream hydraulic geometry relationships. Where the ratio of stream power to sediment size (Ω/D84) exceeds 10,000 kg/s3, downstream hydraulic geometry is well developed; where the ratio falls below 10,000 kg/s3, downstream hydraulic geometry relationships are poorly developed. These limitations on downstream hydraulic geometry have important implications for channel engineering and simulations of landscape change.

  10. Targeting Gli Transcription Activation by Small Molecule Suppresses Tumor Growth

    PubMed Central

    Bosco-Clément, Geneviève; Zhang, Fang; Chen, Zhao; Zhou, Hai-Meng; Li, Hui; Mikami, Iwao; Hirata, Tomomi; Yagui-Beltran, Adam; Lui, Natalie; Do, Hanh T.; Cheng, Tiffany; Tseng, Hsin-Hui; Choi, Helen; Fang, Li-Tai; Kim, Il-Jin; Yue, Dongsheng; Wang, Changli; Zheng, Qingfeng; Fujii, Naoaki; Mann, Michael; Jablons, David M.; He, Biao

    2014-01-01

    Targeted inhibition of Hedgehog signaling at the cell membrane has been associated with anti-cancer activity in preclinical and early clinical studies. Hedgehog signaling involves activation of Gli transcription factors that can also be induced by alternative pathways. In this study we identified an interaction between Gli proteins and a transcription co-activator TAF9, and validated its functional relevance in regulating Gli transactivation. We also describe a novel, synthetic small molecule, FN1-8, that efficiently interferes with Gli/TAF9 interaction and down-regulate Gli/TAF9 dependent transcriptional activity. More importantly, FN1-8 suppresses cancer cell proliferation in vitro and inhibits tumor growth in vivo. Our results suggest that blocking Gli transactivation, a key control point of multiple oncogenic pathways, may be an effective anti-cancer strategy. PMID:23686308

  11. Active targets for the study of nuclei far from stability

    NASA Astrophysics Data System (ADS)

    Beceiro-Novo, S.; Ahn, T.; Bazin, D.; Mittig, W.

    2015-09-01

    Weakly bound nuclear systems can be considered to represent a good testing-ground of our understanding of non-perturbative quantum systems. Reactions leading to bound and unbound states in systems with very unbalanced neutron-to-proton ratios are used to understand the properties of these systems. Radioactive beams with energies from below the Coulomb barrier up to several hundreds MeV/nucleon are now available, and with these beams, a broad variety of studies of nuclei near the drip-line can be performed. To compensate for the low intensity of secondary beams as compared to primary beams, thick targets and high efficiency detection is necessary. In this context, a new generation of detectors was developed, called active target detectors: the detector gas is used as target, and the determination of the reaction vertex in three dimensions allows for good resolution even with thick targets. The reaction products can be measured over essentially 4 π. The physics explored with these detectors together with the technology developed will be described.

  12. Hyperpolarization-activated ion channels as targets for nitric oxide signalling in deep cerebellar nuclei

    PubMed Central

    Wilson, Gary W; Garthwaite, John

    2010-01-01

    Most biological effects of nitric oxide (NO) in the brain are mediated by guanylyl cyclase-coupled NO receptors, whose activation results in increased intracellular cGMP levels. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels, which bind cGMP or cAMP directly, were recently suggested to be a target. The aim here was to test this possibility more directly. Neurones of the rat deep cerebellar nuclei were selected for this purpose, their suitability being attested by immunocytochemistry showing that the principal neurones expressed guanylyl cyclase protein and that NO synthase-containing fibres were abundant in the neuropil. Using whole-cell voltage-clamp recording, HCN channels in the neurones were activated in response to isoprenaline and exogenous cAMP but only occasionally did they respond to NO, although exogenous cGMP was routinely effective. With the less invasive sharp microelectrode recording technique, however, exogenous NO modulated the channels reproducibly, as measured by the size of the HCN channel-mediated voltage sag following hyperpolarization. Moreover, NO also blunted the subsequent rebound depolarizing potentials, consistent with it increasing the hyperpolarization-activated current. Optimizing the whole-cell solution to improve the functioning of NO-activated guanylyl cyclase failed to restore NO sensitivity. Minimizing cellular dialysis by using the perforated-patch technique, however, was successful. The results provide evidence that HCN channels are potential downstream mediators of NO signalling in deep cerebellar nuclei neurones and suggest that the more general importance of this transduction pathway may have been overlooked previously because of unsuitable recording methods. PMID:20529121

  13. A numerically optimized active shield for improved TMS targeting

    PubMed Central

    Hernandez-Garcia, Luis; Hall, Timothy; Gomez, Luis; Michielssen, Eric

    2010-01-01

    Transcranial magnetic stimulation (TMS) devices suffer of poor targeting and penetration depth. A new approach to designing TMS coils is introduced in order to improve the focus of the stimulation region through the use of actively shielded probes. Iterative optimization techniques were used to design different active shielding coils for TMS probes. The new approach aims to increase the amount of energy deposited in a thin cylindrical region below the probe relative to the energy deposited elsewhere in the region (“sharpness”), while simultaneously increase the induced electric field deep in the target region relative to the surface (“penetration”). After convergence, the resulting designs showed that there is a clear tradeoff between sharpness and penetration that can be controlled by the choice of a tuning parameter. The resulting designs were tested on a realistic human head conductivity model, taking the contribution from surface charges into account. The design of choice reduced penetration depths by 16.7%. The activated surface area was reduced by 24.1 % and the volume of the activation was reduced from 42.6% by the shield. Restoring the lost penetration could be achieved by increasing the total power to the coil by 16.3%, but in that case, the stimulated volume reduction was only 13.1% and there was a slight increase in the stimulated surface area (2.9 %) PMID:20965451

  14. Factor XI and Contact Activation as Targets for Antithrombotic Therapy

    PubMed Central

    Gailani, David; Bane, Charles E.; Gruber, Andras

    2015-01-01

    Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012

  15. HER2 activating mutations are targets for colorectal cancer treatment

    PubMed Central

    Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

    2015-01-01

    The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

  16. miR-155 targets Caspase-3 mRNA in activated macrophages.

    PubMed

    De Santis, Rebecca; Liepelt, Anke; Mossanen, Jana C; Dueck, Anne; Simons, Nadine; Mohs, Antje; Trautwein, Christian; Meister, Gunter; Marx, Gernot; Ostareck-Lederer, Antje; Ostareck, Dirk H

    2016-01-01

    To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation. PMID:26574931

  17. Development of AN Active 238UF6 Gas Target

    NASA Astrophysics Data System (ADS)

    Eckardt, C.; Enders, J.; Freudenberger, M.; Göök, A.; von Neumann-Cosel, P.; Oberstedt, A.; Oberstedt, S.

    2014-09-01

    Detailed studies of the fission process, e.g., the search for parity nonconservation (PNC) effects, the energy dependence of fission modes or the population of fission isomers, depend on high quality data, therefore requiring high luminosities. An active gas target containing uranium may overcome the deterioration of energy and angular resolution caused by large solid target thicknesses. A single Frisch-grid ionization chamber has been built to test a mixture of standard counting gases (e.g., argon) with depleted uranium hexafluoride (238UF6), utilizing a triple alpha source to evaluate signal quality and drift velocity. For mass fractions of up to 4 percent of 238U the drift velocity increases with rising UF6 content, while a good signal quality and energy resolution is preserved.

  18. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum.

    PubMed

    Wang, Jigang; Zhang, Chong-Jing; Chia, Wan Ni; Loh, Cheryl C Y; Li, Zhengjun; Lee, Yew Mun; He, Yingke; Yuan, Li-Xia; Lim, Teck Kwang; Liu, Min; Liew, Chin Xia; Lee, Yan Quan; Zhang, Jianbin; Lu, Nianci; Lim, Chwee Teck; Hua, Zi-Chun; Liu, Bin; Shen, Han-Ming; Tan, Kevin S W; Lin, Qingsong

    2015-01-01

    The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing. PMID:26694030

  19. Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum

    PubMed Central

    Wang, Jigang; Zhang, Chong-Jing; Chia, Wan Ni; Loh, Cheryl C. Y.; Li, Zhengjun; Lee, Yew Mun; He, Yingke; Yuan, Li-Xia; Lim, Teck Kwang; Liu, Min; Liew, Chin Xia; Lee, Yan Quan; Zhang, Jianbin; Lu, Nianci; Lim, Chwee Teck; Hua, Zi-Chun; Liu, Bin; Shen, Han-Ming; Tan, Kevin S. W.; Lin, Qingsong

    2015-01-01

    The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasite's haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing. PMID:26694030

  20. Modeling the target acquisition performance of active imaging systems.

    PubMed

    Espinola, Richard L; Jacobs, Eddie L; Halford, Carl E; Vollmerhausen, Richard; Tofsted, David H

    2007-04-01

    Recent development of active imaging system technology in the defense and security community have driven the need for a theoretical understanding of its operation and performance in military applications such as target acquisition. In this paper, the modeling of active imaging systems, developed at the U.S. Army RDECOM CERDEC Night Vision & Electronic Sensors Directorate, is presented with particular emphasis on the impact of coherent effects such as speckle and atmospheric scintillation. Experimental results from human perception tests are in good agreement with the model results, validating the modeling of coherent effects as additional noise sources. Example trade studies on the design of a conceptual active imaging system to mitigate deleterious coherent effects are shown. PMID:19532626

  1. Modeling the target acquisition performance of active imaging systems

    NASA Astrophysics Data System (ADS)

    Espinola, Richard L.; Jacobs, Eddie L.; Halford, Carl E.; Vollmerhausen, Richard; Tofsted, David H.

    2007-04-01

    Recent development of active imaging system technology in the defense and security community have driven the need for a theoretical understanding of its operation and performance in military applications such as target acquisition. In this paper, the modeling of active imaging systems, developed at the U.S. Army RDECOM CERDEC Night Vision & Electronic Sensors Directorate, is presented with particular emphasis on the impact of coherent effects such as speckle and atmospheric scintillation. Experimental results from human perception tests are in good agreement with the model results, validating the modeling of coherent effects as additional noise sources. Example trade studies on the design of a conceptual active imaging system to mitigate deleterious coherent effects are shown.

  2. Calcium-Activated Potassium Channels: Potential Target for Cardiovascular Diseases.

    PubMed

    Dong, De-Li; Bai, Yun-Long; Cai, Ben-Zhi

    2016-01-01

    Ca(2+)-activated K(+) channels (KCa) are classified into three subtypes: big conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) KCa channels. The three types of KCa channels have distinct physiological or pathological functions in cardiovascular system. BKCa channels are mainly expressed in vascular smooth muscle cells (VSMCs) and inner mitochondrial membrane of cardiomyocytes, activation of BKCa channels in these locations results in vasodilation and cardioprotection against cardiac ischemia. IKCa channels are expressed in VSMCs, endothelial cells, and cardiac fibroblasts and involved in vascular smooth muscle proliferation, migration, vessel dilation, and cardiac fibrosis. SKCa channels are widely expressed in nervous and cardiovascular system, and activation of SKCa channels mainly contributes membrane hyperpolarization. In this chapter, we summarize the physiological and pathological roles of the three types of KCa channels in cardiovascular system and put forward the possibility of KCa channels as potential target for cardiovascular diseases. PMID:27038376

  3. The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA.

    PubMed Central

    Baggio, L; Morrison, M

    1996-01-01

    Previous studies have suggested that regulation of the enzymes of ammonia assimilation in human colonic Bacteroides species is coordinated differently than in other eubacteria. The gene encoding an NAD(P)H-dependent glutamate dehydrogenase (gdhA) in Bacteroides thetaiotaomicron was cloned and expressed in Escherichia coli by mutant complementation from the recombinant plasmid pANS100. Examination of the predicted GdhA amino acid sequence revealed that this enzyme possesses motifs typical of the family I-type hexameric GDH proteins. Northern blot analysis with a gdhA-specific probe indicated that a single transcript with an electrophoretic mobility of approximately 1.6 kb was produced in both B. thetaiotaomicron and E. coli gdhA+ transformants. Although gdhA transcription was unaffected, no GdhA enzyme activity could be detected in E. coli transformants when smaller DNA fragments from pANS100, which contained the entire gdhA gene, were analyzed. Enzyme activity was restored if these E. coli strains were cotransformed with a second plasmid, which contained a 3-kb segment of DNA located downstream of the gdhA coding region. Frameshift mutagenesis within the DNA downstream of gdhA in pANS100 also resulted in the loss of GdhA enzyme activity. Collectively, these results are interpreted as evidence for the role of an additional gene product(s) in modulating the activity of GDH enzyme activity. Insertional mutagenesis experiments which led to disruption of the gdhA gene on the B. thetaiotaomicron chromosome indicated that gdhA mutants were not glutamate auxotrophs, but attempts to isolate similar mutants with insertion mutations in the region downstream of the gdhA gene were unsuccessful. PMID:8955404

  4. A Deterministic Approach to Active Debris Removal Target Selection

    NASA Astrophysics Data System (ADS)

    Lidtke, A.; Lewis, H.; Armellin, R.

    2014-09-01

    Many decisions, with widespread economic, political and legal consequences, are being considered based on space debris simulations that show that Active Debris Removal (ADR) may be necessary as the concerns about the sustainability of spaceflight are increasing. The debris environment predictions are based on low-accuracy ephemerides and propagators. This raises doubts about the accuracy of those prognoses themselves but also the potential ADR target-lists that are produced. Target selection is considered highly important as removal of many objects will increase the overall mission cost. Selecting the most-likely candidates as soon as possible would be desirable as it would enable accurate mission design and allow thorough evaluation of in-orbit validations, which are likely to occur in the near-future, before any large investments are made and implementations realized. One of the primary factors that should be used in ADR target selection is the accumulated collision probability of every object. A conjunction detection algorithm, based on the smart sieve method, has been developed. Another algorithm is then applied to the found conjunctions to compute the maximum and true probabilities of collisions taking place. The entire framework has been verified against the Conjunction Analysis Tools in AGIs Systems Toolkit and relative probability error smaller than 1.5% has been achieved in the final maximum collision probability. Two target-lists are produced based on the ranking of the objects according to the probability they will take part in any collision over the simulated time window. These probabilities are computed using the maximum probability approach, that is time-invariant, and estimates of the true collision probability that were computed with covariance information. The top-priority targets are compared, and the impacts of the data accuracy and its decay are highlighted. General conclusions regarding the importance of Space Surveillance and Tracking for the

  5. Improved drug targeting of cancer cells by utilizing actively targetable folic acid-conjugated albumin nanospheres.

    PubMed

    Shen, Zheyu; Li, Yan; Kohama, Kazuhiro; Oneill, Brian; Bi, Jingxiu

    2011-01-01

    Folic acid-conjugated albumin nanospheres (FA-AN) have been developed to provide an actively targetable drug delivery system for improved drug targeting of cancer cells with reduced side effects. The nanospheres were prepared by conjugating folic acid onto the surface of albumin nanospheres using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as a catalyst. To test the efficacy of these nanospheres as a potential delivery platform, doxorubicin-loaded albumin nanospheres (DOX-AN) and doxorubicin-loaded FA-AN (FA-DOX-AN) were prepared by entrapping DOX (an anthracycline, antibiotic drug widely used in cancer chemotherapy that works by intercalating DNA) into AN and FA-AN nanoparticles. Cell uptake of the DOX was then measured. The results show that FA-AN was incorporated into HeLa cells (tumor cells) only after 2.0h incubation, whereas HeLa cells failed to incorporate albumin nanospheres without conjugated folic acid after 4.0h incubation. When HeLa cells were treated with the DOX-AN, FA-DOX-AN nanoparticles or free DOX, cell viability decreased with increasing culture time (i.e. cell death increases with time) over a 70h period. Cell viability was always the lowest for free DOX followed by FA-DOX-AN4 and then DOX-AN. In a second set of experiments, HeLa cells washed to remove excess DOX after an initial incubation for 2h were incubated for 70h. The corresponding cell viability was slightly higher when the cells were treated with FA-DOX-AN or free DOX whilst cells treated with DOX-AN nanoparticles remained viable. The above experiments were repeated for non-cancerous, aortic smooth muscle cells (AoSMC). As expected, cell viability of the HeLa cells (with FA receptor alpha, FRα) and AoSMC cells (without FRα) decreased rapidly with time in the presence of free DOX, but treatment with FA-DOX-AN resulted in selective killing of the tumor cells. These results indicated that FA-AN may be used as a promising actively targetable drug delivery system to improve drug

  6. Active imaging system performance model for target acquisition

    NASA Astrophysics Data System (ADS)

    Espinola, Richard L.; Teaney, Brian; Nguyen, Quang; Jacobs, Eddie L.; Halford, Carl E.; Tofsted, David H.

    2007-04-01

    The U.S. Army RDECOM CERDEC Night Vision & Electronic Sensors Directorate has developed a laser-range-gated imaging system performance model for the detection, recognition, and identification of vehicle targets. The model is based on the established US Army RDECOM CERDEC NVESD sensor performance models of the human system response through an imaging system. The Java-based model, called NVLRG, accounts for the effect of active illumination, atmospheric attenuation, and turbulence effects relevant to LRG imagers, such as speckle and scintillation, and for the critical sensor and display components. This model can be used to assess the performance of recently proposed active SWIR systems through various trade studies. This paper will describe the NVLRG model in detail, discuss the validation of recent model components, present initial trade study results, and outline plans to validate and calibrate the end-to-end model with field data through human perception testing.

  7. Novel strategies for ultrahigh specific activity targeted nanoparticles

    SciTech Connect

    Zhou, Dong

    2012-12-13

    We have developed novel strategies optimized for preparing high specific activity radiolabeled nanoparticles, targeting nuclear imaging of low abundance biomarkers. Several compounds have been labeled with F-18 and Cu-64 for radiolabeling of SCK-nanoparticles via Copper(I) catalyzed or copper-free alkyne-azide cyclolization. Novel strategies have been developed to achieve ultrahigh specific activity with administrable amount of dose for human study using copper-free chemistry. Ligands for carbonic anhydrase 12 (CA12), a low abundance extracellular biomarker for the responsiveness of breast cancer to endocrine therapie, have been labeled with F-18 and Cu-64, and one of them has been evaluated in animal models. The results of this project will lead to major improvements in the use of nanoparticles in nuclear imaging and will significantly advance their potential for detecting low abundance biomarkers of medical importance.

  8. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  9. Targeting energy metabolic and oncogenic signaling pathways in triple-negative breast cancer by a novel adenosine monophosphate-activated protein kinase (AMPK) activator.

    PubMed

    Lee, Kuen-Haur; Hsu, En-Chi; Guh, Jih-Hwa; Yang, Hsiao-Ching; Wang, Dasheng; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2011-11-11

    The antitumor activities of the novel adenosine monophosphate-activated protein kinase (AMPK) activator, OSU-53, were assessed in in vitro and in vivo models of triple-negative breast cancer. OSU-53 directly stimulated recombinant AMPK kinase activity (EC(50), 0.3 μM) and inhibited the viability and clonogenic growth of MDA-MB-231 and MDA-MB-468 cells with equal potency (IC(50), 5 and 2 μM, respectively) despite lack of LKB1 expression in MDA-MB-231 cells. Nonmalignant MCF-10A cells, however, were unaffected. Beyond AMPK-mediated effects on mammalian target of rapamycin signaling and lipogenesis, OSU-53 also targeted multiple AMPK downstream pathways. Among these, the protein phosphatase 2A-dependent dephosphorylation of Akt is noteworthy because it circumvents the feedback activation of Akt that results from mammalian target of rapamycin inhibition. OSU-53 also modulated energy homeostasis by suppressing fatty acid biosynthesis and shifting the metabolism to oxidation by up-regulating the expression of key regulators of mitochondrial biogenesis, such as a peroxisome proliferator-activated receptor γ coactivator 1α and the transcription factor nuclear respiratory factor 1. Moreover, OSU-53 suppressed LPS-induced IL-6 production, thereby blocking subsequent Stat3 activation, and inhibited hypoxia-induced epithelial-mesenchymal transition in association with the silencing of hypoxia-inducible factor 1a and the E-cadherin repressor Snail. In MDA-MB-231 tumor-bearing mice, daily oral administration of OSU-53 (50 and 100 mg/kg) suppressed tumor growth by 47-49% and modulated relevant intratumoral biomarkers of drug activity. However, OSU-53 also induced protective autophagy that attenuated its antiproliferative potency. Accordingly, cotreatment with the autophagy inhibitor chloroquine increased the in vivo tumor-suppressive activity of OSU-53. OSU-53 is a potent, orally bioavailable AMPK activator that acts through a broad spectrum of antitumor activities. PMID

  10. Targeted training modifies oscillatory brain activity in schizophrenia patients

    PubMed Central

    Popov, Tzvetan G.; Carolus, Almut; Schubring, David; Popova, Petia; Miller, Gregory A.; Rockstroh, Brigitte S.

    2015-01-01

    Effects of both domain-specific and broader cognitive remediation protocols have been reported for neural activity and overt performance in schizophrenia (SZ). Progress is limited by insufficient knowledge of relevant neural mechanisms. Addressing neuronal signal resolution in the auditory system as a mechanism contributing to cognitive function and dysfunction in schizophrenia, the present study compared effects of two neuroplasticity-based training protocols targeting auditory–verbal or facial affect discrimination accuracy and a standard rehabilitation protocol on magnetoencephalographic (MEG) oscillatory brain activity in an auditory paired-click task. SZ were randomly assigned to either 20 daily 1-hour sessions over 4 weeks of auditory–verbal training (N = 19), similarly intense facial affect discrimination training (N = 19), or 4 weeks of treatment as usual (TAU, N = 19). Pre-training, the 57 SZ showed smaller click-induced posterior alpha power modulation than did 28 healthy comparison participants, replicating Popov et al. (2011b). Abnormally small alpha decrease 300–800 ms around S2 improved more after targeted auditory–verbal training than after facial affect training or TAU. The improvement in oscillatory brain dynamics with training correlated with improvement on a measure of verbal learning. Results replicate previously reported effects of neuroplasticity-based psychological training on oscillatory correlates of auditory stimulus differentiation, encoding, and updating and indicate specificity of cortical training effects. PMID:26082889

  11. Inhibition of mammalian target of rapamycin activation in the rostral anterior cingulate cortex attenuates pain-related aversion in rats.

    PubMed

    Lu, Bo; Jiang, Jingyan; Sun, Jianliang; Xiao, Chun; Meng, Bo; Zheng, Jinwei; Li, Xiaoyu; Wang, Ruichun; Wu, Guorong; Chen, Junping

    2016-09-01

    Pain is a complex experience that comprises both sensory and affective dimensions. Mammalian target of rapamycin (mTOR) plays an important role in the modulation of neuronal plasticity associated with the pathogenesis of pain sensation. However, the role of mTOR in pain affect is unclear. Using a formalin-induced conditioned place avoidance (F-CPA) test, the current study investigated the effects of the mTOR specific inhibitor rapamycin on noxious stimulation induced aversion in the rostral anterior cingulate cortex (rACC). Intraplantar injection of 5% formalin was associated with significant activation of mTOR, as well as p70 ribosomal S6 protein (p70S6K), its downstream effector, in the rACC. The inhibition of mTOR activation with rapamycin disrupted pain-related aversion; however, this inhibition did not affect formalin-induced spontaneous nociceptive behaviors in rats. These findings demonstrated for the first time that mTOR and its downstream pathway in the rACC contribute to the induction of pain-related negative emotion. PMID:27163752

  12. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  13. Canine adenovirus downstream processing protocol.

    PubMed

    Puig, Meritxell; Piedra, Jose; Miravet, Susana; Segura, María Mercedes

    2014-01-01

    Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration-diafiltration. A Benzonase(®) digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38-45 %. PMID:24132487

  14. Development of an Active 238UF6 Gas Target

    NASA Astrophysics Data System (ADS)

    Eckardt, C.; Enders, J.; Freudenberger, M.; Gook, A.; von Neumann-Cosel, P.; Oberstedt, A.; Oberstedt, S.

    At the superconducting 130 MeV Darmstadt electron linac S-DALINAC a new source of spin-polarized electrons using a GaAs cathode has been installed, opening the path for experiments with polarized electron and photon beams for nuclear structure studies at low momentum transfers, e.g., the search for forward-backward asymmetries originating from parity non-conservation (PNC) in the photon-induced fission process of 238U.Detailed studies of different properties, e.g., the energy dependence of fission modes, the population of fission isomers, or the search for (PNC) effects in the photon-induced fission process of 238U, depends on high quality data, therefore needing high luminosities. An active gas target containing uranium may overcome the problem that large solid target thicknesses cause poor energy and angular resolution.A single Frisch-grid ionization chamber has been built to test a mixture of standard counting gases (e.g., argon) with depleted uraniumhexafluoride (238UF6) using a triple alpha source, evaluating signal quality and drift velocity. For mass fractions up to 2 percent of 238U in the counting gas. The drift velocity increases with rising UF6 content, while a good signal quality and energy resolution is preserved.

  15. Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition

    PubMed Central

    Kwon, Hyung-Joon; Choi, Go-Eun; Ryu, Sangryeol; Kwon, Soon Jae; Kim, Sun Chang; Booth, Claire; Nichols, Kim E.; Kim, Hun Sik

    2016-01-01

    NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K-Akt signal, primarily mediated by NKG2D or DNAM-1, for optimal p65 phosphorylation and NF-κB activation. Vav1 controls downstream p65 phosphorylation and NF-κB activation. Synergistic signalling is defective in X-linked lymphoproliferative disease (XLP1) NK cells entailing 2B4 dysfunction and required for p65 phosphorylation by PI3K-Akt signal, suggesting stepwise signalling checkpoint for NF-κB activation. Thus, our study provides a framework explaining how signals from different activating receptors are coordinated to determine specificity and magnitude of NF-κB activation and NK cell responses. PMID:27221592

  16. Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition.

    PubMed

    Kwon, Hyung-Joon; Choi, Go-Eun; Ryu, Sangryeol; Kwon, Soon Jae; Kim, Sun Chang; Booth, Claire; Nichols, Kim E; Kim, Hun Sik

    2016-01-01

    NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K-Akt signal, primarily mediated by NKG2D or DNAM-1, for optimal p65 phosphorylation and NF-κB activation. Vav1 controls downstream p65 phosphorylation and NF-κB activation. Synergistic signalling is defective in X-linked lymphoproliferative disease (XLP1) NK cells entailing 2B4 dysfunction and required for p65 phosphorylation by PI3K-Akt signal, suggesting stepwise signalling checkpoint for NF-κB activation. Thus, our study provides a framework explaining how signals from different activating receptors are coordinated to determine specificity and magnitude of NF-κB activation and NK cell responses. PMID:27221592

  17. Azo-reductase activated budesodine prodrugs for colon targeting.

    PubMed

    Marquez Ruiz, Juan F; Kedziora, Kinga; O'Reilly, Mary; Maguire, Jacqueline; Keogh, Brian; Windle, Henry; Kelleher, Dermot P; Gilmer, John F

    2012-12-15

    Budesodine is a synthetic glurocorticoid that undergoes substantial first pass metabolism, limiting systemic exposure. Its use in treatment of inflammatory bowel disease would benefit from a targeting strategy that could lead to a local topical effect, improving safety and increasing anti-inflammatory efficacy. A two-step prodrug strategy involving azoreduction/cyclization that we developed previously for prednisolone is here applied with some variations to budesonide. The budesodine prodrugs were tested using an in vitro azoreductase assay simulating human colonic microflora. The kinetics of amino steroid ester cyclization and its pH dependence was also evaluated. The stability of the prodrugs systems in simulated human duodenal and gastric fluid was evaluated to determine the likelihood of intact intestinal transit. The propionic acid derived prodrug 3 undergoes rapid activation by Clostridium perfingens and its putative reduction product cyclizes with acceptable rapidity when synthesized independently. These properties of 3 suggest that it has potential in management of ulcerative colitis. PMID:23122819

  18. Activating frataxin expression by repeat-targeted nucleic acids

    PubMed Central

    Li, Liande; Matsui, Masayuki; Corey, David R.

    2016-01-01

    Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression. PMID:26842135

  19. A bispecific HER2-targeting FynomAb with superior antitumor activity and novel mode of action.

    PubMed

    Brack, Simon; Attinger-Toller, Isabella; Schade, Babette; Mourlane, Frédéric; Klupsch, Kristina; Woods, Richard; Hachemi, Helen; von der Bey, Ulrike; Koenig-Friedrich, Susann; Bertschinger, Julian; Grabulovski, Dragan

    2014-08-01

    Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. PMID:24994770

  20. Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

    SciTech Connect

    Yang, Ji Hye; Shin, Bo Yeon; Han, Jae Yun; Kim, Mi Gwang; Wi, Ji Eun; Kim, Young Woo; Cho, Il Je; Kim, Sang Chan; Shin, Sang Mi; Ki, Sung Hwan

    2014-01-15

    Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes. - Highlights: • We investigated the effect of isorhamnetin on Nrf2 activation. • Isorhamnetin increased Nrf2

  1. MiR-200 modulates coelomocytes antibacterial activities and LPS priming via targeting Tollip in Apostichopus japonicus.

    PubMed

    Lv, Zhimeng; Li, Chenghua; Zhang, Pengjuan; Wang, Zhenhui; Zhang, Weiwei; Jin, Chun-Hua

    2015-08-01

    In order to explore the potential roles of microRNAs (miRNAs) in regulating Toll-like receptor (TLR) pathways, we identified Toll interacting protein as a putative target of miR-200 in Apostichopus japonicus coelomocytes by RNA-seq screening (denoted as AjTollip). The positive expression profiles of miR-200 and AjTollip were detected in both LPS exposure primary coelomocytes and Vibrio splendidus challenge sea cucumber. Co-infection miR-200 mimics significantly elevated the expression of AjTollip and its down-stream molecules. In contrast, miR-200 inhibitor significantly repressed the expression of these TLR-pathway members. More importantly, miR-200 displayed not only to enhance coelomocytes antibacterial activities, but to suppress LPS priming in vitro. Overall, all these results will enhance our understanding on miR-200 regulatory roles in anti-bacterial process in sea cucumber. PMID:25910848

  2. OxLDL-Dependent Activation of Arginase II Is Dependent on the LOX-1 Receptor and Downstream RhoA Signaling

    PubMed Central

    Ryoo, Sungwoo; Bhunia, Anil; Chang, Fumin; Shoukas, Artin; Berkowitz, Dan E; Romer, Lewis H

    2010-01-01

    Aims Arginase II regulates NOS activity by competing for the substrate L-arginine. Oxidized LDL (OxLDL) is a proatherogenic molecule that activates arginase II. We tested the hypotheses that OxLDL-dependent arginase II activation occurs through a specific receptor, and via a Rho GTPase effector mechanism that is inhibited by statins. Methods and Results Arginase II activation by OxLDL was attenuated following preincubation with the LOX-1 receptor-blocking antibody JTX92. This also prevented the dissociation of arginase II from microtubules. LOX-1−/− mice failed to exhibit the increased arginase II activity seen in WT mice fed a high cholesterol diet. Furthermore, endothelium from LOX−/− mice failed to demonstrate the diet-dependent reduction in NO and increase in ROS that were observed in WT mice. OxLDL induced Rho translocation to the membrane and Rho activation, and these effects were inhibited by pretreatment with JTX92 or statins. Transfection with siRNA for RhoA, or inhibition of ROCK both decreased OxLDL-stimulated arginase II activation. Preincubation with simvastatin or lovastatin blocked OxLDL-induced dissociation of arginase II from microtubules and prevented microtubule depolymerization. Conclusions This study provides a new focus for preventive therapy for atherosclerotic disease by delineating a clearer path from OxLDL through the endothelial cell LOX-1 receptor, RhoA, and ROCK, to the activation of arginase II, downregulation of NO, and vascular dysfunction. PMID:21130456

  3. Early Growth Response 4 Is Involved in Cell Proliferation of Small Cell Lung Cancer through Transcriptional Activation of Its Downstream Genes

    PubMed Central

    Yoshimaru, Tetsuro; Daizumoto, Kei; Sone, Saburo; Nishioka, Yasuhiko; Katagiri, Toyomasa

    2014-01-01

    Small cell lung cancer (SCLC) is aggressive, with rapid growth and frequent bone metastasis; however, its detailed molecular mechanism remains poorly understood. Here, we report the critical role of early growth factor 4 (EGR4), a DNA-binding, zinc-finger transcription factor, in cell proliferation of SCLC. EGR4 overexpression in HEK293T cells conferred significant upregulation of specific splice variants of the parathyroid hormone-related protein (PTHrP) gene, resulting in enhancement of the secretion of PTHrP protein, a known mediator of osteolytic bone metastasis. More importantly, depletion of EGR4 expression by siRNA significantly suppressed growth of the SCLC cell lines, SBC-5, SBC-3 and NCI-H1048. On the other hand, introduction of EGR4 into NIH3T3 cells significantly enhanced cell growth. We identified four EGR4 target genes, SAMD5, RAB15, SYNPO and DLX5, which were the most significantly downregulated genes upon depletion of EGR4 expression in all of the SCLC cells examined, and demonstrated the direct recruitment of EGR4 to their promoters by ChIP and luciferase reporter analysis. Notably, knockdown of the expression of these genes by siRNA remarkably suppressed the growth of all the SCLC cells. Taken together, our findings suggest that EGR4 likely regulates the bone metastasis and proliferation of SCLC cells via transcriptional regulation of several target genes, and may therefore be a promising target for the development of anticancer drugs for SCLC patients. PMID:25411851

  4. DARHT-II Downstream Transport Beamline

    SciTech Connect

    Westenskow, G A; Bertolini, L R; Duffy, P T; Paul, A C

    2001-06-06

    This paper describes the mechanical design of the downstream beam transport line for the second axis of the Dual Axis Radiographic Hydrodynamic Test (DARHT II) Facility. The DARHT-II project is a collaboration between LANL, LBNL and LLNL. DARHT II is a 18.4-MeV, 2000-Amperes, 2-{micro}sec linear induction accelerator designed to generate short bursts of x-rays for the purpose of radiographing dense objects. The downstream beam transport line is approximately 22-meter long region extending from the end of the accelerator to the bremsstrahlung target. Within this proposed transport line there are 12 conventional solenoid, quadrupole and dipole magnets; as well as several specialty magnets, which transport and focus the beam to the target and to the beam dumps. There are two high power beam dumps, which are designed to absorb 80-kJ per pulse during accelerator start-up and operation. Aspects of the mechanical design of these elements are presented.

  5. Myricetin suppresses p21-activated kinase 1 in human breast cancer MCF-7 cells through downstream signaling of the β-catenin pathway.

    PubMed

    Jiao, De; Zhang, Xue Dong

    2016-07-01

    As a main active compound in the bark of waxberry (Myrica rubra), myricetin is a macrocyclic diarylheptanoid, and can trigger the apoptosis of HeLa and PC3 cells. The aim of the present study was to elucidate the anticancer effect of myricetin on human breast cancer MCF-7 cells and to explore the possible mechanisms of action. MCF-7 cells were treated with different concentrations of myricetin (0-80 µM) for 12, 24 and 48 h. In the present study, we found that myricetin suppressed the cell viability of the MCF-7 cells at least partly through the induction of apoptosis as determined by MTT assay and flow cytometry. Western blot analysis revealed that myricetin effectively suppressed the protein expression of p21-activated kinase 1 (PAK1), MEK and phosphorylated extracellular mitogen-activated protein kinase (ERK1/2). In addition, treatment of myricetin activated glycogen synthase kinase-3β (GSK3β) and Bax protein expression, and inhibited β-catenin/cyclin D1/proliferating cell nuclear antigen (PCNA)/survivin and promoted caspase-3 activity in the MCF-7 cells. These results demonstrated that myricetin suppressed the cell viability of human breast cancer MCF-7 cells through PAK1/MEK/ERK/GSK3β/β-catenin/cyclin D1/PCNA/survivin/Bax-caspase-3 signaling. PMID:27122002

  6. Proatherogenic macrophage activities are targeted by the flavonoid quercetin.

    PubMed

    Lara-Guzman, Oscar J; Tabares-Guevara, Jorge H; Leon-Varela, Yudy M; Álvarez, Rafael M; Roldan, Miguel; Sierra, Jelver A; Londoño-Londoño, Julian A; Ramirez-Pineda, Jose R

    2012-11-01

    Many studies have demonstrated that the flavonoid quercetin protects against cardiovascular disease (CVD) and related risk factors. Atherosclerosis, the underlying cause of CVD, is also attenuated by oral quercetin administration in animal models. Although macrophages are key players during fatty streak formation and plaque progression and aggravation, little is known about the effects of quercetin on atherogenic macrophages. Here, we report that primary bone marrow-derived macrophages internalized less oxidized low-density lipoprotein (oxLDL) and accumulated less intracellular cholesterol in the presence of quercetin. This reduction of foam cell formation correlated with reduced surface expression of the oxLDL receptor CD36. Quercetin also targeted the lipopolysaccharide-dependent, oxLDL-independent pathway of lipid droplet formation in macrophages. In oxLDL-stimulated macrophages, quercetin inhibited reactive oxygen species production and interleukin (IL)-6 secretion. In a system that evaluated cholesterol crystal-induced IL-1β secretion via nucleotide-binding domain and leucine-rich repeat containing protein 3 inflammasome activation, quercetin also exhibited an inhibitory effect. Dyslipidemic apolipoprotein E-deficient mice chronically treated with intraperitoneal quercetin injections had smaller atheromatous lesions, reduced lipid deposition, and less macrophage and T cell inflammatory infiltrate in the aortic roots than vehicle-treated animals. Serum levels of total cholesterol and the lipid peroxidation product malondialdehyde were also reduced in these mice. Our results demonstrate that quercetin interferes with both key proatherogenic activities of macrophages, namely foam cell formation and pro-oxidant/proinflammatory responses, and these effects may explain the atheroprotective properties of this common flavonoid. PMID:22869926

  7. Target of rapamycin activation predicts lifespan in fruit flies

    PubMed Central

    Scialò, Filippo; Sriram, Ashwin; Naudí, Alba; Ayala, Victoria; Jové, Mariona; Pamplona, Reinald; Sanz, Alberto

    2015-01-01

    Aging and age-related diseases are one of the most important health issues that the world will confront during the 21st century. Only by understanding the proximal causes will we be able to find treatments to reduce or delay the onset of degenerative diseases associated with aging. Currently, the prevalent paradigm in the field is the accumulation of damage. However, a new theory that proposes an alternative explanation is gaining momentum. The hyperfunction theory proposes that aging is not a consequence of a wear and tear process, but a result of the continuation of developmental programs during adulthood. Here we use Drosophila melanogaster, where evidence supporting both paradigms has been reported, to identify which parameters that have been previously related with lifespan best predict the rate of aging in wild type flies cultured at different temperatures. We find that mitochondrial function and mitochondrial reactive oxygen species (mtROS) generation correlates with metabolic rate, but not with the rate of aging. Importantly, we find that activation of nutrient sensing pathways (i.e. insulin-PI3K/Target of rapamycin (Tor) pathway) correlates with lifespan, but not with metabolic rate. Our results, dissociate metabolic rate and lifespan in wild type flies and instead link nutrient sensing signaling with longevity as predicted by the hyperfunction theory. PMID:26259964

  8. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling

    PubMed Central

    Braccini, Laura; Ciraolo, Elisa; Campa, Carlo C.; Perino, Alessia; Longo, Dario L.; Tibolla, Gianpaolo; Pregnolato, Marco; Cao, Yanyan; Tassone, Beatrice; Damilano, Federico; Laffargue, Muriel; Calautti, Enzo; Falasca, Marco; Norata, Giuseppe D.; Backer, Jonathan M.; Hirsch, Emilio

    2015-01-01

    In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. PMID:26100075

  9. PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling.

    PubMed

    Braccini, Laura; Ciraolo, Elisa; Campa, Carlo C; Perino, Alessia; Longo, Dario L; Tibolla, Gianpaolo; Pregnolato, Marco; Cao, Yanyan; Tassone, Beatrice; Damilano, Federico; Laffargue, Muriel; Calautti, Enzo; Falasca, Marco; Norata, Giuseppe D; Backer, Jonathan M; Hirsch, Emilio

    2015-01-01

    In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis. PMID:26100075

  10. 13 CFR 124.509 - What are non-8(a) business activity targets?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... activity targets and are expressed as a percentage of total revenue. The targets call for an increase in... obtain non-8(a) revenues. (e) Waiver of sole source prohibition. (1) The AA/BD, or his or her designee... activity targets? 124.509 Section 124.509 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION...

  11. Pre-target oscillatory brain activity and the attentional blink.

    PubMed

    Petro, Nathan M; Keil, Andreas

    2015-12-01

    Reporting the second of two targets within a stream of distracting words during rapid serial visual presentation (RSVP) is impaired when the targets are separated by a single distractor word, a deficit in temporal attention that has been referred to as the attentional blink (AB). Recent conceptual and empirical work has pointed to pre-target brain states as potential mediators of the AB effect. The current study examined differences in pre-target electrophysiology between correctly and incorrectly reported trials, considering amplitude and phase measures of alpha oscillations as well as the steady-state visual evoked potential (ssVEP) evoked by the RSVP stream. For incorrectly reported trials, relatively lower alpha-band power and greater ssVEP inter-trial phase locking were observed during extended time periods preceding presentation of the first target. These results suggest that facilitated processing of the pre-target distracter stream indexed by reduced alpha and heightened phase locking characterizes a dynamic brain state that predicts lower accuracy in terms of reporting the second target under strict temporal constraints. Findings align with hypotheses in which the AB effect is attributed to neurocognitive factors such as fluctuations in pre-target attention or to cognitive strategies applied at the trial level. PMID:26341931

  12. 78 FR 35612 - Agency Information Collection Activities; Comment Request; Targeted Teacher Shortage Areas...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-13

    ... collection of information from Chief State School Officers to support and document the request for teacher... Agency Information Collection Activities; Comment Request; Targeted Teacher Shortage Areas Nationwide... to this notice will be considered public records. Title of Collection: Targeted Teacher...

  13. Bypassing Protein Corona Issue on Active Targeting: Zwitterionic Coatings Dictate Specific Interactions of Targeting Moieties and Cell Receptors.

    PubMed

    Safavi-Sohi, Reihaneh; Maghari, Shokoofeh; Raoufi, Mohammad; Jalali, Seyed Amir; Hajipour, Mohammad J; Ghassempour, Alireza; Mahmoudi, Morteza

    2016-09-01

    Surface functionalization strategies for targeting nanoparticles (NP) to specific organs, cells, or organelles, is the foundation for new applications of nanomedicine to drug delivery and biomedical imaging. Interaction of NPs with biological media leads to the formation of a biomolecular layer at the surface of NPs so-called as "protein corona". This corona layer can shield active molecules at the surface of NPs and cause mistargeting or unintended scavenging by the liver, kidney, or spleen. To overcome this corona issue, we have designed biotin-cysteine conjugated silica NPs (biotin was employed as a targeting molecule and cysteine was used as a zwitterionic ligand) to inhibit corona-induced mistargeting and thus significantly enhance the active targeting capability of NPs in complex biological media. To probe the targeting yield of our engineered NPs, we employed both modified silicon wafer substrates with streptavidin (i.e., biotin receptor) to simulate a target and a cell-based model platform using tumor cell lines that overexpress biotin receptors. In both cases, after incubation with human plasma (thus forming a protein corona), cellular uptake/substrate attachment of the targeted NPs with zwitterionic coatings were significantly higher than the same NPs without zwitterionic coating. Our results demonstrated that NPs with a zwitterionic surface can considerably facilitate targeting yield of NPs and provide a promising new type of nanocarriers in biological applications. PMID:27526263

  14. Multimodality PET/MRI agents targeted to activated macrophages.

    PubMed

    Tu, Chuqiao; Ng, Thomas S C; Jacobs, Russell E; Louie, Angelique Y

    2014-02-01

    The recent emergence of multimodality imaging, particularly the combination of PET and MRI, has led to excitement over the prospect of improving detection of disease. Iron oxide nanoparticles have become a popular platform for the fabrication of PET/MRI probes owing to their advantages of high MRI detection sensitivity, biocompatibility, and biodegradability. In this article, we report the synthesis of dextran-coated iron oxide nanoparticles (DIO) labeled with the positron emitter (64)Cu to generate a PET/MRI probe, and modified with maleic anhydride to increase the negative surface charge. The modified nanoparticulate PET/MRI probe (MDIO-(64)Cu-DOTA) bears repetitive anionic charges on the surface that facilitate recognition by scavenger receptor type A (SR-A), a ligand receptor found on activated macrophages but not on normal vessel walls. MDIO-(64)Cu-DOTA has an average iron oxide core size of 7-8 nm, an average hydrodynamic diameter of 62.7 nm, an r1 relaxivity of 16.8 mM(-1) s(-1), and an r 2 relaxivity of 83.9 mM(-1) s(-1) (37 °C, 1.4 T). Cell studies confirmed that the probe was nontoxic and was specifically taken up by macrophages via SR-A. In comparison with the nonmodified analog, the accumulation of MDIO in macrophages was substantially improved. These characteristics demonstrate the promise of MDIO-(64)Cu-DOTA for identification of vulnerable atherosclerotic plaques via the targeting of macrophages. PMID:24166283

  15. Magnetoresponsive squalenoyl gemcitabine composite nanoparticles for cancer active targeting.

    PubMed

    Arias, José L; Reddy, L Harivardhan; Couvreur, Patrick

    2008-07-15

    heterogeneous structure of these nanoparticles could confer them both magnetic field responsiveness and potential applicability as a drug carrier for active targeting to solid tumors. PMID:18540685

  16. Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin.

    PubMed

    Saito, Kota; Araki, Yasuhiro; Kontani, Kenji; Nishina, Hiroshi; Katada, Toshiaki

    2005-03-01

    The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression. PMID:15809346

  17. Utilizing the folate receptor for active targeting of cancer nanotherapeutics

    PubMed Central

    Zwicke, Grant L.; Mansoori, G. Ali; Jeffery, Constance J.

    2012-01-01

    The development of specialized nanoparticles for use in the detection and treatment of cancer is increasing. Methods are being proposed and tested that could target treatments more directly to cancer cells, which could lead to higher efficacy and reduced toxicity, possibly even eliminating the adverse effects of damage to the immune system and the loss of quick replicating cells. In this mini-review we focus on recent studies that employ folate nanoconjugates to target the folate receptor. Folate receptors are highly overexpressed on the surface of many tumor types. This expression can be exploited to target imaging molecules and therapeutic compounds directly to cancerous tissues. PMID:23240070

  18. Target Assembly to Check Boresight Alignment of Active Sensors

    NASA Technical Reports Server (NTRS)

    Ramos-Izquierdo, Luis; Scott, V. Stanley; Riris, Haris; Cavanaugh, John; Liiva, Peter; Rodriguez, Michael

    2011-01-01

    A compact and portable target assembly (Fig. 1) has been developed to measure the boresite alignment of LRO's Lunar Orbiter Laser Altimeter (LOLA) instrument at the spacecraft level. The concept for this target assembly has evolved over many years with earlier versions used to test the Mars Observer Laser Altimeter (MOLA), the Geoscience Laser Altimeter System (GLAS), and the Mercury Laser Altimeter (MLA) space-based instruments.

  19. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  20. Identification of orthologous target pairs with shared active compounds and comparison of organism-specific activity patterns.

    PubMed

    Dimova, Dilyana; Stumpfe, Dagmar; Bajorath, Jürgen

    2015-11-01

    A systematic search for active small molecules shared by orthologous targets was carried out, leading to the identification of 803 compound-based orthologous target pairs covering a total of 938 orthologues, 358 unique targets and 98 organisms. Many orthologous target pairs were found to have substantial compound coverage, enabling the introduction of an orthologous target pairs classification including 'organism cliffs' and 'potency-retaining' pairs. A total of 158 orthologous target pairs involving human orthologues were identified, which were typically associated with drug discovery-relevant targets, organism combinations and compound data. Orthologous target pairs with human orthologues included 83 potency-retaining orthologous target pairs covering a variety of targets and organisms. On the basis of these orthologous target pairs, the compound search was further extended and 1149 potent compounds were identified that only had reported activities for non-human orthologues of 48 therapeutic targets, but not their human counterparts, hence providing a large pool of candidate compounds for further evaluation. The complete set of orthologous target pairs identified in our analysis, the orthologous target pairs classification including associated data and all candidate compounds are made freely available. PMID:25931211

  1. GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings

    PubMed Central

    Jiang, Hong; Liu, Wei; Zhan, Shi-Kun; Pan, Yi-Xin; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang; Pan, Si-Jian

    2016-01-01

    Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent. PMID:27532105

  2. Aluminum: New challenges in downstream activities

    NASA Astrophysics Data System (ADS)

    Becker, Miklos N.

    1999-11-01

    During its history, aluminum’s attractive features, such as high strength-to-weight ratio, good electrical mass conductivity, and unique corrosion behavior, have led to a spectacular expansion in its use. The role of aluminum in non-aluminum-based materials is also very important; its contribution to the improvement of magnesium and titanium alloys and to highly complex packaging materials are some of the noteworthy examples. Significant cost reductions on the basic metal production level, near-to-shape fabricating methods, and the well-functioning recycling system are also major contributors to aluminum success. Imminent challenges for the industry are the need for products with very close tolerances on a mass fabricating repetitive basis and just-in-time delivery to original-equipment manufacturers and small users through distributors. A significant part of the challenges remains in the applications area, particularly automotive and aerospace.

  3. Wave and particle evolution downstream of quasi-perpendicular shocks

    NASA Technical Reports Server (NTRS)

    Mckean, M. E.; Omidi, N.; Krauss-Varban, D.; Karimabadi, H.

    1995-01-01

    Distributions of ions heated in quasi-perpendicular bow shocks have large perpendicular temperature anisotropies that provide free energy for the growth of Alfven ion cyclotron (AIC) and mirror waves. These modes are often obsreved in the Earth's magnetosheath. Using two-dimensional hybrid simulations, we show that these waves are produced near the shock front and convected downstream rather than being produced locally downstream. The wave activity reduces the proton anisotropy to magnetosheath levels within a few tens of gyroradii of the shock but takes significantly longer to reduce the anisotropy of He(++) ions. The waves are primarily driven by proton anisotropy and the dynamics of the helium ions is controlled by the proton waves. Downstream of high Mach number shocks, mirror waves compete effectively with AIC waves. Downstream of low Mach number shocks, AIC waves dominate.

  4. The R2R3-MYB–Like Regulatory Factor EOBI, Acting Downstream of EOBII, Regulates Scent Production by Activating ODO1 and Structural Scent-Related Genes in Petunia[C][W

    PubMed Central

    Spitzer-Rimon, Ben; Farhi, Moran; Albo, Boaz; Cna’ani, Alon; Ben Zvi, Michal Moyal; Masci, Tania; Edelbaum, Orit; Yu, Yixun; Shklarman, Elena; Ovadis, Marianna; Vainstein, Alexander

    2012-01-01

    Flower scent is a highly dynamic trait, under developmental, spatial, and diurnal regulation. The mechanism governing scent production is only beginning to be unraveled. In petunia (Petunia hybrida), EMISSION OF BENZENOIDS II (EOBII) controls transcription of both the shikimate pathway-regulating MYB factor ODORANT1 (ODO1) and phenylpropanoid scent-related structural genes. A promoter-activation screen identified an R2R3-MYB–like regulatory factor of phenylpropanoid volatile biosynthesis acting downstream of EOBII, designated EOBI. EOBI silencing led to downregulation of ODO1 and numerous structural scent-related genes from both the shikimate and phenylpropanoid pathways. The ability of EOBI to directly activate ODO1, as revealed by electrophoretic mobility shift assay and yeast one-hybrid analysis, place EOBI upstream of ODO1 in regulating substrate availability for volatile biosynthesis. Interestingly, ODO1-silenced transgenic petunia flowers accumulated higher EOBI transcript levels than controls, suggesting a complex feedback loop between these regulatory factors. The accumulation pattern of EOBI transcript relative to EOBII and ODO1, and the effect of up/downregulation of EOBII on transcript levels of EOBI and ODO1, further support these factors' hierarchical relationships. The dependence of scent production on EOBI expression and its direct interaction with both regulatory and structural genes provide evidence for EOBI’s wide-ranging involvement in the production of floral volatiles. PMID:23275577

  5. Selective Activation of Neuronal Targets With Sinusoidal Electric Stimulation

    PubMed Central

    Freeman, Daniel K.; Eddington, Donald K.; Rizzo, Joseph F.

    2010-01-01

    Electric stimulation of the CNS is being evaluated as a treatment modality for a variety of neurological, psychiatric, and sensory disorders. Despite considerable success in some applications, existing stimulation techniques offer little control over which cell types or neuronal substructures are activated by stimulation. The ability to more precisely control neuronal activation would likely improve the clinical outcomes associated with these applications. Here, we show that specific frequencies of sinusoidal stimulation can be used to preferentially activate certain retinal cell types: photoreceptors are activated at 5 Hz, bipolar cells at 25 Hz, and ganglion cells at 100 Hz. In addition, low-frequency stimulation (≤25 Hz) did not activate passing axons but still elicited robust synaptically mediated responses in ganglion cells; therefore, elicited neural activity is confined to within a focal region around the stimulating electrode. Our results suggest that sinusoidal stimulation provides significantly improved control over elicited neural activity relative to conventional pulsatile stimulation. PMID:20810683

  6. Nonimaging active system determination of target shape through turbulent medium

    NASA Astrophysics Data System (ADS)

    Chandler, Susan M.; Lukesh, Gordon W.

    2001-01-01

    Image reconstruction techniques for atmospheric applications often work best with an initial estimate of the object support. This paper examines the ability of a non-imaging laser pointing system to obtain an estimate of target size and shape based on the statistics of the return signal. Fundamental limits on system pointing, such as the tracking errors, corrupt a simple raster scan that would provide gross object shape form the convolution of the far-field pattern with the target. Using techniques developed previously for the estimation of pointing performance, it is possible to distinguish between simple shapes such as bars, circles and T's based on the statistics of the received time signal. Simulated space objects, such as those illuminated during field experiments, may also be distinguished.

  7. 13 CFR 124.509 - What are non-8(a) business activity targets?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... obtain non-8(a) revenues. (e) Waiver of sole source prohibition. (1) The AA/BD, or his or her designee... activity targets? 124.509 Section 124.509 Business Credit and Assistance SMALL BUSINESS ADMINISTRATION 8(a... Contractual Assistance § 124.509 What are non-8(a) business activity targets? (a) General. (1) To ensure...

  8. Aptamers: Active Targeting Ligands for Cancer Diagnosis and Therapy

    PubMed Central

    Wu, Xu; Chen, Jiao; Wu, Min; Zhao, Julia Xiaojun

    2015-01-01

    Aptamers, including DNA, RNA and peptide aptamers, are a group of promising recognition units that can specifically bind to target molecules and cells. Due to their excellent specificity and high affinity to targets, aptamers have attracted great attention in various fields in which selective recognition units are required. They have been used in biosensing, drug delivery, disease diagnosis and therapy (especially for cancer treatment). In this review, we summarized recent applications of DNA and RNA aptamers in cancer theranostics. The specific binding ability of aptamers to cancer-related markers and cancer cells ensured their high performance for early diagnosis of cancer. Meanwhile, the efficient targeting ability of aptamers to cancer cells and tissues provided a promising way to deliver imaging agents and drugs for cancer imaging and therapy. Furthermore, with the development of nanoscience and nanotechnology, the conjugation of aptamers with functional nanomaterials paved an exciting way for the fabrication of theranostic agents for different types of cancers, which might be a powerful tool for cancer treatment. PMID:25699094

  9. RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

    PubMed Central

    Wilkinson, Adam C.; Ballabio, Erica; Geng, Huimin; North, Phillip; Tapia, Marta; Kerry, Jon; Biswas, Debabrata; Roeder, Robert G.; Allis, C. David; Melnick, Ari; de Bruijn, Marella F.T.R.; Milne, Thomas A.

    2013-01-01

    Summary The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product. PMID:23352661

  10. Abnormal Ventral and Dorsal Attention Network Activity during Single and Dual Target Detection in Schizophrenia

    PubMed Central

    Jimenez, Amy M.; Lee, Junghee; Wynn, Jonathan K.; Cohen, Mark S.; Engel, Stephen A.; Glahn, David C.; Nuechterlein, Keith H.; Reavis, Eric A.; Green, Michael F.

    2016-01-01

    Early visual perception and attention are impaired in schizophrenia, and these deficits can be observed on target detection tasks. These tasks activate distinct ventral and dorsal brain networks which support stimulus-driven and goal-directed attention, respectively. We used single and dual target rapid serial visual presentation (RSVP) tasks during fMRI with an ROI approach to examine regions within these networks associated with target detection and the attentional blink (AB) in 21 schizophrenia outpatients and 25 healthy controls. In both tasks, letters were targets and numbers were distractors. For the dual target task, the second target (T2) was presented at three different lags after the first target (T1) (lag1 = 100 ms, lag3 = 300 ms, lag7 = 700ms). For both single and dual target tasks, patients identified fewer targets than controls. For the dual target task, both groups showed the expected AB effect with poorer performance at lag 3 than at lags 1 or 7, and there was no group by lag interaction. During the single target task, patients showed abnormally increased deactivation of the temporo-parietal junction (TPJ), a key region of the ventral network. When attention demands were increased during the dual target task, patients showed overactivation of the posterior intraparietal cortex, a key dorsal network region, along with failure to deactivate TPJ. Results suggest inefficient and faulty suppression of salience-oriented processing regions, resulting in increased sensitivity to stimuli in general, and difficulty distinguishing targets from non-targets. PMID:27014135

  11. Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

    PubMed Central

    2011-01-01

    Background Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. Natural phenolic compounds can serve as potent redox cyclers that inhibit microbial growth through destabilization of cellular redox homeostasis and/or antioxidation systems. The aim of this study was to identify benzaldehydes that disrupt the fungal antioxidation system. These compounds could then function as chemosensitizing agents in concert with conventional drugs or fungicides to improve antifungal efficacy. Methods Benzaldehydes were tested as natural antifungal agents against strains of Aspergillus fumigatus, A. flavus, A. terreus and Penicillium expansum, fungi that are causative agents of human invasive aspergillosis and/or are mycotoxigenic. The yeast Saccharomyces cerevisiae was also used as a model system for identifying gene targets of benzaldehydes. The efficacy of screened compounds as effective chemosensitizers or as antifungal agents in formulations was tested with methods outlined by the Clinical Laboratory Standards Institute (CLSI). Results Several benzaldehydes are identified having potent antifungal activity. Structure-activity analysis reveals that antifungal activity increases by the presence of an ortho-hydroxyl group in the aromatic ring. Use of deletion mutants in the oxidative stress-response pathway of S. cerevisiae (sod1Δ, sod2Δ, glr1Δ) and two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus (sakAΔ, mpkCΔ), indicates antifungal activity of the benzaldehydes is through disruption of cellular antioxidation. Certain benzaldehydes, in combination with phenylpyrroles, overcome tolerance of A. fumigatus MAPK mutants to this agent and/or increase sensitivity of fungal pathogens to mitochondrial respiration inhibitory agents. Synergistic chemosensitization greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations. Effective

  12. EGF-receptor phosphorylation and downstream signaling are activated by benzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mammary epithelial cells

    SciTech Connect

    Rodriguez-Fragoso, Lourdes; Melendez, Karla; Hudson, Laurie G.; Lauer, Fredine T.; Burchiel, Scott W.

    2009-03-15

    Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo[a]pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-{gamma}1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 {mu}M), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-{gamma}1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-{gamma}1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the patterns of phosphorylation at EGFR, PLC-{gamma}1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways.

  13. RalA, a GTPase targeted by miR-181a, promotes transformation and progression by activating the Ras-related signaling pathway in chronic myelogenous leukemia

    PubMed Central

    Luo, Xiaochuang; Yang, Juhua; Li, Yumin; Li, Tianfu; Wang, Ruirui; Fei, Jia

    2016-01-01

    BCR/ABL is a well-known activator of multiple signaling pathways. RalA, a Ras downstream signaling molecule and a small GTPase, plays an important role in Bcr-Abl-induced leukemogenesis but the exact mechanism remains elusive. Here, we show that RalA GTPase activity is commonly high in chronic myelogenous leukemia (CML) cell lines and patient samples. Overexpression of RalA results in malignant transformation and progression, and induces resistance to imatinib (IM) in BaF3 and K562 cell lines. RalA reduced survival and led to IM resistance in a xenografted mouse model. Ablation of RalA by either siRNA or miR-181a, a RalA targeting microRNA, attenuated the malignant phenotypes in K562 cells. RBC8, a selective Ral inhibitor, enhanced the inhibitory effects of IM in K562, KCL22 and BaF3-P210 cells. Interestingly, the phospho-specific protein microarray assay revealed that multiple phosphorylation signal proteins were decreased by RalA inhibition, including SAPK, JNK, SRC, VEGFR2, P38 MAPK, c-Kit, JunB, and Keratin18. Among them, P38 MAPK and SAPK/JNK are Ras downstream signaling kinases. Taken together, RalA GTPase might be an important oncogene activating the Ras-related signaling pathway in CML. PMID:26967392

  14. In Vivo Anti-HIV Activity of the Heparin-Activated Serine Protease Inhibitor Antithrombin III Encapsulated in Lymph-Targeting Immunoliposomes

    PubMed Central

    Asmal, Mohammed; Whitney, James B.; Luedemann, Corinne; Carville, Angela; Steen, Robert; Letvin, Norman L.; Geiben-Lynn, Ralf

    2012-01-01

    Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log10 reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention. PMID:23133620

  15. A new approach for determination of neuropathy target esterase activity.

    PubMed

    Sigolaeva, L V; Eremenko, A V; Makower, A; Makhaeva, G F; Malygin, V V; Kurochkin, I N

    1999-05-14

    Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE. PMID:10421495

  16. Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: A novel antiangiogenic therapy

    SciTech Connect

    Kamisasanuki, Taro; Tokushige, Saori; Terasaki, Hiroto; Khai, Ngin Cin; Wang, Yuqing; Sakamoto, Taiji; Kosai, Ken-ichiro

    2011-09-16

    Highlights: {yields} CD9 plays stimulus-independent roles in angiogenesis in vitro and in vivo. {yields} Targeting CD9 expression is effective in an angiogenic disease model. {yields} Targeting CD9 expression predominantly affects activated endothelial cells. {yields} CD9 is involved in endothelial cell proliferation, but not survival. {yields} CD9 is part of angiogenic machinery in endothelial cells during angiogenesis. -- Abstract: The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus

  17. Activated α2-Macroglobulin Regulates Transcriptional Activation of c-MYC Target Genes through Cell Surface GRP78 Protein.

    PubMed

    Gopal, Udhayakumar; Gonzalez-Gronow, Mario; Pizzo, Salvatore Vincent

    2016-05-13

    Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu(98)-Leu(115)). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser(62) Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser(10) In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression. PMID:27002159

  18. A new antibiotic with potent activity targets MscL

    PubMed Central

    Iscla, Irene; Wray, Robin; Blount, Paul; Larkins-Ford, Jonah; Conery, Annie L; Ausubel, Frederick M; Ramu, Soumya; Kavanagh, Angela; Huang, Johnny X; Blaskovich, Mark A; Cooper, Matthew A; Obregon-Henao, Andres; Orme, Ian; Tjandra, Edwin S; Stroeher, Uwe H; Brown, Melissa H; Macardle, Cindy; van Holst, Nick; Ling Tong, Chee; Slattery, Ashley D; Gibson, Christopher T; Raston, Colin L; Boulos, Ramiz A

    2015-01-01

    The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections. PMID:25649856

  19. Targeting of TGF-β-activated protein kinase 1 inhibits chemokine (C-C motif) receptor 7 expression, tumor growth and metastasis in breast cancer

    PubMed Central

    Hung, Wen-Chun; Hou, Ming-Feng

    2015-01-01

    TGF-β-activated protein kinase 1 (TAK1) is a critical mediator in inflammation, immune response and cancer development. Our previous study demonstrated that activation of TAK1 increases the expression of chemokine (C-C motif) receptor 7 (CCR7) and promotes lymphatic invasion ability of breast cancer cells. However, the expression and association of activated TAK1 and CCR7 in breast tumor tissues is unknown and the therapeutic effect by targeting TAK1 is also unclear. We showed that activated TAK1 (as indicated by phospho-TAK1) and its binding protein TAB1 are strongly expressed in breast tumor tissues (77% and 74% respectively). In addition, increase of phospho-TAK1 or TAB1 is strongly associated with over-expression of CCR7. TAK1 inhibitor 5Z-7-Oxozeaenol (5Z-O) inhibited TAK1 activity, suppressed downstream signaling pathways including p38, IκB kinase (IKK) and c-Jun N-terminal kinase (JNK) and reduced CCR7 expression in metastatic MDA-MB-231 cells. In addition, 5Z-O repressed NF-κB- and c-JUN-mediated transcription of CCR7 gene. Knockdown of TAB1 attenuated CCR7 expression and tumor growth in an orthotopic animal study. More importantly, lymphatic invasion and lung metastasis were suppressed. Collectively, our results demonstrate that constitutive activation of TAK1 is frequently found in human breast cancer and this kinase is a potential therapeutic target for this cancer. PMID:25557171

  20. Structure–Activity Studies of Phosphopeptidomimetic Prodrugs Targeting the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 3 (Stat3)

    PubMed Central

    Mandal, Pijus K.; Ren, Zhiyong; Chen, Xiaomin; Kaluarachchi, Kumar; Liao, Warren S.-L.; McMurray, John S.

    2013-01-01

    Signal transducer and activator of transcription 3 (Stat3) transmits signals from growth factors and interleukin-6 family cytokines by binding to their receptors via its Src homology 2 (SH2) domain. This results in phosphorylation of Tyr705, dimerization, translocation to the nucleus, and regulation of transcription of downstream genes. Stat3 is constitutively activated in several human cancers and is a target for anti-cancer drug design. We have shown previously phosphorylation of Tyr705 in intact cancer cells can be inhibited with prodrugs of phosphopeptide mimics targeting the SH2 domain. In a series of prodrugs consisting of bis-pivaloyloxymethyl esters of 4′-phosphonodifluoromethyl cinnamoyl-Haic-Gln-NHBn, appending methyl group to the β-position of the cinnamate increased potency ca. twofold, which paralleled the increase in affinity of the corresponding phosphopeptide models. However, dramatic increases in potency were observed when the C-terminal C(O)NHBn of Gln-NHBn was replaced with a simple methyl group. In this communication we continue to explore the effects of structural modifications of prodrugs on their ability to inhibit Tyr705 phosphorylation. A set of 4-substituted prolines incorporated into β-methyl-4-phosphocinnamoyl-leucinyl-Xaa-4-aminopentamide model peptides exhibited affinities of 88–317 nM by fluorescence polarization (Pro IC50 = 156 nM). In corresponding prodrugs, Pro inhibited constitutive Stat3 phosphorylation at 10 μM in MDA-MB-468 breast tumor cells. However, 4,4-difluoroproline and 4,4-dimethylproline resulted in complete inhibition at 0.5 μM. These results suggest that the prodrug with native proline undergoes metabolism that those with substituted prolines do not. In conclusion, changes in structure with minimal impact on intrinsic affinity can nevertheless have profound effects on the cellular potency of prodrug inhibitors of Stat3. PMID:24707243

  1. Nrf2 activation as target to implement therapeutic treatments

    PubMed Central

    Bocci, Velio; Valacchi, Giuseppe

    2015-01-01

    A chronic increase of oxidative stress is typical of serious pathologies such as myocardial infarction, stroke, chronic limb ischemia, chronic obstructive pulmonary disease (COPD), type II-diabetes, age-related macular degeneration leads to an epic increase of morbidity and mortality in all countries of the world. The initial inflammation followed by an excessive release of reactive oxygen species (ROS) implies a diffused cellular injury that needs to be corrected by an inducible expression of the innate detoxifying and antioxidant system. The transcription factor Nrf2, when properly activated, is able to restore a redox homeostasis and possibly improve human health. PMID:25699252

  2. NRF2 Activation as Target to Implement Therapeutic Treatments

    NASA Astrophysics Data System (ADS)

    Bocci, Velio; Valacchi, Giuseppe

    2015-02-01

    A chronic increase of oxidative stress is typical of serious pathologies such as myocardial infarction, stroke, chronic limb ischemia, chronic obstructive pulmonary disease (COPD), type II-diabetes, age-related macular degeneration leads to an epic increase of morbidity and mortality in all countries of the world. The initial inflammation followed by an excessive release of reactive oxygen species (ROS) implies a diffused cellular injury that needs to be corrected by an inducible expression of the innate detoxifying and antioxidant system. The transcription factor Nrf2, when properly activated, is able to restore a redox homeostasis and possibly improve human health.

  3. Target cell-specific modulation of neuronal activity by astrocytes

    NASA Astrophysics Data System (ADS)

    Kozlov, A. S.; Angulo, M. C.; Audinat, E.; Charpak, S.

    2006-06-01

    Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibitory neurotransmitters, astrocytes exert a complex modulatory control on the olfactory network. glutamate | GABA | inhibition | olfactory bulb | synchronization

  4. Design of the DARHT-II Downstream Beamline

    SciTech Connect

    Westenskow, G A; Bertolini, L R; Chen, Y -J; Fessenden, T J; Paul, A C; Watson, J A

    2002-06-07

    This paper describes the design of the downstream beam transport line for the second axis of the Dual Axis Radiographic Hydrodynamic Test (DARHT-II) Facility. The DARHT-II project is a collaboration between LANL, LBNL and LLNL. DARHT II is a 18.4-MeV, 2000-Amperes, 2-{micro}sec linear induction accelerator designed to generate short bursts of x-rays for the purpose of radiographing dense objects. The downstream beam transport line is approximately 22-meter long region extending from the end of the accelerator to the bremsstrahlung target. The principal element of the beam transport section is the fast deflector, or kicker system, used to generate four micropulses from the primary accelerator beam. Within this proposed transport line there are also several conventional solenoid, quadrupole and dipole magnets which transport and focus the beam to the target and to the beam dumps.

  5. Interpersonal stroking touch is targeted to C tactile afferent activation.

    PubMed

    Croy, I; Luong, A; Triscoli, C; Hofmann, E; Olausson, H; Sailer, U

    2016-01-15

    C tactile fibers are a specialized group of fibers innervating the non-glabrous skin that are tuned to light gentle stroking applied with velocities between 1 and 10 cm/s. Those fibers add to the sensation of interpersonal caressing and pleasant touch. It is unclear whether people spontaneously apply touch that is tuned to optimally activate those fibers. This was investigated in three studies. In study one, 45 participants (21.8 ± 2.3 years, 24 women) were asked to stroke an artificial arm. In study two, 32 participants (28.3 ± 8.7 years, 16 women) were asked to stroke their partner. In study three, 11 parents (29.4 ± 5.7 years, 6 women) were asked to stroke their babies. Stroking velocity was tracked in all conditions. Stroking velocities were significantly slower in the partner touch and baby touch condition than in the artificial arm condition and all of the participants stroking their partner or baby used velocities that can activate C tactile fibers. We conclude that human social stroking is optimized for C tactile stimulation. PMID:26433145

  6. Decoding Target Distance and Saccade Amplitude from Population Activity in the Macaque Lateral Intraparietal Area (LIP)

    PubMed Central

    Bremmer, Frank; Kaminiarz, Andre; Klingenhoefer, Steffen; Churan, Jan

    2016-01-01

    Primates perform saccadic eye movements in order to bring the image of an interesting target onto the fovea. Compared to stationary targets, saccades toward moving targets are computationally more demanding since the oculomotor system must use speed and direction information about the target as well as knowledge about its own processing latency to program an adequate, predictive saccade vector. In monkeys, different brain regions have been implicated in the control of voluntary saccades, among them the lateral intraparietal area (LIP). Here we asked, if activity in area LIP reflects the distance between fovea and saccade target, or the amplitude of an upcoming saccade, or both. We recorded single unit activity in area LIP of two macaque monkeys. First, we determined for each neuron its preferred saccade direction. Then, monkeys performed visually guided saccades along the preferred direction toward either stationary or moving targets in pseudo-randomized order. LIP population activity allowed to decode both, the distance between fovea and saccade target as well as the size of an upcoming saccade. Previous work has shown comparable results for saccade direction (Graf and Andersen, 2014a,b). Hence, LIP population activity allows to predict any two-dimensional saccade vector. Functional equivalents of macaque area LIP have been identified in humans. Accordingly, our results provide further support for the concept of activity from area LIP as neural basis for the control of an oculomotor brain-machine interface.

  7. Neural activity promotes long-distance, target-specific regeneration of adult retinal axons.

    PubMed

    Lim, Jung-Hwan A; Stafford, Benjamin K; Nguyen, Phong L; Lien, Brian V; Wang, Chen; Zukor, Katherine; He, Zhigang; Huberman, Andrew D

    2016-08-01

    Axons in the mammalian CNS fail to regenerate after injury. Here we show that if the activity of mouse retinal ganglion cells (RGCs) is increased by visual stimulation or using chemogenetics, their axons regenerate. We also show that if enhancement of neural activity is combined with elevation of the cell-growth-promoting pathway involving mammalian target of rapamycin (mTOR), RGC axons regenerate long distances and re-innervate the brain. Analysis of genetically labeled RGCs revealed that this regrowth can be target specific: RGC axons navigated back to their correct visual targets and avoided targets incorrect for their function. Moreover, these regenerated connections were successful in partially rescuing a subset of visual behaviors. Our findings indicate that combining neural activity with activation of mTOR can serve as powerful tool for enhancing axon regeneration, and they highlight the remarkable capacity of CNS neurons to re-establish accurate circuit connections in adulthood. PMID:27399843

  8. Target design considerations for high specific activity [{sup 11}C]O{sub 2}

    SciTech Connect

    Ferrieri, R.A.; Alexoff, D.L.; Schlyer, D.J.; McDonald, K.; Wolf, A.P.

    1993-12-31

    In the routine preparation of {sup 11}C-labeled compounds through N-[{sup 11}C]-methylation using [{sup 11}C]H{sub 3}I, total masses are always higher than synthesis mass contribution, suggesting that the target system contributes carrier carbon to the final product mass. This conclusion prompted this evaluation of target materials and target design for [{sup 11}C]O{sub 2} production. Ultimately, one is faced with the sprospect of compromising between [{sup 11}C]O{sub 2} specific activity and the amount that can be extracted from the target after a reasonable irradiation time.

  9. MiR-29a Reduces TIMP-1 Production by Dermal Fibroblasts via Targeting TGF-β Activated Kinase 1 Binding Protein 1, Implications for Systemic Sclerosis

    PubMed Central

    Ciechomska, Marzena; O’Reilly, Steven; Suwara, Monika; Bogunia-Kubik, Katarzyna; van Laar, Jacob M.

    2014-01-01

    Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by skin and internal organs fibrosis due to accumulation of extra cellular matrix (ECM) proteins. Tissue inhibitor of metalloproteinases 1 (TIMP-1) plays a key role in ECM deposition. Aim To investigate the role of miR-29a in regulation of TAB1-mediated TIMP-1 production in dermal fibroblasts in systemic sclerosis. Methods Healthy control (HC) and SSc fibroblasts were cultured from skin biopsies. The expression of TIMP-1, MMP-1 and TGF-β activated kinase 1 binding protein 1 (TAB1) was measured following miR-29a transfection using ELISA, qRT-PCR, and Western Blotting. The functional effect of miR-29a on dermal fibroblasts was assessed in collagen gel assay. In addition, HeLa cells were transfected with 3′UTR of TAB1 plasmid cloned downstream of firefly luciferase gene to assess TAB1 activity. HC fibroblasts and HeLa cells were also transfected with Target protectors in order to block the endogenous miR-29a activity. Results We found that TAB1 is a novel target gene of miR-29a, also regulating downstream TIMP-1 production. TAB1 is involved in TGF-β signal transduction, a key cytokine triggering TIMP-1 production. To confirm that TAB1 is a bona fide target gene of miR-29a, we used a TAB1 3′UTR luciferase assay and Target protector system. We showed that miR-29a not only reduced TIMP-1 secretion via TAB1 repression, but also increased functional MMP-1 production resulting in collagen degradation. Blocking TAB1 activity by pharmacological inhibition or TAB1 knockdown resulted in TIMP-1 reduction, confirming TAB1-dependent TIMP-1 regulation. Enhanced expression of miR-29a was able to reverse the profibrotic phenotype of SSc fibroblasts via downregulation of collagen and TIMP-1. Conclusions miR-29a repressed TAB1-mediated TIMP-1 production in dermal fibroblasts, demonstrating that miR-29a may be a therapeutic target in SSc. PMID:25549087

  10. Design and Analysis of Hammerhead Ribozyme Activity Against an Artificial Gene Target

    PubMed Central

    Carter, James; Nawtaisong, Pruksa; Balaraman, Velmurugan; Fraser, Malcolm J.

    2014-01-01

    In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like the dengue virus RNA genomes. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays. PMID:24318886

  11. CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress.

    PubMed

    Fekry, Baharan; Jeffries, Kristen A; Esmaeilniakooshkghazi, Amin; Ogretmen, Besim; Krupenko, Sergey A; Krupenko, Natalia I

    2016-08-01

    Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response. PMID:27302066

  12. Oncogenic mutations in adenomatous polyposis coli (Apc) activate mechanistic target of rapamycin complex 1 (mTORC1) in mice and zebrafish

    PubMed Central

    Valvezan, Alexander J.; Huang, Jian; Lengner, Christopher J.; Pack, Michael; Klein, Peter S.

    2014-01-01

    Truncating mutations in adenomatous polyposis coli (APC) are strongly linked to colorectal cancers. APC is a negative regulator of the Wnt pathway and constitutive Wnt activation mediated by enhanced Wnt–β-catenin target gene activation is believed to be the predominant mechanism responsible for APC mutant phenotypes. However, recent evidence suggests that additional downstream effectors contribute to APC mutant phenotypes. We previously identified a mechanism in cultured human cells by which APC, acting through glycogen synthase kinase-3 (GSK-3), suppresses mTORC1, a nutrient sensor that regulates cell growth and proliferation. We hypothesized that truncating Apc mutations should activate mTORC1 in vivo and that mTORC1 plays an important role in Apc mutant phenotypes. We find that mTORC1 is strongly activated in apc mutant zebrafish and in intestinal polyps in Apc mutant mice. Furthermore, mTORC1 activation is essential downstream of APC as mTORC1 inhibition partially rescues Apc mutant phenotypes including early lethality, reduced circulation and liver hyperplasia. Importantly, combining mTORC1 and Wnt inhibition rescues defects in morphogenesis of the anterior-posterior axis that are not rescued by inhibition of either pathway alone. These data establish mTORC1 as a crucial, β-catenin independent effector of oncogenic Apc mutations and highlight the importance of mTORC1 regulation by APC during embryonic development. Our findings also suggest a new model of colorectal cancer pathogenesis in which mTORC1 is activated in parallel with Wnt/β-catenin signaling. PMID:24092877

  13. Evaluation of active and passive polarimetric electro-optic imagery for civilian and military targets discrimination

    NASA Astrophysics Data System (ADS)

    Lavigne, Daniel A.; Breton, Mélanie; Pichette, Mario; Larochelle, Vincent; Simard, Jean-Robert

    2008-04-01

    Electro-optic (EO) imaging systems are commonly used to detect civilian and military targets during surveillance operations and search and rescue missions. Adding the polarization of light as additional information to such active and passive EO imaging systems may increase the target discrimination performance, as man made objects are known to depolarized light in different manner than natural background. However, while the polarization of light has been used and studied in the past for numerous applications, the understanding of the polarization phenomenology taking place with targets used in cluttered backgrounds requires additional experimentations. Specifically, the target contrast enhancement obtained by analyzing the polarization of the reflected light from either a direct polarized laser source as encountered in active imagers, or from natural ambient illumination, needs further investigation. This paper describes an investigation of the use of polarization-based imaging sensors to discriminate civilian and military targets against different backgrounds. Measurements were carried out using two custom-designed active and passive imaging systems operating in the near infrared (NIR) and the long-wave infrared (LWIR) spectral bands. Polarimetric signatures were acquired during two distinct trials that occurred in 2007, using specific civilian and military targets such as cars and military vehicles. Results demonstrate to what extent and under which illumination and environmental conditions the exploitation of active and passive polarimetric images is suitable to enable target detection and recognition for some events of interest, according to various specific scenarios.

  14. Targeted Proteomics Approaches To Monitor Microbial Activity In Basalt Aquifer

    NASA Astrophysics Data System (ADS)

    Paszczynski, A. J.; Paidisetti, R.

    2007-12-01

    Microorganisms play a major role in biogeochemical cycles of the Earth. Information regarding microbial community composition can be very useful for environmental monitoring since the short generation times of microorganisms allows them to respond rapidly to changing environmental conditions. Microbial mediated attenuation of toxic chemicals offers great potential for the restoration of contaminated environments in an ecologically acceptable manner. Current knowledge regarding the structure and functional activities of microbial communities is limited, but more information is being acquired every day through many genomic- and proteomic- based methods. As of today, only a small fraction of the Earth's microorganisms has been cultured, and so most of the information regarding the biodegradation and therapeutic potentials of these uncultured microorganisms remains unknown. Sequence analysis of DNA and/or RNA has been used for identifying specific microorganisms, to study the community composition, and to monitor gene expression providing limited information about metabolic state of given microbial system. Proteomic studies can reveal information regarding the real-time metabolic state of the microbial communities thereby aiding in understanding their interaction with the environment. In research described here the involvement of microbial communities in the degradation of anthropogenic contaminants such as trichloroethylene (TCE) was studied using mass spectrometry-based proteomics. The co- metabolic degradation of TCE in the groundwater of the Snake River Plain Aquifer at the Test Area North (TAN) site of Idaho National Laboratory (INL) was monitored by the characterization of peptide sequences of enzymes such as methane monooxygenases (MMOs). MMOs, expressed by methanotrophic bacteria are involved in the oxidation of methane and non-specific co-metabolic oxidation of TCE. We developed a time- course cell lysis method to release proteins from complex microbial

  15. [Development of the Saxon Health Target "Active aging - aging in health, autonomy, and participation"].

    PubMed

    Brockow, T; Schulze, J; Fürst, F; Sawatzki, R; Wegge, J; Kliegel, M; Zwingenberger, W; Thönges, B; Eberhard, C; Resch, K-L

    2009-07-01

    In Saxony, the consequences of demographic aging are observable already today. To manage the implications on the health sector, the Saxon Health Targets Steering Committee decided in March 2008 to develop a health target "Active Aging - Aging in Health, Autonomy, and Participation". Target development was based on a 7-level approach (fields of action, main goals, target areas, targets, strategies, intervention measures, indicators for evaluation). A quantitative content analysis was used to reveal 10 potential relevant fields of action, three of which were selected for target development. Targets were developed by 53 stakeholders in multiprofessional working groups. Criteria-based analyses were performed to assure appropriate scientific evidence and feasibility of targets and intervention measures. Over a period of 9 months, 24 targets were defined referring to the main goals "needs-based health care structures", "multiprofessional qualification", "self-rated health" and "intergenerational solidarity". Thirteen targets were developed into recommendations for specific intervention measures. Most of the proposed interventions aim to modify health-related structures or psychosocial determinants of health in the elderly. The best recommendations for intervention measures shall be implemented in cooperation with interested decision-makers. PMID:19565198

  16. Small activating RNA binds to the genomic target site in a seed-region-dependent manner

    PubMed Central

    Meng, Xing; Jiang, Qian; Chang, Nannan; Wang, Xiaoxia; Liu, Chujun; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2016-01-01

    RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition. PMID:26873922

  17. Downstream System for the Second Axis of the DARHT Facility

    SciTech Connect

    Chen, Y-J; Bertolini, L; Caporaso, G J; Chambers, F W; Cook, E G; Falabella, S; Goldin, F J; Guethlein, G; Ho, D D-M; McCarrick, J F; Nelson, S D; Neurath, R; Paul, A C; Pincosy, P A; Poole, B R; Richardson, R A; Sampayan, S; Wang, L-F; Watson, J A; Westenskow, G A; Weir, J T

    2002-07-15

    This paper presents the physics design of the DARHT-II downstream system, which consists of a diagnostic beam stop, a fast, high-precision kicker system and the x-ray converter target assembly. The beamline configuration, the transverse resistive wall instability and the ion hose instability modeling are presented. They also discuss elimination of spot size dilution during kicker switching and implementation of the foil-barrier scheme to minimize the backstreaming ion focusing effects. Finally, they present the target converter's configuration, and the simulated DARHT-II x-ray spot sizes and doses. Some experimental results, which support the physics design, are also presented.

  18. Identification of novel target genes specifically activated by deregulated E2F in human normal fibroblasts.

    PubMed

    Kitamura, Hodaka; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P; Okuno, Junko; Shimizu, Emi; Kurayoshi, Kenta; Kugawa, Kazuyuki; Toh, Hiroyuki; Ohtani, Kiyoshi

    2015-09-01

    The transcription factor E2F is the principal target of the tumor suppressor pRB. E2F plays crucial roles not only in cell proliferation by activating growth-related genes but also in tumor suppression by activating pro-apoptotic and growth-suppressive genes. We previously reported that, in human normal fibroblasts, the tumor suppressor genes ARF, p27(Kip1) and TAp73 are activated by deregulated E2F activity induced by forced inactivation of pRB, but not by physiological E2F activity induced by growth stimulation. In contrast, growth-related E2F targets are activated by both E2F activities, underscoring the roles of deregulated E2F in tumor suppression in the context of dysfunctional pRB. In this study, to further understand the roles of deregulated E2F, we explored new targets that are specifically activated by deregulated E2F using DNA microarray. The analysis identified nine novel targets (BIM, RASSF1, PPP1R13B, JMY, MOAP1, RBM38, ABTB1, RBBP4 and RBBP7), many of which are involved in the p53 and RB tumor suppressor pathways. Among these genes, the BIM gene was shown to be activated via atypical E2F-responsive promoter elements and to contribute to E2F1-mediated apoptosis. Our results underscore crucial roles of deregulated E2F in growth suppression to counteract loss of pRB function. PMID:26201719

  19. Drug target identification using network analysis: Taking active components in Sini decoction as an example

    PubMed Central

    Chen, Si; Jiang, Hailong; Cao, Yan; Wang, Yun; Hu, Ziheng; Zhu, Zhenyu; Chai, Yifeng

    2016-01-01

    Identifying the molecular targets for the beneficial effects of active small-molecule compounds simultaneously is an important and currently unmet challenge. In this study, we firstly proposed network analysis by integrating data from network pharmacology and metabolomics to identify targets of active components in sini decoction (SND) simultaneously against heart failure. To begin with, 48 potential active components in SND against heart failure were predicted by serum pharmacochemistry, text mining and similarity match. Then, we employed network pharmacology including text mining and molecular docking to identify the potential targets of these components. The key enriched processes, pathways and related diseases of these target proteins were analyzed by STRING database. At last, network analysis was conducted to identify most possible targets of components in SND. Among the 25 targets predicted by network analysis, tumor necrosis factor α (TNF-α) was firstly experimentally validated in molecular and cellular level. Results indicated that hypaconitine, mesaconitine, higenamine and quercetin in SND can directly bind to TNF-α, reduce the TNF-α-mediated cytotoxicity on L929 cells and exert anti-myocardial cell apoptosis effects. We envisage that network analysis will also be useful in target identification of a bioactive compound. PMID:27095146

  20. Simulation of a Novel Active Target for Neutron-Unbound State Measurements

    NASA Astrophysics Data System (ADS)

    Frank, Nathan; MoNA Collaboration

    2013-10-01

    Measurement of nuclei at extreme ratios of protons to neutrons is challenging due to the low production rate. New facilities will increase the production of neutron-rich isotopes, but still not reach the neutron dripline for heavier nuclei. We simulated a carbon-based active target system that could be constructed to both increase statistics while preserving the experimental resolution. This simulation is an adaptation of the in-house MoNA Collaboration C + + based simulation tool to extract the decay energy of neutron-unbound states. A number of experiments of this type have been carried out at the National Superconducting Cyclotron Laboratory (NSCL). In most experiments, we produce neutron-unbound nuclei by bombarding a Beryllium target with a radioactive beam. The nucleus of interest immediately decays into a charged particle and one or more neutrons. In this simulation, we have constructed a carbon-based active target that provides a measurement of energy loss, which is used to calculate the nuclear interaction point within the target. This additional information is used to improve the resolution or preserve the resolution of a thinner target while increasing statistics. This presentation will cover some aspects of the simulation process as well as show a resolution improvement of up to about 4 with a ~700 mg/cm2 active target compared to a Be-target. The simulation utilized experimental settings from published work. Work supported by National Science Foundation Grant #0969173.

  1. Drug target identification using network analysis: Taking active components in Sini decoction as an example.

    PubMed

    Chen, Si; Jiang, Hailong; Cao, Yan; Wang, Yun; Hu, Ziheng; Zhu, Zhenyu; Chai, Yifeng

    2016-01-01

    Identifying the molecular targets for the beneficial effects of active small-molecule compounds simultaneously is an important and currently unmet challenge. In this study, we firstly proposed network analysis by integrating data from network pharmacology and metabolomics to identify targets of active components in sini decoction (SND) simultaneously against heart failure. To begin with, 48 potential active components in SND against heart failure were predicted by serum pharmacochemistry, text mining and similarity match. Then, we employed network pharmacology including text mining and molecular docking to identify the potential targets of these components. The key enriched processes, pathways and related diseases of these target proteins were analyzed by STRING database. At last, network analysis was conducted to identify most possible targets of components in SND. Among the 25 targets predicted by network analysis, tumor necrosis factor α (TNF-α) was firstly experimentally validated in molecular and cellular level. Results indicated that hypaconitine, mesaconitine, higenamine and quercetin in SND can directly bind to TNF-α, reduce the TNF-α-mediated cytotoxicity on L929 cells and exert anti-myocardial cell apoptosis effects. We envisage that network analysis will also be useful in target identification of a bioactive compound. PMID:27095146

  2. Human gaze stabilization during natural activities: translation, rotation, magnification, and target distance effects.

    PubMed

    Crane, B T; Demer, J L

    1997-10-01

    Stability of images on the retina was determined in 14 normal humans in response to rotational and translational perturbations during self-generated pitch and yaw, standing, walking, and running on a treadmill. The effects on image stability of target distance, vision, and spectacle magnification were examined. During locomotion the horizontal and vertical velocity of images on the retina was <4 degrees /s for a visible target located beyond 4 m. Image velocity significantly increased to >4 degrees /s during self-generated motion. For all conditions of standing and locomotion, angular vestibulo-ocular reflex (AVOR) gain was less than unity and varied significantly by activity, by target distance, and among subjects. There was no significant correlation(P > 0.05) between AVOR gain and image stability during standing and walking despite significant variation among subjects. This lack of correlation is likely due to translation of the orbit. The degree of orbital translation and rotation varied significantly with activity and viewing condition in a manner suggesting an active role in gaze stabilization. Orbital translation was consistently antiphase with rotation at predominant frequencies <4 Hz. When orbital translation was neglected in computing gaze, computed image velocities increased. The compensatory effect of orbital translation allows gaze stabilization despite subunity AVOR gain during natural activities. Orbital translation decreased during close target viewing, whereas orbital rotation decreased while wearing telescopic spectacles. As the earth fixed target was moved closer, image velocity on the retina significantly increased (P < 0.05) for all activities except standing. Latency of the AVOR increased slightly with decreasing target distance but remained <10 ms for even the closest target. This latency was similar in darkness or light, indicating that the visual pursuit tracking is probably not important in gaze stabilization. Trials with a distant target

  3. SAGA is an essential in vivo target of the yeast acidic activator Gal4p

    PubMed Central

    Bhaumik, Sukesh R.; Green, Michael R.

    2001-01-01

    Despite major advances in characterizing the eukaryotic transcriptional machinery, the function of promoter-specific transcriptional activators (activators) is still not understood. For example, in no case have the direct in vivo targets of a transcriptional activator been unambiguously identified, nor has it been resolved whether activators have a single essential target or multiple redundant targets. Here we address these issues for the prototype acidic activator yeast Gal4p. Gal4p binds to the upstream activating sequence (UAS) of GAL1 and several other GAL genes and stimulates transcription in the presence of galactose. Previous studies have shown that GAL1 transcription is dependent on the yeast SAGA (Spt/Ada/GCN5/acetyltransferase) complex. Using formaldehyde-based in vivo cross-linking, we show that the Gal4p activation domain recruits SAGA to the GAL1 UAS. If SAGA is not recruited to the UAS, the preinitiation complex (PIC) fails to assemble at the GAL1 core promoter, and transcription does not occur. SAGA, but not other transcription components, is also recruited by the Gal4p activation domain to a plasmid containing minimal Gal4p-binding sites. Recruitment of SAGA by Gal4p and stimulation of PIC assembly is dependent on several SAGA subunits but not the SAGA histone acetyl-transferase (HAT) GCN5. Based on these and other results, we conclude that SAGA is an essential target of Gal4p that, following recruitment to the UAS, facilitates PIC assembly and transcription. PMID:11485988

  4. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2014-04-01 2014-04-01 false Skeet, trap, target, and similar shooting activities. 478.35 Section 478.35 Alcohol, Tobacco Products, and Firearms BUREAU OF... similar shooting activities. Licensing and recordkeeping requirements, including permissible...

  5. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2011-04-01 2010-04-01 true Skeet, trap, target, and similar shooting activities. 478.35 Section 478.35 Alcohol, Tobacco Products, and Firearms BUREAU OF... similar shooting activities. Licensing and recordkeeping requirements, including permissible...

  6. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2013-04-01 2013-04-01 false Skeet, trap, target, and similar shooting activities. 478.35 Section 478.35 Alcohol, Tobacco Products, and Firearms BUREAU OF... similar shooting activities. Licensing and recordkeeping requirements, including permissible...

  7. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2012-04-01 2010-04-01 true Skeet, trap, target, and similar shooting activities. 478.35 Section 478.35 Alcohol, Tobacco Products, and Firearms BUREAU OF... similar shooting activities. Licensing and recordkeeping requirements, including permissible...

  8. 27 CFR 478.35 - Skeet, trap, target, and similar shooting activities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 3 2010-04-01 2010-04-01 false Skeet, trap, target, and similar shooting activities. 478.35 Section 478.35 Alcohol, Tobacco Products, and Firearms BUREAU OF... similar shooting activities. Licensing and recordkeeping requirements, including permissible...

  9. Early kinetic window of target T cell susceptibility to CD25+ regulatory T cell activity.

    PubMed

    Sojka, Dorothy K; Hughson, Angela; Sukiennicki, Teresa L; Fowell, Deborah J

    2005-12-01

    Peripheral tolerance is maintained in part by thymically derived CD25+CD4+ T cells (regulatory T cells (Tregs)). Their mechanism of action has not been well characterized. Therefore, to get a better understanding of Treg action, we investigated the kinetics of murine Treg activity in vitro. Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient only in the first 6-10 h of culture. Visualization of this time frame, using a sensitive single-cell assay for IL-2, revealed the early elaboration of target cell IL-2 producers in the first 6 h despite the presence of CD25+CD4+ Tregs. However, after 6 h, a rapid rise in the number of IL-2 producers in the absence of Tregs was dramatically abrogated by the presence of Tregs. Importantly, the timing of suppression was dictated by the kinetics of target T cell activation suggesting that early target T cell signals may alter susceptibility to suppression. Modulating target T cell activation signals with provision of CD28, IL-2, or high Ag dose all abrogated suppression of proliferation late in culture. However, only CD28 signals enabled target T cells to resist the early Treg-induced down-regulation of IL-2. Therefore the quality of early target T cell activation signals, in particular engagement of CD28, represents an important control point in the balance between vulnerability and resistance to Treg suppression. PMID:16301632

  10. Pitchfork and Gprasp2 Target Smoothened to the Primary Cilium for Hedgehog Pathway Activation.

    PubMed

    Jung, Bomi; Padula, Daniela; Burtscher, Ingo; Landerer, Cedric; Lutter, Dominik; Theis, Fabian; Messias, Ana C; Geerlof, Arie; Sattler, Michael; Kremmer, Elisabeth; Boldt, Karsten; Ueffing, Marius; Lickert, Heiko

    2016-01-01

    The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation. PMID:26901434

  11. Pitchfork and Gprasp2 Target Smoothened to the Primary Cilium for Hedgehog Pathway Activation

    PubMed Central

    Jung, Bomi; Padula, Daniela; Burtscher, Ingo; Landerer, Cedric; Lutter, Dominik; Theis, Fabian; Messias, Ana C.; Geerlof, Arie; Sattler, Michael; Kremmer, Elisabeth; Boldt, Karsten; Ueffing, Marius; Lickert, Heiko

    2016-01-01

    The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation. PMID:26901434

  12. EM23, a natural sesquiterpene lactone, targets thioredoxin reductase to activate JNK and cell death pathways in human cervical cancer cells

    PubMed Central

    Chen, Wen-Bo; Wang, Guo-Cai; Ma, Dong-Lei; Wong, Nai Sum; Xiao, Hao; Liu, Qiu-Ying; Zhou, Guang-Xiong; Li, Yao-Lan; Li, Man-Mei; Wang, Yi-Fei; Liu, Zhong

    2016-01-01

    Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants and found to have potential anticancer activities. However, the intracellular molecular targets of SLs and the underlying molecular mechanisms have not been well elucidated. In this study, we observed that EM23, a natural SL, exhibited anti-cancer activity in human cervical cancer cell lines by inducing apoptosis as indicated by caspase 3 activation, XIAP downregulation and mitochondrial dysfunction. Mechanistic studies indicated that EM23-induced apoptosis was mediated by reactive oxygen species (ROS) and the knockdown of thioredoxin (Trx) or thioredoxin reductase (TrxR) resulted in a reduction in apoptosis. EM23 attenuated TrxR activity by alkylation of C-terminal redox-active site Sec498 of TrxR and inhibited the expression levels of Trx/TrxR to facilitate ROS accumulation. Furthermore, inhibition of Trx/TrxR system resulted in the dissociation of ASK1 from Trx and the downstream activation of JNK. Pretreatment with ASK1/JNK inhibitors partially rescued cells from EM23-induced apoptosis. Additionally, EM23 inhibited Akt/mTOR pathway and induced autophagy, which was observed to be proapoptotic and mediated by ROS. Together, these results reveal a potential molecular mechanism for the apoptotic induction observed with SL compound EM23, and emphasize its putative role as a therapeutic agent for human cervical cancer. PMID:26758418

  13. EM23, a natural sesquiterpene lactone, targets thioredoxin reductase to activate JNK and cell death pathways in human cervical cancer cells.

    PubMed

    Shao, Fang-Yuan; Wang, Sheng; Li, Hong-Yu; Chen, Wen-Bo; Wang, Guo-Cai; Ma, Dong-Lei; Wong, Nai Sum; Xiao, Hao; Liu, Qiu-Ying; Zhou, Guang-Xiong; Li, Yao-Lan; Li, Man-Mei; Wang, Yi-Fei; Liu, Zhong

    2016-02-01

    Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants and found to have potential anticancer activities. However, the intracellular molecular targets of SLs and the underlying molecular mechanisms have not been well elucidated. In this study, we observed that EM23, a natural SL, exhibited anti-cancer activity in human cervical cancer cell lines by inducing apoptosis as indicated by caspase 3 activation, XIAP downregulation and mitochondrial dysfunction. Mechanistic studies indicated that EM23-induced apoptosis was mediated by reactive oxygen species (ROS) and the knockdown of thioredoxin (Trx) or thioredoxin reductase (TrxR) resulted in a reduction in apoptosis. EM23 attenuated TrxR activity by alkylation of C-terminal redox-active site Sec498 of TrxR and inhibited the expression levels of Trx/TrxR to facilitate ROS accumulation. Furthermore, inhibition of Trx/TrxR system resulted in the dissociation of ASK1 from Trx and the downstream activation of JNK. Pretreatment with ASK1/JNK inhibitors partially rescued cells from EM23-induced apoptosis. Additionally, EM23 inhibited Akt/mTOR pathway and induced autophagy, which was observed to be proapoptotic and mediated by ROS. Together, these results reveal a potential molecular mechanism for the apoptotic induction observed with SL compound EM23, and emphasize its putative role as a therapeutic agent for human cervical cancer. PMID:26758418

  14. Folate-conjugated polymer micelles for active targeting to cancer cells: preparation, in vitro evaluation of targeting ability and cytotoxicity

    NASA Astrophysics Data System (ADS)

    You, Jian; Li, Xin; de Cui, Fu; Du, Yong-Zhong; Yuan, Hong; Hu, Fu qiang

    2008-01-01

    To obtain an active-targeting carrier to cancer cells, folate-conjugated stearic acid grafted chitosan oligosaccharide (Fa-CSOSA) was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. The substitution degree is 22.1%. The critical micelle concentrations (CMCs) of Fa-CSOSA were 0.017 and 0.0074 mg ml-1 in distilled water and PBS (pH 7.4), respectively. The average volume size range of Fa-CSOSA micelles was 60-120 nm. The targeting ability of Fa-CSOSA micelles was investigated against two kinds of cell lines (A549 and Hela), which have different amounts of folate receptors in their surface. The results indicated that Fa-CSOSA micelles presented a targeting ability to the cells (Hela) with a higher expression of folate receptor during a short-time incubation (<6 h). As incubation proceeded, the special spatial structure of the micelles gradually plays a main role in cellular internalization of the micelles. Good internalization of the micelles into both Hela and A549 cells was shown. Then, paclitaxel (PTX) was encapsulated into the micelles, and the content of PTX in the micelles was about 4.8% (w/w). The average volume size range of PTX-loaded micelles was 150-340 nm. Furthermore, the anti-tumor efficacy in vitro was investigated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method. The IC50 of Taxol (a clinical formulation containing PTX) on A549 and Hela cells was 7.0 and 11.0 µg ml-1, respectively. The cytotoxicity of PTX-loaded micelles was improved sharply (IC50 on A549: 0.32 µg ml-1 IC50 on Hela: 0.268 µg ml-1). This is attributed to the increased intracellular delivery of the drug. The Fa-CSOSA micelles that are presented may be a promising active-targeting carrier candidate via folate mediation.

  15. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9.

    PubMed

    Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

    2015-06-01

    Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations. PMID:25985872

  16. Targeting CXCR1/2 Significantly Reduces Breast Cancer Stem Cell Activity and Increases the Efficacy of Inhibiting HER2 via HER2-dependent and -independent Mechanisms

    PubMed Central

    Singh, Jagdeep K.; Farnie, Gillian; Bundred, Nigel J.; Simões, Bruno M; Shergill, Amrita; Landberg, Göran; Howell, Sacha; Clarke, Robert B.

    2012-01-01

    Purpose Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are predicted to be responsible for tumour initiation, maintenance and metastases. Interleukin-8 (IL-8) is upregulated in breast cancer and associated with poor prognosis. Breast cancer cell line studies indicate that IL-8 via its cognate receptors, CXCR1 and CXCR2, is important in regulating breast CSC activity. We investigated the role of IL-8 in the regulation of CSC activity using patient-derived breast cancers and determined the potential benefit of combining CXCR1/2 inhibition with HER2-targeted therapy. Experimental design CSC activity of metastatic and invasive human breast cancers (n=19) was assessed ex vivo using the mammosphere colony forming assay. Results Metastatic fluid IL-8 level correlated directly with mammosphere formation (r=0.652; P<0.05; n=10). Recombinant IL-8 directly increased mammosphere formation/self-renewal in metastatic and invasive breast cancers (n=17). IL-8 induced activation of EGFR/HER2 and downstream signalling pathways and effects were abrogated by inhibition of SRC, EGFR/HER2, PI3K or MEK. Furthermore, lapatinib inhibited the mammosphere-promoting effect of IL-8 in both HER2-positive and negative patient-derived cancers. CXCR1/2 inhibition also blocked the effect of IL-8 on mammosphere formation and added to the efficacy of lapatinib in HER2-positive cancers. Conclusions These studies establish a role for IL-8 in the regulation of patient-derived breast CSC activity and demonstrate that IL-8/CXCR1/2 signalling is partly mediated via a novel SRC and EGFR/HER2-dependent pathway. Combining CXCR1/2 inhibitors with current HER2-targeted therapies has potential as an effective therapeutic strategy to reduce CSC activity in breast cancer and improve the survival of HER2-positive patients. PMID:23149820

  17. A human tRNA synthetase is a potent PARP1-activating effector target for resveratrol.

    PubMed

    Sajish, Mathew; Schimmel, Paul

    2015-03-19

    Resveratrol is reported to extend lifespan and provide cardio-neuro-protective, anti-diabetic, and anti-cancer effects by initiating a stress response that induces survival genes. Because human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS) translocates to the nucleus under stress conditions, we considered the possibility that the tyrosine-like phenolic ring of resveratrol might fit into the active site pocket to effect a nuclear role. Here we present a 2.1 Å co-crystal structure of resveratrol bound to the active site of TyrRS. Resveratrol nullifies the catalytic activity and redirects TyrRS to a nuclear function, stimulating NAD(+)-dependent auto-poly-ADP-ribosylation of poly(ADP-ribose) polymerase 1 (PARP1). Downstream activation of key stress signalling pathways are causally connected to TyrRS-PARP1-NAD(+) collaboration. This collaboration is also demonstrated in the mouse, and is specifically blocked in vivo by a resveratrol-displacing tyrosyl adenylate analogue. In contrast to functionally diverse tRNA synthetase catalytic nulls created by alternative splicing events that ablate active sites, here a non-spliced TyrRS catalytic null reveals a new PARP1- and NAD(+)-dependent dimension to the physiological mechanism of resveratrol. PMID:25533949

  18. Pharmacological Targeting of AMP-Activated Protein Kinase and Opportunities for Computer-Aided Drug Design.

    PubMed

    Miglianico, Marie; Nicolaes, Gerry A F; Neumann, Dietbert

    2016-04-14

    As a central regulator of metabolism, the AMP-activated protein kinase (AMPK) is an established therapeutic target for metabolic diseases. Beyond the metabolic area, the number of medical fields that involve AMPK grows continuously, expanding the potential applications for AMPK modulators. Even though indirect AMPK activators are used in the clinics for their beneficial metabolic outcome, the few described direct agonists all failed to reach the market to date, which leaves options open for novel targeting methods. As AMPK is not actually a single molecule and has different roles depending on its isoform composition, the opportunity for isoform-specific targeting has notably come forward, but the currently available modulators fall short of expectations. In this review, we argue that with the amount of available structural and ligand data, computer-based drug design offers a number of opportunities to undertake novel and isoform-specific targeting of AMPK. PMID:26510622

  19. Profiling of Multiple Targets of Artemisinin Activated by Hemin in Cancer Cell Proteome.

    PubMed

    Zhou, Yiqing; Li, Weichao; Xiao, Youli

    2016-04-15

    The antimalarial drug artemisinin is found to have diverse biological activities ranging from anti-inflammatory to anticancer properties; however, as of today, the cellular targets and mechanism of action of this important compound have remained elusive. Here, we report the global protein target profiling of artemisinin in the HeLa cancer cell proteome using a chemical proteomics approach. In the presence of hemin, multiple proteins were targeted by artemisinin probe through covalent modification. Further studies revealed that reducing of hemin to heme by protein thiols was essential for endoperoxide activation and subsequent protein alkylation. Artemisinin may exert its synergistic therapeutic anticancer effects via modulation of a variety of cellular pathways through acting on multiple targets. PMID:26854499

  20. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets.

    PubMed

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  1. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    PubMed Central

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein–protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  2. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

    PubMed Central

    Bialek, Julia K.; Dunay, Gábor A.; Voges, Maike; Schäfer, Carola; Spohn, Michael; Stucka, Rolf; Hauber, Joachim; Lange, Ulrike C.

    2016-01-01

    CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5’ long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination. PMID:27341108

  3. Target interaction profiling of midostaurin and its metabolites in neoplastic mast cells predicts distinct effects on activation and growth

    PubMed Central

    Peter, Barbara; Winter, Georg E.; Blatt, Katharina; Bennett, Keiryn L.; Stefanzl, Gabriele; Rix, Uwe; Eisenwort, Gregor; Hadzijusufovic, Emir; Gridling, Manuela; Dutreix, Catherine; Hoermann, Gregor; Schwaab, Juliana; Radia, Deepti; Roesel, Johannes; Manley, Paul W.; Reiter, Andreas; Superti-Furga, Giulio; Valent, Peter

    2016-01-01

    Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multi-kinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction-profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC50 values. Midostaurin and CGP62221 also produced growth-inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, that accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug-competition experiments revealed that midostaurin interacts with KIT and several additional kinase-targets. The key downstream-regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE-receptor-downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs. PMID:26349526

  4. Target interaction profiling of midostaurin and its metabolites in neoplastic mast cells predicts distinct effects on activation and growth.

    PubMed

    Peter, B; Winter, G E; Blatt, K; Bennett, K L; Stefanzl, G; Rix, U; Eisenwort, G; Hadzijusufovic, E; Gridling, M; Dutreix, C; Hoermann, G; Schwaab, J; Radia, D; Roesel, J; Manley, P W; Reiter, A; Superti-Furga, G; Valent, P

    2016-02-01

    Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multikinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC(50) values. Midostaurin and CGP62221 also produced growth inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, which accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug competition experiments revealed that midostaurin interacts with KIT and several additional kinase targets. The key downstream regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE receptor downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation, which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs. PMID:26349526

  5. Coupling of downstream RNA polymerase-promoter interactions with formation of catalytically competent transcription initiation complex

    PubMed Central

    Mekler, Vladimir; Minakhin, Leonid; Borukhov, Sergei; Mustaev, Arkady; Severinov, Konstantin

    2014-01-01

    Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT-rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis. PMID:25311862

  6. Targeting the RAS pathway by mitogen-activated protein kinase inhibitors.

    PubMed

    Kiessling, Michael K; Rogler, Gerhard

    2015-01-01

    Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer. PMID:26691679

  7. Targeting, Endocytosis, and Lysosomal Delivery of Active Enzymes to Model Human Neurons by ICAM-1-Targeted Nanocarriers

    PubMed Central

    Hsu, Janet; Hoenicka, Janet; Muro, Silvia

    2016-01-01

    Purpose Delivery of therapeutics to neurons is paramount to treat neurological conditions, including many lysosomal storage disorders. However, key aspects of drug-carrier behavior in neurons are relatively unknown: the occurrence of non-canonical endocytic pathways (present in other cells); whether carriers that traverse the blood-brain barrier are, contrarily, retained within neurons; if neuron-surface receptors are accessible to bulky carriers compared to small ligands; or if there are differences regarding neuronal compartments (neuron body vs. neurites) pertaining said parameters. We have explored these questions using model polymer nanocarriers targeting intercellular adhesion molecule-1 (ICAM-1). Methods Differentiated human neuroblastoma cells were incubated with anti-ICAM-coated polystyrene nanocarriers and analyzed by fluorescence microscopy. Results ICAM-1 expression and nanocarrier binding was enhanced in altered (TNFα) vs. control conditions. While small ICAM-1 ligands (anti-ICAM) preferentially accessed the cell body, anti-ICAM nanocarriers bound with faster kinetics to neurites, yet reached similar saturation over time. Anti-ICAM nanocarriers were also endocytosed with faster kinetics and lower saturation levels in neurites. Non-classical cell adhesion molecule (CAM) endocytosis ruled uptake, and neurite-to-cell body transport was inferred. Nanocarriers trafficked to lysosomes, delivering active enzymes (dextranase) with substrate reduction in a lysosomal-storage disease model. Conclusion ICAM-1-targeting holds potential for intracellular delivery of therapeutics to neurons. PMID:25319100

  8. Active radar guides missile to its target: receptor-based targeted treatment of hepatocellular carcinoma by nanoparticulate systems.

    PubMed

    Yan, Jing-Jun; Liao, Jia-Zhi; Lin, Ju-Sheng; He, Xing-Xing

    2015-01-01

    Patients with hepatocellular carcinoma (HCC) usually present at advanced stages and do not benefit from surgical resection, so drug therapy should deserve a prominent place in unresectable HCC treatment. But chemotherapy agents, such as doxorubicin, cisplatin, and paclitaxel, frequently encounter important problems such as low specificity and non-selective biodistribution. Recently, the development of nanotechnology led to significant breakthroughs to overcome these problems. Decorating the surfaces of nanoparticulate-based drug carriers with homing devices has demonstrated its potential in concentrating chemotherapy agents specifically to HCC cells. In this paper, we reviewed the current status of active targeting strategies for nanoparticulate systems based on various receptors such as asialoglycoprotein receptor, transferrin receptor, epidermal growth factor receptor, folate receptor, integrin, and CD44, which are abundantly expressed on the surfaces of hepatocytes or liver cancer cells. Furthermore, we pointed out their merits and defects and provided theoretical references for further research. PMID:25424700

  9. Extracellularly activated nanocarriers: A new paradigm of tumor targeted drug delivery

    PubMed Central

    Gullotti, Emily; Yeo, Yoon

    2009-01-01

    One of the main goals of nanomedicine is to develop a nanocarrier that can selectively deliver anti-cancer drugs to the targeted tumors. Extensive efforts have resulted in several tumor-targeted nanocarriers, some of which are approved for clinical use. Most nanocarriers achieve tumor-selective accumulation through the enhanced permeability and retention effect. Targeting molecules such as antibodies, peptides, ligands, or nucleic acids attached to the nanocarriers further enhance their recognition and internalization by the target tissues. While both the stealth and targeting features are important for effective and selective drug delivery to the tumors, achieving both features simultaneously is often found to be difficult. Some of the recent targeting strategies have the potential to overcome this challenge. These strategies utilize the unique extracellular environment of tumors to change the long-circulating nanocarriers to release the drug or interact with cells in a tumor-specific manner. This review discusses the new targeting strategies with recent examples, which utilize the environmental stimuli to activate the nanocarriers. Traditional strategies for tumor-targeted nanocarriers are briefly discussed with an emphasis on their achievements and challenges. PMID:19366234

  10. Developing, implementing, and evaluating a condom promotion program targeting sexually active adolescents.

    PubMed

    Alstead, M; Campsmith, M; Halley, C S; Hartfield, K; Goldbaum, G; Wood, R W

    1999-12-01

    This article describes the development, implementation, and evaluation of the Condom Campaign, a 1995 HIV prevention program promoting condom use among sexually active adolescents in three King County, Washington, urban communities. This program employed three main strategies: (a) mobilizing all levels of the target communities to support and guide program development and implementation; (b) creating and implementing a mass media campaign targeting sexually active teenagers that promoted correct condom use and favorable attitudes toward condoms; and (c) recruiting public agencies, community organizations, and businesses to distribute condoms from bins and vending machines. We evaluated the program through a series of cross-sectional interviews conducted in the three communities chosen for their elevated levels of adolescent sexual risk behavior. Overall, 73% of target youth reported exposure to the Condom Campaign; exposure did not differ by age, gender, race, or level of sexual experience. Levels of sexual activity remained stable throughout the media campaign. PMID:10693646

  11. Prazosin displays anticancer activity against human prostate cancers: targeting DNA and cell cycle.

    PubMed

    Lin, Ssu-Chia; Chueh, Shih-Chieh; Hsiao, Che-Jen; Li, Tsia-Kun; Chen, Tzu-Hsuan; Liao, Cho-Hwa; Lyu, Ping-Chiang; Guh, Jih-Hwa

    2007-10-01

    Quinazoline-based alpha1-adrenoceptor antagonists, in particular doxazosin and terazosin, are suggested to display antineoplastic activity against prostate cancers. However, there are few studies elucidating the effect of prazosin. In this study, prazosin displayed antiproliferative activity superior to that of other alpha1-blockers, including doxazosin, terazosin, tamsulosin, and phentolamine. Prazosin induced G2 checkpoint arrest and subsequent apoptosis in prostate cancer PC-3, DU-145, and LNCaP cells. In p53-null PC-3 cells, prazosin induced an increase in DNA strand breaks and ATM/ATR checkpoint pathways, leading to the activation of downstream signaling cascades, including Cdc25c phosphorylation at Ser216, nuclear export of Cdc25c, and cyclin-dependent kinase (Cdk) 1 phosphorylation at Tyr15. The data, together with sustained elevated cyclin A levels (other than cyclin B1 levels), suggested that Cdk1 activity was inactivated by prazosin. Moreover, prazosin triggered mitochondria-mediated and caspase-executed apoptotic pathways in PC-3 cells. The oral administration of prazosin significantly reduced tumor mass in PC-3-derived cancer xenografts in nude mice. In summary, we suggest that prazosin is a potential antitumor agent that induces cell apoptosis through the induction of DNA damage stress, leading to Cdk1 inactivation and G2 checkpoint arrest. Subsequently, mitochondria-mediated caspase cascades are triggered to induce apoptosis in PC-3 cells. PMID:17971903

  12. IκB Kinase ε Targets Interferon Regulatory Factor 1 in Activated T Lymphocytes

    PubMed Central

    Sgarbanti, Marco; Marsili, Giulia; Remoli, Anna Lisa; Stellacci, Emilia; Mai, Antonello; Rotili, Dante; Perrotti, Edvige; Acchioni, Chiara; Orsatti, Roberto; Iraci, Nunzio; Ferrari, Mathieu; Borsetti, Alessandra

    2014-01-01

    IκB kinase ε (IKK-ε) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present study, we investigated IKK-ε activation following T cell receptor (TCR)/CD28 stimulation of primary CD4+ T cells and its role in the stimulation of a type I IFN response. IKK-ε was activated following TCR/CD28 stimulation of primary CD4+ T cells; however, in T cells treated with poly(I·C), TCR/CD28 costimulation blocked induction of IFN-β transcription. We demonstrated that IKK-ε phosphorylated the transcription factor IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4+ T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-κB RelA subunit and interfered with PCAF-mediated acetylation of NF-κB RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-ε activation, which in turn contributes to suppression of IFN-β production. PMID:24396068

  13. Histone acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo

    PubMed Central

    Kuo, Min-Hao; Zhou, Jianxin; Jambeck, Per; Churchill, Mair E.A.; Allis, C. David

    1998-01-01

    Gcn5p is a transcriptional coactivator required for correct expression of various genes in yeast. Several transcriptional regulators, including Gcn5p, possess intrinsic histone acetyltransferase (HAT) activity in vitro. However, whether the HAT activity of any of these proteins is required for gene activation remains unclear. Here, we demonstrate that the HAT activity of Gcn5p is critical for transcriptional activation of target genes in vivo. Core histones are hyperacetylated in cells overproducing functional Gcn5p, and promoters of Gcn5p-regulated genes are associated with hyperacetylated histones upon activation by low-copy Gcn5p. Point mutations within the Gcn5p catalytic domain abolish both promoter-directed histone acetylation and Gcn5p-mediated transcriptional activation. These data provide the first in vivo evidence that promoter-specific histone acetylation, catalyzed by functional Gcn5p, plays a critical role in gene activation. PMID:9499399

  14. AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication.

    PubMed

    Weber, Nicholas D; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S; Roychoudhury, Pavitra; Schiffer, Joshua T; Aubert, Martine; Jerome, Keith R

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  15. AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

    PubMed Central

    Weber, Nicholas D.; Stone, Daniel; Sedlak, Ruth Hall; De Silva Feelixge, Harshana S.; Roychoudhury, Pavitra; Schiffer, Joshua T.; Aubert, Martine; Jerome, Keith R.

    2014-01-01

    Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy. PMID:24827459

  16. Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo.

    PubMed

    Morelli, E; Leone, E; Cantafio, M E Gallo; Di Martino, M T; Amodio, N; Biamonte, L; Gullà, A; Foresta, U; Pitari, M R; Botta, C; Rossi, M; Neri, A; Munshi, N C; Anderson, K C; Tagliaferri, P; Tassone, P

    2015-11-01

    Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA (miR)-125b in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells in vitro. Apoptotic and autophagy-associated cell death were triggered in MM cells on miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in severe combined immunodeficient/non-obese diabetic mice induced significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor-suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials. PMID:25987254

  17. Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo

    PubMed Central

    Morelli, E; Leone, E; Cantafio, M E Gallo; Di Martino, M T; Amodio, N; Biamonte, L; Gullà, A; Foresta, U; Pitari, M R; Botta, C; Rossi, M; Neri, A; Munshi, N C; Anderson, K C; Tagliaferri, P; Tassone, P

    2015-01-01

    Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA (miR)-125b in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells in vitro. Apoptotic and autophagy-associated cell death were triggered in MM cells on miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in severe combined immunodeficient/non-obese diabetic mice induced significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor-suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials. PMID:25987254

  18. Observation of Children's Physical Activity Levels in Primary School: Is the School an Ideal Setting for Meeting Government Activity Targets?

    ERIC Educational Resources Information Center

    Waring, Michael; Warburton, Peter; Coy, Martin

    2007-01-01

    Given the commitment (and funding) by the British government to promote physical activity among all ages, and despite the inevitable political manipulation of physical education (PE) and school sport, there is now an ideal opportunity to focus on primary schools as a key target group for the future. This study determined the physical activity…

  19. IMPACT OF TARGET MATERIAL ACTIVATION ON PERSONNEL EXPOSURE AND RADIOACTIVE CONTAMINATION IN THE NATIONAL IGNITION FACILITY

    SciTech Connect

    Khater, H; Epperson, P; Thacker, R; Beale, R; Kohut, T; Brereton, S

    2009-06-30

    Detailed activation analyses are performed for the different materials under consideration for use in the target capsules and hohlraums used during the ignition campaign on the National Ignition Facility. Results of the target material activation were additionally used to estimate the levels of contamination within the NIF target chamber and the workplace controls necessary for safe operation. The analysis examined the impact of using Be-Cu and Ge-doped CH capsules on the external dose received by workers during maintenance activities. Five days following a 20 MJ shot, dose rates inside the Target Chamber (TC) due to the two proposed capsule materials are small ({approx} 1 {micro}rem/h). Gold and depleted-uranium (DU) are considered as potential hohlraum materials. Following a shot, gold will most probably get deposited on the TC first wall. On the other hand, while noble-gas precursors from the DU are expected to stay in the TC, most of the noble gases are pumped out of the chamber and end up on the cryopumps. The dose rates inside the TC due to activated gold or DU, at 5 days following a 20 MJ shot, are about 1 mrem/h. Dose rates in the vicinity of the cryo-pumps (containing noble 'fission' gases) drop-off to about 1 mrem/h during the first 12 hours following the shot. Contamination from activation of NIF targets will result in the NIF target chamber exceeding DOE surface contamination limits. Objects removed from the TC will need to be managed as radioactive material. However, the results suggest that airborne contamination from resuspension of surface contamination will not be significant and is at levels that can be managed by negative ventilation when accessing the TC attachments.

  20. Modeling downstream fining in sand-bed rivers. II: Application

    USGS Publications Warehouse

    Wright, S.; Parker, G.

    2005-01-01

    In this paper the model presented in the companion paper, Wright and Parker (2005) is applied to a generic river reach typical of a large, sand-bed river flowing into the ocean in order to investigate the mechanisms controlling longitudinal profile development and downstream fining. Three mechanisms which drive downstream fining are studied: a delta prograding into standing water, sea-level rise, and tectonic subsidence. Various rates of sea-level rise (typical of the late Holocene) and tectonic subsidence are modeled in order to quantify their effects on the degree of profile concavity and downstream fining. Also, several other physical mechanisms which may affect fining are studied, including the relative importance of the suspended versus bed load, the effect of the loss of sediment overbank, and the influence of the delta bottom slope. Finally, sensitivity analysis is used to show that the grain-size distribution at the interface between the active layer and substrate has a significant effect on downstream fining. ?? 2005 International Association of Hydraulic Engineering and Research.

  1. Activation of extracellular regulated kinase and mechanistic target of rapamycin pathway in focal cortical dysplasia.

    PubMed

    Patil, Vinit V; Guzman, Miguel; Carter, Angela N; Rathore, Geetanjali; Yoshor, Daniel; Curry, Daniel; Wilfong, Angus; Agadi, Satish; Swann, John W; Adesina, Adekunle M; Bhattacharjee, Meenakshi B; Anderson, Anne E

    2016-04-01

    Neuropathology of resected brain tissue has revealed an association of focal cortical dysplasia (FCD) with drug-resistant epilepsy (DRE). Recent studies have shown that the mechanistic target of rapamycin (mTOR) pathway is hyperactivated in FCD as evidenced by increased phosphorylation of the ribosomal protein S6 (S6) at serine 240/244 (S(240/244) ), a downstream target of mTOR. Moreover, extracellular regulated kinase (ERK) has been shown to phosphorylate S6 at serine 235/236 (S(235/236) ) and tuberous sclerosis complex 2 (TSC2) at serine 664 (S(664) ) leading to hyperactive mTOR signaling. We evaluated ERK phosphorylation of S6 and TSC2 in two types of FCD (FCD I and FCD II) as a candidate mechanism contributing to mTOR pathway dysregulation. Tissue samples from patients with tuberous sclerosis (TS) served as a positive control. Immunostaining for phospho-S6 (pS6(240/244) and pS6(235/236) ), phospho-ERK (pERK), and phospho-TSC2 (pTSC2) was performed on resected brain tissue with FCD and TS. We found increased pS6(240/244) and pS6(235/236) staining in FCD I, FCD II and TS compared to normal-appearing tissue, while pERK and pTSC2 staining was increased only in FCD IIb and TS tissue. Our results suggest that both the ERK and mTOR pathways are dysregulated in FCD and TS; however, the signaling alterations are different for FCD I as compared to FCD II and TS. PMID:26381727

  2. Activity and radiation protection studies for the W-Ta target of CSNS.

    PubMed

    Yu, Q Z; Liang, T J; Yin, W

    2009-09-01

    The Chinese government initiated a conceptual design for the project of China Spallation Neutron Source (CSNS), which consists of an H-linear accelerator, a rapid cycling synchrotron accelerating the beam to 1.6 GeV, a target station converting proton beam into lower energy (<1 eV) neutron beam optimised to instruments for neutron scattering applications. The facility operates at 25-Hz repetition rate with an initial beam power of 100 kW. In the target station, the target-moderator-reflector (TMR) components are exposed to the intensive fluxes of high-energy hadrons and become highly radioactive as a result of long-time irradiation. In this paper, the activity of the TMR components are calculated using the Monte Carlo code system LAHET&MCNP4C&CINDER'90. Comparisons of some results with that simulated by FLUKA code are also performed. Detailed analyses of the radionuclides and their characters in the tantalum clad tungsten target (W-Ta target) are important for the radiation protection of the CSNS target station. The shielding design of the service cell for the decay gamma ray induced from the W-Ta target and its vessel shows that the ambient dose rate decreases exponentially with increasing heavy concrete thickness. And 80 cm thickness of heavy concrete for each side of the service cell can satisfy the safety requirement. PMID:19770213

  3. Active target studies of the αp-process at CRIB

    NASA Astrophysics Data System (ADS)

    Kahl, D.; Hashimoto, T.; Duy, N. N.; Kubono, S.; Yamaguchi, H.; Binh, D. N.; Chen, A. A.; Cherubini, S.; Hayakawa, S.; He, J. J.; Ishiyama, H.; Iwasa, N.; Khiem, L. H.; Kwon, Y. K.; Michimasa, S.; Nakao, T.; Ota, S.; Teranishi, T.; Tokieda, H.; Wakabayashi, Y.; Yamada, T.; Zhang, L. Y.

    2014-05-01

    The αp-process is a sequence of (α, p)(p, γ) reactions important to the nuclear trajectory to higher masses in type I X-ray bursts. Specifically, the αp-process is schematically pure helium-burning, and thus unlike pure hydrogen-burning processes, does not require slow β+ decays. Explosive helium burning is responsible for the observed short rise-times of X-ray bursts but ultimately gives way to the rp-process as the Coulomb barrier increases. Because the stellar reaction rates of these (α, p) reactions are poorly known over the relevant astrophysical energies, we performed systematic studies of the 18Ne(α,p), 22Mg(α,p) and 30S(α,p) reactions at the Center for Nuclear Study (CNS) low-energy radioactive ion beam separator, called CRIB. We produce the radioactive beams in-flight and scan the center-of-mass energy down into the Gamow Window using a thick target in inverse kinematics. The helium target gas also serves as part of the detector system, an active target, which was newly designed for these measurements. The active target, which uses gas electron multiplier (GEM) foils, allows for higher beam injection rates than previous multi-sampling and tracking proportional counters (MSTPC). We present a summary of our recent results from these active target experiments at CRIB.

  4. Remote Bridge Deflection Measurement Using an Advanced Video Deflectometer and Actively Illuminated LED Targets.

    PubMed

    Tian, Long; Pan, Bing

    2016-01-01

    An advanced video deflectometer using actively illuminated LED targets is proposed for remote, real-time measurement of bridge deflection. The system configuration, fundamental principles, and measuring procedures of the video deflectometer are first described. To address the challenge of remote and accurate deflection measurement of large engineering structures without being affected by ambient light, the novel idea of active imaging, which combines high-brightness monochromatic LED targets with coupled bandpass filter imaging, is introduced. Then, to examine the measurement accuracy of the proposed advanced video deflectometer in outdoor environments, vertical motions of an LED target with precisely-controlled translations were measured and compared with prescribed values. Finally, by tracking six LED targets mounted on the bridge, the developed video deflectometer was applied for field, remote, and multipoint deflection measurement of the Wuhan Yangtze River Bridge, one of the most prestigious and most publicized constructions in China, during its routine safety evaluation tests. Since the proposed video deflectometer using actively illuminated LED targets offers prominent merits of remote, contactless, real-time, and multipoint deflection measurement with strong robustness against ambient light changes, it has great potential in the routine safety evaluation of various bridges and other large-scale engineering structures. PMID:27563901

  5. Active target studies of the αp-process at CRIB

    SciTech Connect

    Kahl, D.; Yamaguchi, H.; Michimasa, S.; Nakao, T.; Ota, S.; Tokieda, H.; Hashimoto, T.; Duy, N. N.; Khiem, L. H.; Kubono, S.; Binh, D. N.; Chen, A. A.; Cherubini, S.; Hayakawa, S.; He, J. J.; Zhang, L. Y.; Ishiyama, H.; Iwasa, N.; Yamada, T.; Kwon, Y. K.; and others

    2014-05-02

    The αp-process is a sequence of (α, p)(p, γ) reactions important to the nuclear trajectory to higher masses in type I X-ray bursts. Specifically, the αp-process is schematically pure helium-burning, and thus unlike pure hydrogen-burning processes, does not require slow β{sup +} decays. Explosive helium burning is responsible for the observed short rise-times of X-ray bursts but ultimately gives way to the rp-process as the Coulomb barrier increases. Because the stellar reaction rates of these (α, p) reactions are poorly known over the relevant astrophysical energies, we performed systematic studies of the {sup 18}Ne(α,p), {sup 22}Mg(α,p) and {sup 30}S(α,p) reactions at the Center for Nuclear Study (CNS) low-energy radioactive ion beam separator, called CRIB. We produce the radioactive beams in-flight and scan the center-of-mass energy down into the Gamow Window using a thick target in inverse kinematics. The helium target gas also serves as part of the detector system, an active target, which was newly designed for these measurements. The active target, which uses gas electron multiplier (GEM) foils, allows for higher beam injection rates than previous multi-sampling and tracking proportional counters (MSTPC). We present a summary of our recent results from these active target experiments at CRIB.

  6. The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

    PubMed Central

    Shen, Hong Ming; Poirier, Michael G.; Allen, Michael J.; North, Justin; Lal, Ratnesh; Widom, Jonathan

    2009-01-01

    The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes. PMID:19380635

  7. Light-controlled active release of photocaged ciprofloxacin for lipopolysaccharide-targeted drug delivery using dendrimer conjugates.

    PubMed

    Wong, Pamela T; Tang, Shengzhuang; Mukherjee, Jhindan; Tang, Kenny; Gam, Kristina; Isham, Danielle; Murat, Claire; Sun, Rachel; Baker, James R; Choi, Seok Ki

    2016-08-16

    We report an active delivery mechanism targeted specifically to Gram(-) bacteria based on the photochemical release of photocaged ciprofloxacin carried by a cell wall-targeted dendrimer nanoconjugate. PMID:27476878

  8. The Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway as a Discovery Target in Stroke.

    PubMed

    Sun, Jing; Nan, Guangxian

    2016-05-01

    Protein kinases are critical modulators of a variety of intracellular and extracellular signal transduction pathways, and abnormal phosphorylation events can contribute to disease progression in a variety of diseases. As a result, protein kinases have emerged as important new drug targets for small molecule therapeutics. The mitogen-activated protein kinase (MAPK) signaling pathway transmits signals from the cell membrane to the nucleus in response to a variety of different stimuli. Because this pathway controls a broad spectrum of cellular processes, including growth, inflammation, and stress responses, it is accepted as a therapeutic target for cancer and peripheral inflammatory disorders. There is also increasing evidence that MAPK is an important regulator of ischemic and hemorrhagic cerebral vascular disease, raising the possibility that it might be a drug discovery target for stroke. In this review, we discuss the MAPK signaling pathway in association with its activation in stroke-induced brain injury. PMID:26842916

  9. A dual active-restrictive approach to incorporating environmental flow targets into existing reservoir operation rules

    NASA Astrophysics Data System (ADS)

    Shiau, Jenq-Tzong; Wu, Fu-Chun

    2010-08-01

    Environmental flow schemes may be implemented through active or restrictive strategies. The former may be applied via reservoir releases, and the latter can be executed by reducing water demands. We present a dual active-restrictive approach to devising the optimal reservoir operation rules that aim to secure off-stream water supplies while maximizing environmental benefits. For the active part, a multicomponent environmental flow target (including the minimum and monthly flows) is incorporated in the operation rules. For the restrictive counterpart, we use a novel demands partitioning and prioritizing (DPP) approach to reallocating the demands of various sectors. The DPP approach partitions the existing off-stream demand and newly incorporated environmental demand and reassembles the two as the first- and second-priority demands. Water is reallocated to each demand according to the ratios derived from the prioritized demands. The proposed approach is coupled with a multicriteria optimization framework to seek the optimal operation rules for the existing Feitsui Reservoir system (Taiwan) under various scenarios. The best overall performance is achieved by an optimal dual strategy whose operational parameters are all determined by optimization. The optimal environmental flow target may well be a top-priority constant base flow rather than the variable quantities. The active strategy would outperform the restrictive one. For the former, a top-priority base flow target is essential; for the latter, the off-stream demand can become vanishingly small in compensation for the eliminated base flow target, thus promoting the monthly flow target as nearly the top-priority demand. For either the active or restrictive strategy, a prioritized environmental flow demand would provide a path toward the optimal overall performance. A significantly improved overall performance over the existing operation rules is unlikely if the active and restrictive parameters are both favorable

  10. A Network-Based Multi-Target Computational Estimation Scheme for Anticoagulant Activities of Compounds

    PubMed Central

    Li, Canghai; Chen, Lirong; Song, Jun; Tang, Yalin; Xu, Xiaojie

    2011-01-01

    Background Traditional virtual screening method pays more attention on predicted binding affinity between drug molecule and target related to a certain disease instead of phenotypic data of drug molecule against disease system, as is often less effective on discovery of the drug which is used to treat many types of complex diseases. Virtual screening against a complex disease by general network estimation has become feasible with the development of network biology and system biology. More effective methods of computational estimation for the whole efficacy of a compound in a complex disease system are needed, given the distinct weightiness of the different target in a biological process and the standpoint that partial inhibition of several targets can be more efficient than the complete inhibition of a single target. Methodology We developed a novel approach by integrating the affinity predictions from multi-target docking studies with biological network efficiency analysis to estimate the anticoagulant activities of compounds. From results of network efficiency calculation for human clotting cascade, factor Xa and thrombin were identified as the two most fragile enzymes, while the catalytic reaction mediated by complex IXa:VIIIa and the formation of the complex VIIIa:IXa were recognized as the two most fragile biological matter in the human clotting cascade system. Furthermore, the method which combined network efficiency with molecular docking scores was applied to estimate the anticoagulant activities of a serial of argatroban intermediates and eight natural products respectively. The better correlation (r = 0.671) between the experimental data and the decrease of the network deficiency suggests that the approach could be a promising computational systems biology tool to aid identification of anticoagulant activities of compounds in drug discovery. Conclusions This article proposes a network-based multi-target computational estimation method for

  11. Profiling Hepatic microRNAs in Zebrafish: Fluoxetine Exposure Mimics a Fasting Response That Targets AMP-Activated Protein Kinase (AMPK)

    PubMed Central

    Craig, Paul M.; Trudeau, Vance L.; Moon, Thomas W.

    2014-01-01

    This study examined the similarities in microRNA profiles between fasted and fluoxetine (FLX) exposed zebrafish and downstream target transcripts and biological pathways. Using a custom designed microarray targeting 270 zebrafish miRNAs, we identified 9 differentially expressed miRNAs targeting transcripts in biological pathways associated with anabolic metabolism, such as adipogenesis, cholesterol biosynthesis, triacylglycerol synthesis, and insulin signaling. Exposure of female zebrafish to 540 ng/L FLX, an environmentally relevant concentration and a known metabolic repressor, increased specific miRNAs indicating greater inhibition of these pathways in spite of continued feeding. Further examination revealed two specific miRNAs, dre-let-7d and dre-miR-140-5p, were predicted in silico to bind to a primary regulator of metabolism, adenosine monophosphate-activated protein kinase (AMPK), and more specifically the two isoforms of the catalytic subunit, AMPKα1 and α2, respectively. Real-time analysis of the relative transcript abundance of the α1 and α2 mRNAs indicated a significant inverse relationship between specific miRNA and target transcript. This suggests that AMPK-related pathways may be compromised during FLX exposure as a result of increased miRNA abundance. The mechanism by which FLX regulates miRNA abundance is unknown but may be direct at the liver. The serotonin transporter, slc6a4, is the target of FLX and other selective serotonin reuptake inhibitors (SSRI) and it was found to be expressed in the liver, although treatment did not alter expression of this transporter. Exposure to FLX disrupts key hepatic metabolic pathways, which may be indicative of reduced overall fitness and these effects may be linked to specific miRNA abundance. This has important implications for the heath of fish because concentrations of SSRIs in aquatic ecosystems are continually increasing. PMID:24751937

  12. The Role of Specificity, Targeted Learning Activities, and Prior Knowledge for the Effects of Relevance Instructions

    ERIC Educational Resources Information Center

    Roelle, Julian; Lehmkuhl, Nina; Beyer, Martin-Uwe; Berthold, Kirsten

    2015-01-01

    In 2 experiments we examined the role of (a) specificity, (b) the type of targeted learning activities, and (c) learners' prior knowledge for the effects of relevance instructions on learning from instructional explanations. In Experiment 1, we recruited novices regarding the topic of atomic structure (N = 80) and found that "specific"…

  13. REDV Peptide Conjugated Nanoparticles/pZNF580 Complexes for Actively Targeting Human Vascular Endothelial Cells.

    PubMed

    Shi, Changcan; Li, Qian; Zhang, Wencheng; Feng, Yakai; Ren, Xiangkui

    2015-09-16

    Herein, we demonstrate that the REDV peptide modified nanoparticles (NPs) can serve as a kind of active targeting gene carrier to condensate pZNF580 for specific promotion of the proliferation of endothelial cells (ECs). First, we synthesized a series of biodegradable amphiphilic copolymers by ring-opening polymerization reaction and graft modification with REDV peptide. Second, we prepared active targeting NPs via self-assembly of the amphiphilic copolymers using nanoprecipitation technology. After condensation with negatively charged pZNF580, the REDV peptide modified NPs/pZNF580 complexes were formed finally. Due to the binding affinity toward ECs of the specific peptide, these REDV peptide modified NPs/pZNF580 complexes could be recognized and adhered specifically by ECs in the coculture system of ECs and human artery smooth muscle cells (SMCs) in vitro. After expression of ZNF580, as the key protein to promote the proliferation of ECs, the relative ZNF580 protein level increased from 15.7% to 34.8%. The specificity in actively targeting ECs of the REDV peptide conjugated NPs/pZNF580 complexes was still retained in the coculture system. These findings in the present study could facilitate the development of actively targeting gene carriers for the endothelialization of artificial blood vessels. PMID:26373583

  14. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNT) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family ...

  15. The chromatin remodelling factor Brg-1 interacts with β-catenin to promote target gene activation

    PubMed Central

    Barker, Nick; Hurlstone, Adam; Musisi, Hannah; Miles, Antony; Bienz, Mariann; Clevers, Hans

    2001-01-01

    Wnt-induced formation of nuclear Tcf–β-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of β-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. Here we demonstrate an interaction between β-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodelling complexes. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, we observe genetic interactions between the Brg-1 and β-catenin homologues in flies. We conclude that β-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodelling as a prerequisite for transcriptional activation. PMID:11532957

  16. How to Target Activated Ras Proteins: Direct Inhibition vs. Induced Mislocalization.

    PubMed

    Brock, Ethan J; Ji, Kyungmin; Reiners, John J; Mattingly, Raymond R

    2016-01-01

    Oncogenic Ras proteins are a driving force in a significant set of human cancers and wildtype, unmutated Ras proteins likely contribute to the malignant phenotype of many more. The overall challenge of targeting activated Ras proteins has great promise to treat cancer, but this goal has yet to be achieved. Significant efforts and resources have been committed to inhibiting Ras, but these energies have so far made little impact in the clinic. Direct attempts to target activated Ras proteins have faced many obstacles, including the fundamental nature of the gain-of-function oncogenic activity being produced by a loss-of-function at the biochemical level. Nevertheless, there has been very promising recent pre-clinical progress. The major strategy that has so far reached the clinic aimed to inhibit activated Ras indirectly through blocking its post-translational modification and inducing its mislocalization. While these efforts to indirectly target Ras through inhibition of farnesyl transferase (FTase) were rationally designed, this strategy suffered from insufficient attention to the distinctions between the isoforms of Ras. This led to subsequent failures in large-scale clinical trials targeting K-Ras driven lung, colon, and pancreatic cancers. Despite these setbacks, efforts to indirectly target activated Ras through inducing its mislocalization have persisted. It is plausible that FTase inhibitors may still have some utility in the clinic, perhaps in combination with statins or other agents. Alternative approaches for inducing mislocalization of Ras through disruption of its palmitoylation cycle or interaction with chaperone proteins are in early stages of development. PMID:26423696

  17. Preclinical Activity of ARQ 087, a Novel Inhibitor Targeting FGFR Dysregulation.

    PubMed

    Hall, Terence G; Yu, Yi; Eathiraj, Sudharshan; Wang, Yunxia; Savage, Ronald E; Lapierre, Jean-Marc; Schwartz, Brian; Abbadessa, Giovanni

    2016-01-01

    Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920). PMID:27627808

  18. Targeted delivery of CCR2 antagonist to activated pulmonary endothelium prevents metastasis.

    PubMed

    Roblek, Marko; Calin, Manuela; Schlesinger, Martin; Stan, Daniela; Zeisig, Reiner; Simionescu, Maya; Bendas, Gerd; Borsig, Lubor

    2015-12-28

    Enhanced levels of the inflammatory chemokine CCL2 are known to correlate with increased tumorigenesis and metastases, and thereby poor prognosis for cancer patients. The CCL2-CCR2 chemokine axis was shown to facilitate the metastatic initiation through the recruitment of inflammatory monocytes and the activation of endothelial cells at metastatic sites. Both steps are required for efficient cancer cell trans-endothelial migration and seeding in the targeted tissue. The translation of preclinical evidence proved to be challenging due to systemic effects of chemokine inhibition and limited target specificity. Here we tested an approach of a targeted delivery of the CCR2 antagonist Teijin Compound 1 to metastatic sites. VCAM-1 binding peptide tagged liposomes carrying the CCR2 antagonist enabled a specific delivery to cancer cell-activated endothelium. The subsequent binding of target-sensitive liposomes triggered the release of the Teijin Compound 1 and thereby local inhibition of CCR2 in the lungs. Blocking of CCR2 resulted in reduced induction of the lungs vascular permeability, and thereby reduced tumor cell extravasation. However, the recruitment of inflammatory monocytes to the pre-metastatic lungs remained unaltered. Endothelial VCAM-1 targeted delivery of the CCR2 antagonist resulted in inhibition of pulmonary metastases both in a murine (MC-38GFP cells) and a human xenograft (patient-derived cells) model. Thus, timely- and spatially-defined inhibition of CCR2 signaling represents a potential therapeutic approach for treatment of metastasis without affecting homeostatic functions. PMID:26522070

  19. Mechanistic Target of Rapamycin Complex 1/S6 Kinase 1 Signals Influence T Cell Activation Independently of Ribosomal Protein S6 Phosphorylation

    PubMed Central

    Salmond, Robert J.; Brownlie, Rebecca J.; Meyuhas, Oded

    2015-01-01

    Ag-dependent activation of naive T cells induces dramatic changes in cellular metabolism that are essential for cell growth, division, and differentiation. In recent years, the serine/threonine kinase mechanistic target of rapamycin (mTOR) has emerged as a key integrator of signaling pathways that regulate these metabolic processes. However, the role of specific downstream effectors of mTOR function in T cells is poorly understood. Ribosomal protein S6 (rpS6) is an essential component of the ribosome and is inducibly phosphorylated following mTOR activation in eukaryotic cells. In the current work, we addressed the role of phosphorylation of rpS6 as an effector of mTOR function in T cell development, growth, proliferation, and differentiation using knockin and TCR transgenic mice. Surprisingly, we demonstrate that rpS6 phosphorylation is not required for any of these processes either in vitro or in vivo. Indeed, rpS6 knockin mice are completely sensitive to the inhibitory effects of rapamycin and an S6 kinase 1 (S6K1)–specific inhibitor on T cell activation and proliferation. These results place the mTOR complex 1-S6K1 axis as a crucial determinant of T cell activation independently of its ability to regulate rpS6 phosphorylation. PMID:26453749

  20. Cost estimation for the active debris removal of multiple priority targets

    NASA Astrophysics Data System (ADS)

    Braun, Vitali; Wiedemann, Carsten; Schulz, Eugen

    The increasing number of space debris objects, especially in distinct low Earth orbit (LEO) altitudes between 600 and 1000 km, leads to an increase in the potential collision risk between the objects and threatens active satellites in that region. Several recent studies show that active debris removal (ADR) has to be performed in order to prevent a collisional cascading effect, also known as the Kessler syndrome. In order to stabilize the population growth in the critical LEO region, a removal of five prioritized objects per year has been recognized as a significant figure. Various proposals are addressing the technical issues for ADR missions, including the de-orbiting of objects by means of a service satellite using a chemical or an electric propulsion system. The servicer would rendezvous with a preselected target, perform a docking maneuver and then provide a de-orbit burn to transfer the target on a trajectory where it re-enters the Earth’s atmosphere within a given time frame. In this paper the technical aspects are complemented by a cost estimation model, focusing on multi target missions, which are based on a service satellite capable of de-orbiting more than one target within a single mission. The cost model for ADR includes initial development cost, production cost, launch cost and operation cost as well as the modelling of the propulsion system of the servicer. Therefore, different scenarios are defined for chemical and electric propulsion systems as applied to multi target missions, based on a literature review of concepts currently being under discussion. The costs of multi target missions are compared to a scenario where only one target is removed. Also, the results allow to determine an optimum number of objects to be removed per mission and provide numbers which can be used in future studies, e.g. those related to ADR cost and benefit analyses.

  1. Philippines' downstream sector poised for growth

    SciTech Connect

    Not Available

    1992-05-11

    This paper reports that the Philippines' downstream sector is poised for sharp growth. Despite a slip in refined products demand in recent years, Philippines products demand will rebound sharply by 2000, East-West Center (EWC), Honolulu, predicts. Philippines planned refinery expansions are expected to meet that added demand, EWC Director Fereidun Fesharaki says. Like the rest of the Asia-Pacific region, product specifications are changing, but major refiners in the area expect to meet the changes without major case outlays. At the same time, Fesharaki says, push toward deregulation will further bolster the outlook for the Philippines downstream sector.

  2. 40 CFR 745.226 - Certification of individuals and firms engaged in lead-based paint activities: target housing and...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Lead-Based Paint Activities § 745.226 Certification of individuals and firms engaged in lead-based paint activities: target housing... engaged in lead-based paint activities: target housing and child-occupied facilities. 745.226 Section...

  3. 40 CFR 745.226 - Certification of individuals and firms engaged in lead-based paint activities: target housing and...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Lead-Based Paint Activities § 745.226 Certification of individuals and firms engaged in lead-based paint activities: target housing... engaged in lead-based paint activities: target housing and child-occupied facilities. 745.226 Section...

  4. 40 CFR 745.226 - Certification of individuals and firms engaged in lead-based paint activities: target housing and...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Lead-Based Paint Activities § 745.226 Certification of individuals and firms engaged in lead-based paint activities: target housing... engaged in lead-based paint activities: target housing and child-occupied facilities. 745.226 Section...

  5. 40 CFR 745.226 - Certification of individuals and firms engaged in lead-based paint activities: target housing and...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Lead-Based Paint Activities § 745.226 Certification of individuals and firms engaged in lead-based paint activities: target housing... engaged in lead-based paint activities: target housing and child-occupied facilities. 745.226 Section...

  6. Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages.

    PubMed Central

    Büscher, D; Hipskind, R A; Krautwald, S; Reimann, T; Baccarini, M

    1995-01-01

    Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation. PMID:7799956

  7. Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells.

    PubMed

    Khalil, R A; Menice, C B; Wang, C L; Morgan, K G

    1995-06-01

    Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes. PMID:7538916

  8. Phishing for suitable targets in the Netherlands: routine activity theory and phishing victimization.

    PubMed

    Leukfeldt, E Rutger

    2014-08-01

    This article investigates phishing victims, especially the increased or decreased risk of victimization, using data from a cybercrime victim survey in the Netherlands (n=10,316). Routine activity theory provides the theoretical perspective. According to routine activity theory, several factors influence the risk of victimization. A multivariate analysis was conducted to assess which factors actually lead to increased risk of victimization. The model included background and financial data of victims, their Internet activities, and the degree to which they were "digitally accessible" to an offender. The analysis showed that personal background and financial characteristics play no role in phishing victimization. Among eight Internet activities, only "targeted browsing" led to increased risk. As for accessibility, using popular operating systems and web browsers does not lead to greater risk, while having up-to-date antivirus software as a technically capable guardian has no effect. The analysis showed no one, clearly defined group has an increased chance of becoming a victim. Target hardening may help, but opportunities for prevention campaigns aimed at a specific target group or dangerous online activities are limited. Therefore, situational crime prevention will have to come from a different angle. Banks could play the role of capable guardian. PMID:25080013

  9. Imaging Caspase-3 Activation as a Marker of Apoptosis-Targeted Treatment Response in Cancer

    PubMed Central

    Chen, Delphine L.; Engle, Jacquelyn T.; Griffin, Elizabeth A.; Miller, J. Philip; Chu, Wenhua; Zhou, Dong; Mach, Robert H.

    2016-01-01

    Purpose We tested whether positron emission tomography (PET) with the caspase-3 targeted isatin analog [18F]WC-4-116 could image caspase-3 activation in response to an apoptosis-inducing anticancer therapy. Procedures [18F]WC-4-116 uptake was determined in etoposide-treated EL4 cells. Biodistribution studies with [18F]WC-4-116 and [18F]ICMT-18, a non-caspase-3-targeted tracer, as well as [18F]WC-4-116 microPET imaging assessed responses in Colo205 tumor bearing mice treated with death receptor 5 (DR5) targeted agonist antibodies. Immunohistochemical staining and enzyme assays confirmed caspase-3 activation. Two-way analysis of variance or Student’s t-test assessed for treatment-related changes in tracer uptake. Results [18F]WC-4-116 increased 8 ± 2-fold in etoposide-treated cells. The [18F]WC-4-116 %ID/g also increased significantly in tumors with high caspase-3 enzyme activity (p < 0.05). [18F]ICMT-18 tumor uptake did not differ in tumors with high or low caspase-3 enzyme activity. Conclusions [18F]WC-4-116 uptake in vivo reflects increased caspase-3 activation and may be useful for detecting caspase-3 mediated apoptosis treatment responses in cancer. PMID:25344147

  10. Modeling and production of 240Am by deuteron-induced activation of a 240Pu target

    NASA Astrophysics Data System (ADS)

    Finn, Erin C.; McNamara, Bruce; Greenwood, Larry; Wittman, Richard; Soderquist, Charles; Woods, Vincent; VanDevender, Brent; Metz, Lori; Friese, Judah

    2015-04-01

    A novel reaction pathway for production of 240Am is reported. Models of reaction cross-sections in EMPIRE II suggest that deuteron-induced activation of a 240Pu target produces maximum yields of 240Am from 11.5 MeV incident deuterons. This activation had not been previously reported in the literature. A 240Pu target was activated under the modeled optimum conditions to produce 240Am. The modeled cross-section for the 240Pu(d, 2n)240Am reaction is on the order of 20-30 mbarn, but the experimentally estimated value is 5.6 ± 0.2 mbarn. We discuss reasons for the discrepancy as well as production of other Am isotopes that contaminate the final product.

  11. Modeling and production of 240Am by deuteron-induced activation of a 240Pu target

    SciTech Connect

    Finn, Erin C.; McNamara, Bruce K.; Greenwood, Lawrence R.; Wittman, Richard S.; Soderquist, Chuck Z.; Woods, Vincent T.; VanDevender, Brent A.; Metz, Lori A.; Friese, Judah I.

    2015-02-01

    A novel reaction pathway for production of 240Am is reported. Models of reaction cross-sections in EMPIRE II suggests that deuteron-induced activation of a 240Pu target produces maximum yields of 240Am from 11.5 MeV incident deuterons. This activation had not been previously reported in the literature. A 240Pu target was activated under the modeled optimum conditions to produce 240Am. The modeled cross-section for the 240Pu(d, 2n)240Am reaction is on the order of 20-30 mbarn, but the experimentally estimated value is 5.3 ± 0.2 mbarn. We discuss reasons for the discrepancy as well as production of other Am isotopes that contaminate the final product.

  12. Modulating antibiotic activity towards respiratory bacterial pathogens by co-medications: a multi-target approach.

    PubMed

    Vandevelde, Nathalie M; Tulkens, Paul M; Van Bambeke, Françoise

    2016-07-01

    Non-antibiotic drugs can modulate bacterial physiology and/or antibiotic activity, opening perspectives for innovative therapeutic strategies. Focusing on respiratory pathogens and considering in vitro, in vivo, and clinical data, here we examine the effect of these drugs on the expression of resistance mechanisms, biofilm formation, and intracellular survival, as well as their influence on the activity of antibiotics on bacteria. Beyond the description of the effects observed, we also comment on concentrations that are active and discuss the mechanisms of drug-drug or drug-target interactions. This discussion should be helpful in defining useful targets for adjuvant therapy and establishing the corresponding pharmacophores for further drug fine-tuning. PMID:27094105

  13. Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes.

    PubMed

    Adibekian, Alexander; Martin, Brent R; Chang, Jae Won; Hsu, Ku-Lung; Tsuboi, Katsunori; Bachovchin, Daniel A; Speers, Anna E; Brown, Steven J; Spicer, Timothy; Fernandez-Vega, Virneliz; Ferguson, Jill; Hodder, Peter S; Rosen, Hugh; Cravatt, Benjamin F

    2012-06-27

    The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo. PMID:22690931

  14. Confirming Target Engagement for Reversible Inhibitors In Vivo by Kinetically Tuned Activity-Based Probes

    PubMed Central

    Adibekian, Alexander; Martin, Brent R.; Chang, Jae Won; Hsu, Ku-Lung; Tsuboi, Katsunori; Bachovchin, Daniel A.; Speers, Anna E.; Brown, Steven J.; Spicer, Timothy; Fernandez-Vega, Virneliz; Ferguson, Jill; Hodder, Peter S.; Rosen, Hugh; Cravatt, Benjamin F.

    2012-01-01

    The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo. PMID:22690931

  15. Utilizing G2/M retention effect to enhance tumor accumulation of active targeting nanoparticles

    PubMed Central

    Hu, Guanlian; Cun, Xingli; Ruan, Shaobo; Shi, Kairong; Wang, Yang; Kuang, Qifang; Hu, Chuan; Xiao, Wei; He, Qin; Gao, Huile

    2016-01-01

    In recent years, active targeting strategies by ligand modification have emerged to enhance tumor accumulation of NP, but their clinical application was strictly restricted due to the complex preparation procedures, poor stability and serious toxicity. An effective and clinical translational strategy is required to satisfy the current problems. Interestingly, the internalization of NP is intimately related with cell cycle and the expression of receptors is not only related with cancer types but also cell cycle progression. So the cellular uptake of ligand modified NP may be related with cell cycle. However, few investigations were reported about the relationship between cell cycle and the internalization of ligand modified NP. Herein, cellular uptake of folic acid (FA) modified NP after utilizing chemotherapeutic to retain the tumor cells in G2/M phase was studied and a novel strategy was designed to enhance the active targeting effect. In our study, docetaxel (DTX) notably synchronized cells in G2/M phase and pretreatment with DTX highly improved in vitro and in vivo tumor cell targeting effect of FA decorated NP (FANP). Since FA was a most common used tumor active targeting ligand, we believe that this strategy possesses broader prospects in clinical application for its simplicity and effectiveness. PMID:27273770

  16. Peroxisome proliferator-activated receptor α, a potential therapeutic target for alcoholic liver disease

    PubMed Central

    Nan, Yue-Min; Wang, Rong-Qi; Fu, Na

    2014-01-01

    Alcoholic liver injury represents a progressive process with a range of consequences including hepatic steatosis, steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Targeting key molecular regulators involved in the development of alcoholic liver injury may be of great value in the prevention of liver injury. Peroxisome proliferator-activated receptor α (PPARα) plays a pivotal role in modulation of hepatic lipid metabolism, oxidative stress, inflammatory response and fibrogenesis. As such, PPARα may be a potential therapeutic target for the treatment of alcoholic liver disease. PMID:25009377

  17. Anti-complementary constituents of Houttuynia cordata and their targets in complement activation cascade.

    PubMed

    Jiang, Yun; Lu, Yan; Zhang, Yun-Yi; Chen, Dao-Feng

    2014-01-01

    Activity-guided fractionation for complement inhibitors led to the isolation of 23 known compounds from Houttuynia cordata Thunb. Seven flavonoids, two alkaloids, one coumarin and two phenols showed anti-complementary activity. Preliminary inhibitory mechanism of four flavonoids, including quercitrin, afzelin, isoquercitrin and quercetin in the complement activation cascade were examined for the first time. The results indicated that the target components of flavonols are different from those of flavonosides, and the glycoside moieties may be necessary to block C3 and C4 components. PMID:24423008

  18. Activation, Heating and Exposure Rates for Mo‐99 Experiments with 25‐Disk Targets

    SciTech Connect

    Kelsey, Charles T. IV

    2012-05-09

    , target housing weldment and target assembly), (2) Shielded box with everything in it except the target assembly, (3) Shielded box with nothing in it, (4) Target assembly taken outside of shielded box, (5) Target disks in cradle (target assembly with thermocouple weldment and flange removed), (6) Empty cradle, and (7) Target disks alone. Decay photon spectra from the CINDER2008 calculations were used as sources for the exposure rate calculations in the same model used for the flux calculations with beam on. As components were removed to simulate the seven cases considered the material compositions were changed to air and their respective sources were turned off. The MCNPX model geometry is plotted in Figure 1. The left and right detector locations for cases 1, 2 and 3 were 30 cm from the shielded box walls and 30 cm from the beam pipe openings in the left and right sides of the model (they are not in the beam line). A zoomed in plot of the target assembly alone is in Figure 2. Exposure rates for the seven cases are plotted as a function of time after irradiation in Figures 3, 4 and 5. To aid in comparison between the cases, all of these figures have been plotted using the same scale. Figures 3 and 4 are respectively the thermal and production test results for cases 1 through 6. Figure 5 includes case 7 results for both. Differences between cases 1 and 2 for both tests are not statistically significant showing that activation of components other than the target assembly, many of which are also shielding the target assembly, dominates exposure rates outside the shielded box. Case 3 shows the contribution from activation of the shield box itself. In front where shielded box wall is thickest box activation accounts for essentially all of the exposure rate outside. Differences between cases 4 and 5 are also minimal, showing that the contribution to target assembly exposure rates from the thermocouple flange and weldment are small compared to the target disks and cradle. From

  19. Maternal diabetes promotes mTORC1 downstream signalling in rabbit preimplantation embryos.

    PubMed

    Gürke, Jacqueline; Schindler, Maria; Pendzialek, S Mareike; Thieme, René; Grybel, Katarzyna J; Heller, Regine; Spengler, Katrin; Fleming, Tom P; Fischer, Bernd; Navarrete Santos, Anne

    2016-05-01

    The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets in in vitro cultured preimplantation rabbit blastocysts and in vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1; CCND1), translation initiation (eukaryotic translation initiation factor 4E; EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: gene SLC7A8) and proliferation (proliferating cell nuclear antigen; PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns of CCND1 and EIF4E were increased in embryos from diabetic rabbits, while the expression of proliferation marker PCNA was decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy. PMID:26836250

  20. Target cell death triggered by cytotoxic T lymphocytes: a target cell mutant distinguishes passive pore formation and active cell suicide mechanisms.

    PubMed Central

    Ucker, D S; Wilson, J D; Hebshi, L D

    1994-01-01

    The role of the target cell in its own death mediated by cytotoxic T lymphocytes (CTL) has been controversial. The ability of the pore-forming granule components of CTL to induce target cell death directly has been taken to suggest an essentially passive role for the target. This view of CTL-mediated killing ascribes to the target the single role of providing an antigenic stimulus to the CTL; this signal results in the vectoral degranulation and secretion of pore-forming elements onto the target. On the other hand, by a number of criteria, target cell death triggered by CTL appears fundamentally different from death resulting from membrane damage and osmotic lysis. CTL-triggered target cell death involves primary internal lesions of the target cell that reflect a physiological cell death process. Orderly nuclear disintegration, including lamin phosphorylation and solubilization, chromatin condensation, and genome digestion, are among the earliest events, preceding the loss of plasma membrane integrity. We have tested directly the involvement of the target cell in its own death by examining whether we could isolate mutants of target cells that have retained the ability to be recognized by and provide an antigenic stimulus to CTL while having lost the capacity to respond by dying. Here, we describe one such mutant, BW87. We have used this CTL-resistant mutant to analyze the mechanisms of CTL-triggered target cell death under a variety of conditions. The identification of a mutable target cell element essential for the cell death response to CTL provides genetic evidence that target cell death reflects an active cell suicide process similar to other physiological cell deaths. PMID:8264610

  1. Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase

    PubMed Central

    Ohya, Ken-ichi; Kajigaya, Sachiko; Kitanaka, Akira; Yoshida, Koji; Miyazato, Akira; Yamashita, Yoshihiro; Yamanaka, Takeo; Ikeda, Uichi; Shimada, Kazuyuki; Ozawa, Keiya; Mano, Hiroyuki

    1999-01-01

    Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (BCR downstream signaling 1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling. PMID:10518561

  2. Changing Neuroestrogens Within the Auditory Forebrain Rapidly Transform Stimulus Selectivity in a Downstream Sensorimotor Nucleus

    PubMed Central

    Remage-Healey, Luke; Joshi, Narendra R.

    2012-01-01

    The activity of sensory circuits is shaped by neuromodulators, which can have downstream consequences for both sensorimotor integration and behavioral output. Recent evidence indicates that brain-derived estrogens (‘neuroestrogens’) can act as local circuit modulators in the songbird auditory forebrain. Specifically, neuroestrogens fluctuate in the auditory caudomedial nidopallium (NCM) during social interactions and in response to song stimuli. Within minutes of elevation, neuroestrogens also enhance auditory response properties of NCM neurons, and acute blockade of estrogen production in NCM disrupts behavioral song discrimination. Here, we test the hypothesis that fluctuating neuroestrogens within NCM influence stimulus selectivity in a downstream sensorimotor nucleus (HVC) that receives indirect auditory input from NCM. Dual extracellular recordings coupled with retrodialysis delivery show that song-selectivity in HVC is rapidly enhanced by increasing neuroestrogens in NCM in adult males. Conversely, inhibiting neuroestrogen production in NCM causes a rapid decline in song-selectivity in HVC, demonstrating the endogenous nature of this modulatory network. By contrast, HVC selectivity is unaffected by neuroestrogen delivery to either nearby caudomedial mesopallium (CMM) or into HVC itself, indicating that neuroestrogen actions are restricted to NCM. In juvenile males, identical E2 treatment in NCM also does not alter HVC selectivity, consistent with a developmental maturation of the auditory network. Lastly, the rapid actions of estrogens leading to enhanced HVC selectivity appear to be mediated by membrane-bound receptors in NCM. These findings indicate that steroid-dependent modulation of sensory processing is not locally restricted and can be transmitted transynaptically to influence downstream sensorimotor and premotor targets. PMID:22699904

  3. Resistance of Cancer Cells to Targeted Therapies Through the Activation of Compensating Signaling Loops.

    PubMed

    von Manstein, Viktoria; Yang, Chul Min; Richter, Diane; Delis, Natalia; Vafaizadeh, Vida; Groner, Bernd

    2013-12-01

    The emergence of low molecular weight kinase inhibitors as "targeted" drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase

  4. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor

    PubMed Central

    Shin, Weon Sup; Han, Jiyou; Kumar, Rajesh; Lee, Gyung Gyu; Sessler, Jonathan L.; Kim, Jong-Hoon; Kim, Jong Seung

    2016-01-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38—a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic (“phototheranostic”). PMID:27374023

  5. Recent Developments in Active Tumor Targeted Multifunctional Nanoparticles for Combination Chemotherapy in Cancer Treatment and Imaging

    PubMed Central

    Glasgow, Micah D. K.; Chougule, Mahavir B.

    2016-01-01

    Nanotechnology and combination therapy are two major fields that show great promise in the treatment of cancer. The delivery of drugs via nanoparticles helps to improve drug’s therapeutic effectiveness while reducing adverse side effects associated with high dosage by improving their pharmacokinetics. Taking advantage of molecular markers over-expressing on tumor tissues compared to normal cells, an “active” molecular marker targeted approach would be beneficial for cancer therapy. These actively targeted nanoparticles would increase drug concentration at the tumor site, improving efficacy while further reducing chemo-resistance. The multidisciplinary approach may help to improve the overall efficacy in cancer therapy. This review article summarizes recent developments of targeted multifunctional nanoparticles in the delivery of various drugs for a combinational chemotherapy approach to cancer treatment and imaging. PMID:26554150

  6. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor.

    PubMed

    Shin, Weon Sup; Han, Jiyou; Kumar, Rajesh; Lee, Gyung Gyu; Sessler, Jonathan L; Kim, Jong-Hoon; Kim, Jong Seung

    2016-01-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38-a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic ("phototheranostic"). PMID:27374023

  7. A Covalent Cysteine-Targeting Kinase Inhibitor of Ire1 Permits Allosteric Control of Endoribonuclease Activity.

    PubMed

    Waller, Daniel D; Jansen, Gregor; Golizeh, Makan; Martel-Lorion, Chloe; Dejgaard, Kurt; Shiao, Tze Chieh; Mancuso, John; Tsantrizos, Youla S; Roy, René; Sebag, Michael; Sleno, Lekha; Thomas, David Y

    2016-05-01

    The unfolded protein response (UPR) initiated by the transmembrane kinase/ribonuclease Ire1 has been implicated in a variety of diseases. Ire1, with its unique position in the UPR, is an ideal target for the development of therapies; however, the identification of specific kinase inhibitors is challenging. Recently, the development of covalent inhibitors has gained great momentum because of the irreversible deactivation of the target. We identified and determined the mechanism of action of the Ire1-inhibitory compound UPRM8. MS analysis revealed that UPRM8 inhibition occurs by covalent adduct formation at a conserved cysteine at the regulatory DFG+2 position in the Ire1 kinase activation loop. Mutational analysis of the target cysteine residue identified both UPRM8-resistant and catalytically inactive Ire1 mutants. We describe a novel covalent inhibition mechanism of UPRM8, which can serve as a lead for the rational design and optimization of inhibitors of human Ire1. PMID:26792008

  8. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor

    NASA Astrophysics Data System (ADS)

    Shin, Weon Sup; Han, Jiyou; Kumar, Rajesh; Lee, Gyung Gyu; Sessler, Jonathan L.; Kim, Jong-Hoon; Kim, Jong Seung

    2016-07-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38—a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic (“phototheranostic”).

  9. Downstream prediction using a nonlinear prediction method

    NASA Astrophysics Data System (ADS)

    Adenan, N. H.; Noorani, M. S. M.

    2013-11-01

    The estimation of river flow is significantly related to the impact of urban hydrology, as this could provide information to solve important problems, such as flooding downstream. The nonlinear prediction method has been employed for analysis of four years of daily river flow data for the Langat River at Kajang, Malaysia, which is located in a downstream area. The nonlinear prediction method involves two steps; namely, the reconstruction of phase space and prediction. The reconstruction of phase space involves reconstruction from a single variable to the m-dimensional phase space in which the dimension m is based on optimal values from two methods: the correlation dimension method (Model I) and false nearest neighbour(s) (Model II). The selection of an appropriate method for selecting a combination of preliminary parameters, such as m, is important to provide an accurate prediction. From our investigation, we gather that via manipulation of the appropriate parameters for the reconstruction of the phase space, Model II provides better prediction results. In particular, we have used Model II together with the local linear prediction method to achieve the prediction results for the downstream area with a high correlation coefficient. In summary, the results show that Langat River in Kajang is chaotic, and, therefore, predictable using the nonlinear prediction method. Thus, the analysis and prediction of river flow in this area can provide river flow information to the proper authorities for the construction of flood control, particularly for the downstream area.

  10. Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling

    NASA Astrophysics Data System (ADS)

    Kondo, Yuki; Ito, Tasuku; Nakagami, Hirofumi; Hirakawa, Yuki; Saito, Masato; Tamaki, Takayuki; Shirasu, Ken; Fukuda, Hiroo

    2014-03-01

    During plant radial growth typically seen in trees, procambial and cambial cells act as meristematic cells in the vascular system to self-proliferate and differentiate into xylem cells. These two processes are regulated by a signalling pathway composed of a peptide ligand and its receptor; tracheary element differentiation inhibitory factor (TDIF) and TDIF RECEPTOR (TDR). Here we show that glycogen synthase kinase 3 proteins (GSK3s) are crucial downstream components of the TDIF signalling pathway suppressing xylem differentiation from procambial cells. TDR interacts with GSK3s at the plasma membrane and activates GSK3s in a TDIF-dependent fashion. Consistently, a specific inhibitor of plant GSK3s strongly induces xylem cell differentiation through BRI1-EMS SUPPRESSOR 1 (BES1), a well-known target transcription factor of GSK3s. Our findings provide insight into the regulation of cell fate determination in meristem maintenance.

  11. The retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector of estrogen action

    PubMed Central

    Abbondanza, Ciro; Medici, Nicola; Nigro, Vincenzo; Rossi, Valentina; Gallo, Luigi; Piluso, Giulio; Belsito, Angela; Roscigno, Annarita; Bontempo, Paola; Puca, Annibale A.; Molinari, Anna Maria; Moncharmont, Bruno; Puca, Giovanni A.

    2000-01-01

    Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co-activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced in vivo, in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control. PMID:10706618

  12. The retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector of estrogen action.

    PubMed

    Abbondanza, C; Medici, N; Nigro, V; Rossi, V; Gallo, L; Piluso, G; Belsito, A; Roscigno, A; Bontempo, P; Puca, A A; Molinari, A M; Moncharmont, B; Puca, G A

    2000-03-28

    Co-immunoprecipitation experiments in cell extract from cultured cells or target tissues indicated that estrogen receptor was complexed with the retinoblastoma binding protein RIZ in a ligand-dependent manner. Mapping of interaction sites indicated that in both proteins the same regions and motifs responsible for the interaction of transcriptional co-activator and nuclear receptors were involved. In cultured cells, estradiol induced a redistribution of RIZ protein within the nucleus and in the cytoplasm. A similar effect was produced in vivo, in prepuberal rat endometrium, by administration of a physiological dose of estradiol. Therefore, RIZ protein could be a specific effector of estrogen action downstream of the hormone-receptor interaction, presumably involved in proliferation control. PMID:10706618

  13. Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

    SciTech Connect

    Verkman, A.S.; Ausiello, D.A.; Jung, C.Y.; Skorecki, K.L.

    1986-08-12

    Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

  14. Microparticle Surface Modifications Targeting Dendritic Cells for Non-Activating Applications

    PubMed Central

    Lewis, Jamal S.; Zaveri, Toral D.; Crooks, Charles P.; Keselowsky, Benjamin G.

    2012-01-01

    Microparticulate systems for delivery of therapeutics to DCs for immunotherapy have gained attention recently. However, reports addressing the optimization of DC-targeting microparticle delivery systems are limited, particularly for cases where the goal is to deliver payload to DCs in a non-activating fashion. Here, we investigate targeting DCs using poly (d lactide-co-glycolide) microparticles (MPs) in a non-stimulatory manner and assess efficacy in vitro and in vivo. We modified MPs by surface immobilizing DC receptor targeting molecules – antibodies (anti-CD11c, anti-DEC-205) or peptides (P-D2, RGD), where anti-CD11c antibody, P-D2 and RGD peptides target integrins and anti-DEC-205 antibody targets the c-type lectin receptor DEC-205. Our results demonstrate the modified MPs are neither toxic nor activating, and DC uptake of MPs in vitro is improved by the anti-DEC-205 antibody, the anti-CD11c antibody and the P-D2 peptide modifications. The P-D2 peptide MP modification significantly improved DC antigen presentation in vitro both at immediate and delayed time points. Notably, MP functionalization with P-D2 peptide and anti-CD11c antibody increased the rate and extent of MP translocation in vivo by DCs and MΦs, with the P-D2 peptide modified MPs demonstrating the highest translocation. This work informs the design of non-activating polymeric microparticulate applications such as vaccines for autoimmune diseases. PMID:22796161

  15. Engineering of hollow mesoporous silica nanoparticles for remarkably enhanced tumor active targeting efficacy.

    PubMed

    Chen, Feng; Hong, Hao; Shi, Sixiang; Goel, Shreya; Valdovinos, Hector F; Hernandez, Reinier; Theuer, Charles P; Barnhart, Todd E; Cai, Weibo

    2014-01-01

    Hollow mesoporous silica nanoparticle (HMSN) has recently gained increasing interests due to their tremendous potential as an attractive nano-platform for cancer imaging and therapy. However, possibly due to the lack of efficient in vivo targeting strategy and well-developed surface engineering techniques, engineering of HMSN for in vivo active tumor targeting, quantitative tumor uptake assessment, multimodality imaging, biodistribution and enhanced drug delivery have not been achieved to date. Here, we report the in vivo tumor targeted positron emission tomography (PET)/near-infrared fluorescence (NIRF) dual-modality imaging and enhanced drug delivery of HMSN using a generally applicable surface engineering technique. Systematic in vitro and in vivo studies have been performed to investigate the stability, tumor targeting efficacy and specificity, biodistribution and drug delivery capability of well-functionalized HMSN nano-conjugates. The highest uptake of TRC105 (which binds to CD105 on tumor neovasculature) conjugated HMSN in the 4T1 murine breast cancer model was ~10%ID/g, 3 times higher than that of the non-targeted group, making surface engineered HMSN a highly attractive drug delivery nano-platform for future cancer theranostics. PMID:24875656

  16. Engineering of Hollow Mesoporous Silica Nanoparticles for Remarkably Enhanced Tumor Active Targeting Efficacy

    PubMed Central

    Chen, Feng; Hong, Hao; Shi, Sixiang; Goel, Shreya; Valdovinos, Hector F.; Hernandez, Reinier; Theuer, Charles P.; Barnhart, Todd E.; Cai, Weibo

    2014-01-01

    Hollow mesoporous silica nanoparticle (HMSN) has recently gained increasing interests due to their tremendous potential as an attractive nano-platform for cancer imaging and therapy. However, possibly due to the lack of efficient in vivo targeting strategy and well-developed surface engineering techniques, engineering of HMSN for in vivo active tumor targeting, quantitative tumor uptake assessment, multimodality imaging, biodistribution and enhanced drug delivery have not been achieved to date. Here, we report the in vivo tumor targeted positron emission tomography (PET)/near-infrared fluorescence (NIRF) dual-modality imaging and enhanced drug delivery of HMSN using a generally applicable surface engineering technique. Systematic in vitro and in vivo studies have been performed to investigate the stability, tumor targeting efficacy and specificity, biodistribution and drug delivery capability of well-functionalized HMSN nano-conjugates. The highest uptake of TRC105 (which binds to CD105 on tumor neovasculature) conjugated HMSN in the 4T1 murine breast cancer model was ~10%ID/g, 3 times higher than that of the non-targeted group, making surface engineered HMSN a highly attractive drug delivery nano-platform for future cancer theranostics. PMID:24875656

  17. Enhancing cancer targeting and anticancer activity by a stimulus-sensitive multifunctional polymer-drug conjugate.

    PubMed

    Tu, Ying; Zhu, Lin

    2015-08-28

    Undesirable physicochemical properties, low tumor targeting, insufficient cell internalization, acquired drug resistance, and severe side effects significantly limit the applications of anticancer drugs. In this study, to improve the tumor targeting and drug efficacy of the poorly water-soluble drug, doxorubicin (DOX), a novel drug delivery platform (PEG-ppTAT-DOX) was developed, which contained a polyethylene glycol (PEG), a matrix metalloproteinase 2 (MMP2)-sensitive peptide linker (pp), a cell penetrating peptide (TAT), and a model drug (doxorubicin). The prepared drug platform possessed several key features, including: (i) the nanoparticle formation via the self-assembly; (ii) prevention of the non-specific interaction via the PEGylation; (iii) tumor targeting via the MMP2-mediated PEG deshielding and exposure of the TAT; (iv) the TAT-mediated cell internalization; (v) the TAT-induced endosomal escape; (vi) the inhibition of P-glycoprotein mediated drug efflux; and (vii) the TAT-medicated nuclear translocation. These cooperative functions ensured the improved tumor targetability, enhanced tumor cell internalization, improved intracellular distribution, and potentiated anticancer activity. Compared to the multi-component nanocarriers, the proposed simple but multifunctional polymer-drug conjugate might have greater potential for tumor-targeted drug delivery and enhanced chemotherapy. PMID:26113423

  18. Cerebellar brain inhibition in the target and surround muscles during voluntary tonic activation.

    PubMed

    Panyakaew, Pattamon; Cho, Hyun Joo; Srivanitchapoom, Prachaya; Popa, Traian; Wu, Tianxia; Hallett, Mark

    2016-04-01

    Motor surround inhibition is the neural mechanism that selectively favours the contraction of target muscles and inhibits nearby muscles to prevent unwanted movements. This inhibition was previously reported at the onset of a movement, but not during a tonic contraction. Cerebellar brain inhibition (CBI) is reduced in active muscles during tonic activation; however, it has not been studied in the surround muscles. CBI was evaluated in the first dorsal interosseus (FDI) muscle as the target muscle, and the abductor digiti minimi, flexor carpi radialis and extensor carpi radialis muscles as surround muscles, during rest and tonic activation of the FDI muscle in 21 subjects. Cerebellar stimulation was performed under magnetic resonance imaging-guided neuronavigation targeting lobule VIII of the cerebellar hemisphere. Stimulus intensities for cerebellar stimulation were based on the resting motor cortex threshold (RMT) and adjusted for the depth difference between the cerebellar and motor cortices. We used 90-120% of the adjusted RMT as the conditioning stimulus intensity during rest. The intensity that generated the best CBI at rest in the FDI muscle was selected for use during tonic activation. During selective tonic activation of the FDI muscle, CBI was significantly reduced only for the FDI muscle, and not for the surround muscles. Unconditioned motor evoked potential sizes were increased in all muscles during FDI muscle tonic activation as compared with rest, despite background electromyography activity increasing only for the FDI muscle. Our study suggests that the cerebellum may play an important role in selective tonic finger movement by reducing its inhibition in the motor cortex only for the relevant agonist muscle. PMID:26900871

  19. A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation.

    PubMed

    Katayama, Shota; Moriguchi, Tetsuo; Ohtsu, Naoki; Kondo, Toru

    2016-05-23

    Targeted transcriptional activation of endogenous genes is important for understanding physiological transcriptional networks, synthesizing genetic circuits, and inducing cellular phenotype changes. The CRISPR/Cas9 system has great potential to achieve this purpose, however, it has not yet been successfully used to efficiently activate endogenous genes and induce changes in cellular phenotype. A powerful method for transcriptional activation by using CRISPR/Cas9 was developed. Replacement of a methylated promoter with an unmethylated one by CRISPR/Cas9 was sufficient to activate the expression of the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in HEK293T cells. Moreover, CRISPR/Cas9-based OLIG2 activation induced the embryonic carcinoma cell line NTERA-2 to express the neuronal marker βIII-tubulin. PMID:27079176

  20. [Significance of mTOR (mammalian target of rapamycin) activity in human lymphomas].

    PubMed

    Márk, Ágnes

    2014-06-01

    Neoplastic processes, tumor growth, and tumor cell proliferation and survival are often due to the altered activation of different signaling pathways. The increased activity of PI3K/AKT/mTOR signaling has been shown to be an important regulator of tumor growth in several solid tumors and in mantle cell lymphomas. The active form of mTOR kinase (mammalian target of rapamycin) is a key signaling molecule, and it exists in two different complexes, mTORC1 and mTORC2. In the present work, mTOR activity was investigated in different lymphoma types, in parallel with clinical data. We also examined in Hodgkin lymphomas (HL) the role of mTOR activity in survival mechanisms such as antiapoptotic protein expression and alterations in the microenvironment. We determined which lymphoma types display characteristic high mTOR activity in our TMA (tissue microarray) study. We observed that mTOR activity is increased in mitotic lymphoid cells compared to interphasic cells. The number of diffuse large B cell lymphoma (DLBCL) and HL cases was extended in a further set of TMA. We observed significantly higher mTOR activity in the non-centrum germinativum derived subtype of DLBCL than in the centrum germinativum derived subtype, which was a prognostic marker; 63% of mTOR active cases showed Rictor overexpression, indicating mTORC2 activity. High mTOR activity was also established in 92% of HL cases, which was linked to mTORC1. This finding was not a prognostic marker, however, it can be useful in targeted therapy. We observed the overexpression of the antiapoptotic protein BCL-xL and NFκB-p50 in the majority of mTOR active HLs. HLs showed high numbers of regulatory T cells in the microenvironment and high expression of galectin-1 in tumor cells and in the extracellular matrix, when compared to reactive lymph nodes. We confirmed that mTOR inhibition had significant antiproliferative and antiapoptotic effects in lymphoma cell lines and in lymphoma xenografts (HL, DLBCL, Burkitt lymphoma

  1. Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting

    PubMed Central

    Lam, Vinh Q.; Akopian, David; Rome, Michael; Henningsen, Doug

    2010-01-01

    The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP–FtsY complex. This leads to accelerated SRP–FtsY complex assembly, and allows the SRP–FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP–FtsY GTPase complex exposes FtsY’s lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting. PMID:20733058

  2. Validation of a target acquisition model for active imager using perception experiments

    NASA Astrophysics Data System (ADS)

    Lapaz, Frédéric; Canevet, Loïc

    2007-10-01

    Active night vision systems based on laser diodes emitters have now reached a technology level allowing military applications. In order to predict the performance of observers using such systems, we built an analytic model including sensor, atmosphere, visualization and eye effects. The perception task has been modelled using the Targeting Task Performance metric (TTP metric) developed by R. Vollmerhausen from the Night Vision and Electronic Sensors Directorate (NVESD). Sensor and atmosphere models have been validated separately. In order to validate the whole model, two identification tests have been set up. The first set submitted to trained observers was made of hybrid images. The target to background contrast, the blur and the noise were added to armoured vehicles signatures in accordance to sensor and atmosphere models. The second set of images was made with the same targets, sensed by a real active sensor during field trials. Images were recorded, showing different vehicles, at different ranges and orientations, under different illumination and acquisition configurations. Indeed, this set of real images was built with three different types of gating: wide illumination, illumination of the background and illumination of the target. Analysis of the perception experiments results showed a good concordance between the two sets of images. The calculation of an identification criterion, related to this set of vehicles in the near infrared, gave the same results in both cases. The impact of gating on observer's performance was also evaluated.

  3. Targeted massively parallel sequencing of angiosarcomas reveals frequent activation of the mitogen activated protein kinase pathway

    PubMed Central

    Murali, Rajmohan; Chandramohan, Raghu; Möller, Inga; Scholz, Simone L.; Berger, Michael; Huberman, Kety; Viale, Agnes; Pirun, Mono; Socci, Nicholas D.; Bouvier, Nancy; Bauer, Sebastian; Artl, Monika; Schilling, Bastian; Schimming, Tobias; Sucker, Antje; Schwindenhammer, Benjamin; Grabellus, Florian; Speicher, Michael R.; Schaller, Jörg; Hillen, Uwe; Schadendorf, Dirk; Mentzel, Thomas; Cheng, Donavan T.; Wiesner, Thomas; Griewank, Klaus G.

    2015-01-01

    Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors (35%) and losses of CDKN2A in 9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas. PMID:26440310

  4. Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

    PubMed Central

    Cruceanu, Margareta; Stephen, Andrew G.; Beuning, Penny J.; Gorelick, Robert J.; Fisher, Robert J.; Williams, Mark C.

    2006-01-01

    We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7’s capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10 nM, 25 nM, and 100 nM concentration, respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC’s nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins, and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. PMID:17034752

  5. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells

    PubMed Central

    Zmora, Pawel; Moldenhauer, Anna-Sophie; Hofmann-Winkler, Heike; Pöhlmann, Stefan

    2015-01-01

    The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host. PMID:26379044

  6. Natural killer group 2D and CD28 receptors differentially activate mammalian/mechanistic target of rapamycin to alter murine effector CD8+ T-cell differentiation.

    PubMed

    McQueen, Bryan; Trace, Kelsey; Whitman, Emily; Bedsworth, Taylor; Barber, Amorette

    2016-03-01

    Memory CD8+ T cells are an essential component of anti-tumour and anti-viral immunity. Activation of the mammalian/mechanistic target of rapamycin (mTOR) pathway has been implicated in regulating the differentiation of effector and memory T cells. However, the mechanisms that control mTOR activity during immunity to tumours and infections are not well known. Activation of co-stimulatory receptors, including CD28 and natural killer group 2D (NKG2D), activate phosphatidylinositol-3 kinase and subsequently may activate the mTOR pathway in CD8+ T cells. This study compared the activation of the mTOR signalling pathway after co-stimulation through CD28 or NKG2D receptors in murine effector CD8+ T cells. Compared with CD28 co-stimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1, as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1. NKG2D co-stimulation also showed increased gene expression of tuberous sclerosis protein 2, a negative regulator of mTORc1, whereas CD28 co-stimulation increased gene expression of Ras homologue enriched in brain, an activator of mTORc1, and hypoxia-inducible factor-1α and vascular endothelial growth factor-α, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-co-stimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, B lymphocyte-induced maturation protein (BLIMP-1), interferon regulatory factor 4, and inhibitor of DNA binding 2, whereas low levels of mTORc1 activation allowed for the expression of Eomes, B-cell lymphoma 6 (BCL6), and inhibitor of DNA binding 3 during NKG2D stimulation, and increased expression of memory markers CD62 ligand and CD127. These data show that compared with CD28, co-stimulation through the NKG2D receptor leads to the differential activation of the mTOR signalling pathway and potentially supports

  7. A mask for high-intensity heavy-ion beams in the MAYA active target

    NASA Astrophysics Data System (ADS)

    Rodríguez-Tajes, C.; Pancin, J.; Damoy, S.; Roger, T.; Babo, M.; Caamaño, M.; Farget, F.; Grinyer, G. F.; Jacquot, B.; Pérez-Loureiro, D.; Ramos, D.; Suzuki, D.

    2014-12-01

    The use of high-intensity and/or heavy-ion beams in active targets and time-projection chambers is often limited by the strong ionization produced by the beam. Besides the difficulties associated with the saturation of the detector and electronics, beam-related signals may hide the physical events of interest or reduce the detector performance. In addition, space-charge effects may deteriorate the homogeneity of the electric drift field and distort the subsequent reconstruction of particle trajectories. In anticipation of future projects involving such conditions, a dedicated beam mask has been developed and tested in the MAYA active target. Experimental results with a 136Xe beam are presented.

  8. Development and performance test of the analysis software for the CRIB active target

    NASA Astrophysics Data System (ADS)

    Lee, Pilsoo; Lee, Chun Sik; Moon, Jun Young; Chae, Kyung Yuk; Cha, Soo Mi; Yamaguchi, Hidetoshi; Nakao, Taro; Kahl, David M.; Kubono, Shigeru; Cherubini, Silvio; Hayakawa, Seiya; Signorini, Cosimo

    2015-02-01

    Software for genuinely event-by-event analysis and event reconstruction of data obtained by using an active target has been developed in the graphical user interface under the CERN ROOT framework. The primary motivation for developing the software was to provide physicists who perform experiments using an active target a more user-friendly environment for the purpose of investigating the performance of detection systems and obtaining ideas about physics from a large amount of experimental data. To test the performance of the software, we analyzed experimental data from a 16N radioactive ion beam experiment for α-decay measurements. As a result of the analysis, we observed the Bragg curve and measured the range of the 16N RI beam in the detector. Data were calibrated against the calculation after comparing the Bragg curve to the one obtained from an energy loss calculation in P-10 gas. We present a detailed description of the analysis software and its test results.

  9. 12C+p resonant elastic scattering in the Maya active target

    NASA Astrophysics Data System (ADS)

    Sambi, S.; Raabe, R.; Borge, M. J. G.; Caamano, M.; Damoy, S.; Fernández-Domínguez, B.; Flavigny, F.; Fynbo, H.; Gibelin, J.; Grinyer, G. F.; Heinz, A.; Jonson, B.; Khodery, M.; Nilsson, T.; Orlandi, R.; Pancin, J.; Perez-Loureiro, D.; Randisi, G.; Ribeiro, G.; Roger, T.; Suzuki, D.; Tengblad, O.; Thies, R.; Datta, U.

    2015-03-01

    In a proof-of-principle measurement, the Maya active target detector was employed for a 12C( p, p) resonant elastic scattering experiment in inverse kinematics. The excitation energy region from 0 to 3MeV above the proton breakup threshold in 13N was investigated in a single measurement. By using the capability of the detector to localize the reaction vertex and record the tracks of the recoiling protons, data covering a large solid angle could be utilized, at the same time keeping an energy resolution comparable with that of direct-kinematics measurements. The excitation spectrum in 13N was fitted using the R-matrix formalism. The level parameters extracted are in good agreement with previous studies. The active target proved its potential for the study of resonant elastic scattering in inverse kinematics with radioactive beams, when detection efficiency is of primary importance.

  10. Activity, assay and target data curation and quality in the ChEMBL database.

    PubMed

    Papadatos, George; Gaulton, Anna; Hersey, Anne; Overington, John P

    2015-09-01

    The emergence of a number of publicly available bioactivity databases, such as ChEMBL, PubChem BioAssay and BindingDB, has raised awareness about the topics of data curation, quality and integrity. Here we provide an overview and discussion of the current and future approaches to activity, assay and target data curation of the ChEMBL database. This curation process involves several manual and automated steps and aims to: (1) maximise data accessibility and comparability; (2) improve data integrity and flag outliers, ambiguities and potential errors; and (3) add further curated annotations and mappings thus increasing the usefulness and accuracy of the ChEMBL data for all users and modellers in particular. Issues related to activity, assay and target data curation and integrity along with their potential impact for users of the data are discussed, alongside robust selection and filter strategies in order to avoid or minimise these, depending on the desired application. PMID:26201396

  11. Aryl Hydrocarbon Receptor Activation by Dioxin Targets Phosphoenolpyruvate Carboxykinase (PEPCK) for ADP-ribosylation via 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible Poly(ADP-ribose) Polymerase (TiPARP)*

    PubMed Central

    Diani-Moore, Silvia; Zhang, Sheng; Ram, Payal; Rifkind, Arleen B.

    2013-01-01

    Effects of the environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) include a wasting syndrome associated with decreased gluconeogenesis. TCDD is a potent activator of the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The relationship between gene activation by the AHR and TCDD toxicities is not well understood. We recently identified a pathway by which the AHR target gene TiPARP (TCDD-inducible poly(ADP-ribose) polymerase) contributes to TCDD suppression of transcription of phosphoenolpyruvate carboxykinase (PEPCK), a key regulator of gluconeogenesis, by consuming NAD+ and decreasing Sirtuin 1 activation of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a transcriptional activator of PEPCK. We report here that TCDD-induced TiPARP also targets PEPCK for ADP-ribosylation. Both cytosolic and mitochondrial forms of PEPCK were found to undergo ADP-ribosylation. Unexpectedly, AHR suppression also enhanced ADP-ribosylation and did so by a poly(ADP-ribose) polymerase-independent mechanism. This report 1) identifies ADP-ribosylation as a new posttranslational modification for PEPCK, 2) describes a pathway by which transcriptional induction of TiPARP by the AHR can lead to a downstream posttranslational change in a TCDD target protein (PEPCK), and 3) reveals that the AHR exerts complex, previously unidentified modulatory effects on ADP-ribosylation. PMID:23770670

  12. Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex

    PubMed Central

    Saayman, Sheena M; Lazar, Daniel C; Scott, Tristan A; Hart, Jonathan R; Takahashi, Mayumi; Burnett, John C; Planelles, Vicente; Morris, Kevin V; Weinberg, Marc S

    2016-01-01

    HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiretroviral therapy (cART) with lifelong treatment being the only option. The “shock and kill” strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a “hotspot” for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChIT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNFα induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a “functional cure” of HIV/AIDS. PMID:26581162

  13. Use of spatially explicit physicochemical data to measure downstream impacts of headwater stream disturbance

    EPA Science Inventory

    Regulatory agencies need methods to quantify the influence of headwater streams on downstream water quality as a result of litigation surrounding jurisdictional criteria and the influence of mountaintop removal coal mining activities. We collected comprehensive, spatially-referen...

  14. Active tumor-targeting luminescent gold clusters with efficient urinary excretion.

    PubMed

    Wang, Xiaojuan; He, Hua; Wang, Yanan; Wang, Junying; Sun, Xing; Xu, Hai; Nau, Werner M; Zhang, Xiaodong; Huang, Fang

    2016-07-28

    We present novel active targeting luminescent gold nanoclusters (AuNCs), which are prepared through a one-pot procedure by using a pentapeptide (CRGDS) for stabilization and tumor recognition. CRGDS-AuNCs exhibit a high tumor-specific retention with an exceptionally high tumor-to-liver uptake ratio of 9.3. Their small hydrodynamic diameter and zwitterionic surface facilitate urinary excretion, which reaches 82% within 24 h after injection. PMID:27354156

  15. Recent developments for an active UF6 gas target for photon-induced fission experiments

    NASA Astrophysics Data System (ADS)

    Freudenberger, M.; Eckardt, C.; Enders, J.; Göök, A.; von Neumann-Cosel, P.; Oberstedt, A.; Oberstedt, S.

    2013-12-01

    Recent developments for an active uranium-hexafluoride-loaded gas target as well as results on the detector gas properties are presented. The gas of choice is a mixture of argon with small amounts of UF6. This contribution presents the experimental setup and focusses on the electron drift velocity with increasing UF6 content. A time-dependent decrease in electron drift velocity is observed in our setup.

  16. Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus.

    PubMed

    Lu, Yingjuan; Parker, Nikki; Kleindl, Paul J; Cross, Vicky A; Wollak, Kristin; Westrick, Elaine; Stinnette, Torian W; Gehrke, Mark A; Wang, Kevin; Santhapuram, Hari Krishna R; You, Fei; Hahn, Spencer J; Vaughn, Jeremy F; Klein, Patrick J; Vlahov, Iontcho R; Low, Philip S; Leamon, Christopher P

    2015-01-01

    Folate receptor (FR)-β has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity. Using FR-expressing macrophages, the effect of EC0565 on mTOR signaling and cellular proliferation was studied. The pharmacokinetics, metabolism and bioavailability of EC0565 were studied in normal rats. The in vivo activity of EC0565 was assessed in rats with adjuvant arthritis, a "macrophage-rich" model with close resemblance to rheumatoid arthritis. EC0565 forms micellar aggregates in physiological buffers and demonstrates good water solubility as well as strong multivalent FR-binding capacity. EC0565 inhibited mTOR signaling in rat macrophages at nanomolar concentrations and induced G0/G1 cell cycle arrest in serum-starved RAW264.7 cells. Subcutaneously administered EC0565 in rats displayed good bioavailability and a relatively long half-life (~12 h). When given at 250 nmol/kg, EC0565 selectively inhibited proliferating cell nuclear antigen expression in thioglycollate-stimulated rat peritoneal cells. With limited dosing regimens, the antiarthritic activity of EC0565 was found superior to that of etanercept, everolimus and a nontargeted everolimus analog. The in vivo activity of EC0565 was also comparable to that of a folate-targeted aminopterin. Folate-targeted mTOR inhibition may be an effective way of suppressing activated macrophages in sites of inflammation, especially in nutrient-deprived conditions, such as in the arthritic joints. Further investigation and improvement upon the physical and biochemical properties of EC0565 are warranted. PMID:26181632

  17. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

    NASA Astrophysics Data System (ADS)

    Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

    2013-08-01

    Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific

  18. Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pathogenic fungi are becoming resistant to currently available drugs. Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. The aim of this study was to identify benzaldehydes that...

  19. Downstream gradients in bioindicator responses: Point source contaminant effects on fish health

    SciTech Connect

    Marshall Adams, S.; Ham, K.D.; Greeley, M.S.; Hinton, D.E.

    1995-12-31

    To investigate potential causal relationships between contaminant exposure and biological responses in fish, a suite of bioindicators ranging from the biochemical to the community-level were measured in fish populations and communities downstream from a bleached kraft mill effluent (BKME) discharge. Downstream gradients in responses were evident in elevated hepatic mixed-function oxygenase activity, several measures of condition and bioenergetic status, growth, the health assessment index, and several fish community-level parameters. A multivariate discriminant analysis procedure, which included many of the individual bioindicators, also demonstrated a gradient in integrated health status of a sentinel fish species in the contaminated river. These downstream response gradients were probably influenced to a greater degree by contaminant discharges than by natural or anthropogenic nutrient sources downstream. Establishing causal relationships between a specific contaminant source and responses in sentinel aquatic organisms becomes relatively more straightforward when downstream gradients in biological responses are observed at multiple levels of biological organization.

  20. Novel small molecules targeting ciliary transport of Smoothened and oncogenic Hedgehog pathway activation

    PubMed Central

    Jung, Bomi; Messias, Ana C.; Schorpp, Kenji; Geerlof, Arie; Schneider, Günter; Saur, Dieter; Hadian, Kamyar; Sattler, Michael; Wanker, Erich E.; Hasenöder, Stefan; Lickert, Heiko

    2016-01-01

    Trafficking of the G protein-coupled receptor (GPCR) Smoothened (Smo) to the primary cilium (PC) is a potential target to inhibit oncogenic Hh pathway activation in a large number of tumors. One drawback is the appearance of Smo mutations that resist drug treatment, which is a common reason for cancer treatment failure. Here, we undertook a high content screen with compounds in preclinical or clinical development and identified ten small molecules that prevent constitutive active mutant SmoM2 transport into PC for subsequent Hh pathway activation. Eight of the ten small molecules act through direct interference with the G protein-coupled receptor associated sorting protein 2 (Gprasp2)-SmoM2 ciliary targeting complex, whereas one antagonist of ionotropic receptors prevents intracellular trafficking of Smo to the PC. Together, these findings identify several compounds with the potential to treat drug-resistant SmoM2-driven cancer forms, but also reveal off-target effects of established drugs in the clinics. PMID:26931153

  1. Apratoxin A Shows Novel Pancreas-Targeting Activity through the Binding of Sec 61.

    PubMed

    Huang, Kuan-Chun; Chen, Zhihong; Jiang, Yimin; Akare, Sandeep; Kolber-Simonds, Donna; Condon, Krista; Agoulnik, Sergei; Tendyke, Karen; Shen, Yongchun; Wu, Kuo-Ming; Mathieu, Steven; Choi, Hyeong-Wook; Zhu, Xiaojie; Shimizu, Hajime; Kotake, Yoshihiko; Gerwick, William H; Uenaka, Toshimitsu; Woodall-Jappe, Mary; Nomoto, Kenichi

    2016-06-01

    Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug. Here, we describe a detailed pathologic analysis that revealed a specific pancreas-targeting activity of apratoxin A, such that severe pancreatic atrophy was observed in apratoxin A-treated animals. Follow-up tissue distribution studies further uncovered a unique drug distribution profile for apratoxin A, showing high drug exposure in pancreas and salivary gland. It has been shown previously that apratoxin A inhibits the protein secretory pathway by preventing cotranslational translocation. However, the molecule targeted by apratoxin A in this pathway has not been well defined. By using a (3)H-labeled apratoxin A probe and specific Sec 61α/β antibodies, we identified that the Sec 61 complex is the molecular target of apratoxin A. We conclude that apratoxin A in vivo toxicity is likely caused by pancreas atrophy due to high apratoxin A exposure. Mol Cancer Ther; 15(6); 1208-16. ©2016 AACR. PMID:27196783

  2. Identification of therapeutic targets in ovarian cancer through active tyrosine kinase profiling

    PubMed Central

    Ocaña, Alberto; Pandiella, Atanasio

    2015-01-01

    The activation status of a set of pro-oncogenic tyrosine kinases in ovarian cancer patient samples was analyzed to define potential therapeutic targets. Frequent activation of HER family receptor tyrosine kinases, especially HER2, was observed. Studies in ovarian cancer cell lines confirmed the activation of HER2. Moreover, knockdown of HER2 caused a strong inhibition of their proliferation. Analyses of the action of agents that target HER2 indicated that the antibody drug conjugate trastuzumab-emtansine (T-DM1) caused a substantial antitumoral effect in vivo and in vitro, and potentiated the action of drugs used in the therapy of ovarian cancer. T-DM1 provoked cell cycle arrest in mitosis, and caused the appearance of aberrant mitotic spindles in cells treated with the drug. Biochemical experiments confirmed accumulation of the mitotic markers phospho-Histone H3 and phospho-BUBR1 in cells treated with the drug. Prolonged treatment of ovarian cancer cells with T-DM1 provoked the appearance of multinucleated cells which later led to cell death. Together, these data indicate that HER2 represents an important oncogene in ovarian cancer, and suggest that targeting this tyrosine kinase with T-DM1 may be therapeutically effective, especially in ovarian tumors with high content of HER2. PMID:26336133

  3. Waveguide invariant active sonar target detection and depth classification in shallow water

    NASA Astrophysics Data System (ADS)

    Goldhahn, Ryan A.

    Reverberation and clutter are two of the principle obstacles to active sonar target detection in shallow water. Diffuse seabed backscatter can obscure low energy target returns, while clutter discretes, specific features of the sea floor, produce temporally compact returns which may be mistaken for targets of interest. Detecting weak targets in the presence of reverberation and discriminating water column targets from bottom clutter are thus critical to good performance in active sonar. Both problems are addressed in this thesis using the time-frequency interference pattern described by a constant known as the waveguide invariant which summarizes in a scalar parameter the dispersive properties of the ocean environment. Conventional active sonar detection involves constant false alarm rate (CFAR) normalization of the reverberation return which does not account for the frequency-selective fading in a wideband pulse caused by multipath propagation. An alternative to conventional reverberation estimation is presented, motivated by striations observed in time-frequency analysis of active sonar data. A mathematical model for these reverberation striations is derived using waveguide invariant theory. This model is then used to motivate waveguide invariant reverberation estimation which involves averaging the time-frequency spectrum along these striations. An evaluation of this reverberation estimate using real Mediterranean data is given and its use in a generalized likelihood ratio test (GLRT) based CFAR detector is demonstrated. CFAR detection using waveguide invariant reverberation estimates is shown to out-perform conventional cell-averaged and frequency-invariant CFAR detection methods in shallow water environments producing strong reverberation returns which exhibit the described striations. Results are presented on simulated and real Mediterranean data from the SCARAB98 experiment. The ability to discriminate between water column targets and clutter discretes is

  4. The value of prevention: managing the risks associated with targeted violence and active shooters.

    PubMed

    Doherty, Matthew

    2016-01-01

    Every time we turn on the news, or open our Internet browsers, a story about an active shooter--at a school, house of worship, public place and even in our workplace--spills onto the page, the author reports. In this article he focuses on how we can prevent these incidents from occurring. What exactly is "targeted violence"--and why is what experts call "behavioral threat assessment" one of the single most effective ways to prevent the next active shooter incident in any organization? PMID:26978957

  5. Cancer Stem Cells: The Potential Targets of Chinese Medicines and Their Active Compounds.

    PubMed

    Hong, Ming; Tan, Hor Yue; Li, Sha; Cheung, Fan; Wang, Ning; Nagamatsu, Tadashi; Feng, Yibin

    2016-01-01

    The pivotal role of cancer stem cells (CSCs) in the initiation and progression of malignancies has been rigorously validated, and the specific methods for identifying and isolating the CSCs from the parental cancer population have also been rapidly developed in recent years. This review aims to provide an overview of recent research progress of Chinese medicines (CMs) and their active compounds in inhibiting tumor progression by targeting CSCs. A great deal of CMs and their active compounds, such as Antrodia camphorate, berberine, resveratrol, and curcumin have been shown to regress CSCs, in terms of reversing drug resistance, inducing cell death and inhibiting cell proliferation as well as metastasis. Furthermore, one of the active compounds in coptis, berbamine may inhibit tumor progression by modulating microRNAs to regulate CSCs. The underlying molecular mechanisms and related signaling pathways involved in these processes were also discussed and concluded in this paper. Overall, the use of CMs and their active compounds may be a promising therapeutic strategy to eradicate cancer by targeting CSCs. However, further studies are needed to clarify the potential of clinical application of CMs and their active compounds as complementary and alternative therapy in this field. PMID:27338343

  6. Cancer Stem Cells: The Potential Targets of Chinese Medicines and Their Active Compounds

    PubMed Central

    Hong, Ming; Tan, Hor Yue; Li, Sha; Cheung, Fan; Wang, Ning; Nagamatsu, Tadashi; Feng, Yibin

    2016-01-01

    The pivotal role of cancer stem cells (CSCs) in the initiation and progression of malignancies has been rigorously validated, and the specific methods for identifying and isolating the CSCs from the parental cancer population have also been rapidly developed in recent years. This review aims to provide an overview of recent research progress of Chinese medicines (CMs) and their active compounds in inhibiting tumor progression by targeting CSCs. A great deal of CMs and their active compounds, such as Antrodia camphorate, berberine, resveratrol, and curcumin have been shown to regress CSCs, in terms of reversing drug resistance, inducing cell death and inhibiting cell proliferation as well as metastasis. Furthermore, one of the active compounds in coptis, berbamine may inhibit tumor progression by modulating microRNAs to regulate CSCs. The underlying molecular mechanisms and related signaling pathways involved in these processes were also discussed and concluded in this paper. Overall, the use of CMs and their active compounds may be a promising therapeutic strategy to eradicate cancer by targeting CSCs. However, further studies are needed to clarify the potential of clinical application of CMs and their active compounds as complementary and alternative therapy in this field. PMID:27338343

  7. Monitoring Target Engagement of Deubiquitylating Enzymes Using Activity Probes: Past, Present, and Future.

    PubMed

    Harrigan, Jeanine; Jacq, Xavier

    2016-01-01

    Deubiquitylating enzymes or DUBs are a class of enzymes that selectively remove the polypeptide posttranslational modification ubiquitin from a number of substrates. Approximately 100 DUBs exist in human cells and are involved in key regulatory cellular processes, which drive many disease states, making them attractive therapeutic targets. Several aspects of DUB biology have been studied through genetic knock-out or knock-down, genomic, or proteomic studies. However, investigation of enzyme activation and regulation requires additional tools to monitor cellular and physiological dynamics. A comparison between genetic ablation and dominant-negative target validation with pharmacological inhibition often leads to striking discrepancies. Activity probes have been used to profile classes of enzymes, including DUBs, and allow functional and dynamic properties to be assigned to individual proteins. The ability to directly monitor DUB activity within a native biological system is essential for understanding the physiological and pathological role of individual DUBs. We will discuss the evolution of DUB activity probes, from in vitro assay development to their use in monitoring DUB activity in cells and in animal tissues, as well as recent progress and prospects for assessing DUB inhibition in vivo. PMID:27613052

  8. Subcellular Targeting of Methylmercury Lyase Enhances Its Specific Activity for Organic Mercury Detoxification in Plants1

    PubMed Central

    Bizily, Scott P.; Kim, Tehryung; Kandasamy, Muthugapatti K.; Meagher, Richard B.

    2003-01-01

    Methylmercury is an environmental pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals. In an effort to remove this toxin in situ, we have been engineering plants that express the bacterial mercury resistance enzymes organomercurial lyase MerB and mercuric ion reductase MerA. In vivo kinetics experiments suggest that the diffusion of hydrophobic organic mercury to MerB limits the rate of the coupled reaction with MerA (Bizily et al., 2000). To optimize reaction kinetics for organic mercury compounds, the merB gene was engineered to target MerB for accumulation in the endoplasmic reticulum and for secretion to the cell wall. Plants expressing the targeted MerB proteins and cytoplasmic MerA are highly resistant to organic mercury and degrade organic mercury at 10 to 70 times higher specific activity than plants with the cytoplasmically distributed wild-type MerB enzyme. MerB protein in endoplasmic reticulum-targeted plants appears to accumulate in large vesicular structures that can be visualized in immunolabeled plant cells. These results suggest that the toxic effects of organic mercury are focused in microenvironments of the secretory pathway, that these hydrophobic compartments provide more favorable reaction conditions for MerB activity, and that moderate increases in targeted MerB expression will lead to significant gains in detoxification. In summary, to maximize phytoremediation efficiency of hydrophobic pollutants in plants, it may be beneficial to target enzymes to specific subcellular environments. PMID:12586871

  9. 40 CFR 745.227 - Work practice standards for conducting lead-based paint activities: target housing and child...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... lead-based paint activities: target housing and child-occupied facilities. 745.227 Section 745.227... practice standards for conducting lead-based paint activities: target housing and child-occupied facilities... section are found in the following: The U.S. Department of Housing and Urban Development (HUD)...

  10. 40 CFR 745.227 - Work practice standards for conducting lead-based paint activities: target housing and child...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... lead-based paint activities: target housing and child-occupied facilities. 745.227 Section 745.227... practice standards for conducting lead-based paint activities: target housing and child-occupied facilities... section are found in the following: The U.S. Department of Housing and Urban Development (HUD)...

  11. 40 CFR 745.227 - Work practice standards for conducting lead-based paint activities: target housing and child...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... lead-based paint activities: target housing and child-occupied facilities. 745.227 Section 745.227... practice standards for conducting lead-based paint activities: target housing and child-occupied facilities... section are found in the following: The U.S. Department of Housing and Urban Development (HUD)...

  12. 40 CFR 745.227 - Work practice standards for conducting lead-based paint activities: target housing and child...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... lead-based paint activities: target housing and child-occupied facilities. 745.227 Section 745.227... practice standards for conducting lead-based paint activities: target housing and child-occupied facilities... section are found in the following: The U.S. Department of Housing and Urban Development (HUD)...

  13. Small Molecule Activators of the Heat Shock Response: Chemical Properties, Molecular Targets, and Therapeutic Promise

    PubMed Central

    West, James D.; Wang, Yanyu; Morano, Kevin A.

    2012-01-01

    All cells have developed various mechanisms to respond and adapt to a variety of environmental challenges, including stresses that damage cellular proteins. One such response, the heat shock response (HSR), leads to the transcriptional activation of a family of molecular chaperone proteins that promote proper folding or clearance of damaged proteins within the cytosol. In addition to its role in protection against acute insults, the HSR also regulates lifespan and protects against protein misfolding that is associated with degenerative diseases of aging. As a result, identifying pharmacological regulators of the HSR has become an active area of research in recent years. Here, we review progress made in identifying small molecule activators of the HSR, what cellular targets these compounds interact with to drive response activation, and how such molecules may ultimately be employed to delay or reverse protein misfolding events that contribute to a number of diseases. PMID:22799889

  14. Tumor-Targeting of EGFR Inhibitors by Hypoxia-Mediated Activation**

    PubMed Central

    Kryeziu, Kushtrim; Pichler, Verena; Roller, Alexander; Berger, Walter; Heffeter, Petra; Kowol, Christian R.

    2015-01-01

    The development of receptor tyrosine-kinase inhibitors (TKIs) was a major step forward in cancer treatment. However, the therapy with TKIs is limited by strong side effects and drug resistance. The aim of this study was the design of novel epidermal growth factor receptor (EGFR) inhibitors that are specifically activated in malignant tissue. Thus, a CoIII-based prodrug strategy for the targeted release of an EGFR inhibitor triggered by hypoxia in the solid tumor was used. New inhibitors with chelating moieties were prepared and tested for their EGFR-inhibitory potential. The most promising candidate was coupled to CoIII and the biological activity tested in cell culture. Indeed, hypoxic activation and subsequent EGFR inhibition was proven. Finally, the compound was tested in vivo, also revealing potent anticancer activity. PMID:25079700

  15. The effect of mining and related activities on the sediment-trace element geochemistry of Lake Coeur d'Alene, Idaho, USA. Part III. Downstream effects: the Spokane River Basin

    NASA Astrophysics Data System (ADS)

    Grosbois, Cecile A.; Horowitz, Arthur J.; Smith, James J.; Elrick, Kent A.

    2001-04-01

    During 1998/1999, surface and subsurface sediment samples were collected along the entire length of the Spokane River from its outlet at the northern end of Lake Coeur d'Alene (CDA), Idaho, to Lake Roosevelt on the Columbia River, Washington. The study was conducted to determine if the trace element enrichments observed in Lake CDA and on the floodplain and in the CDA River extend through the Spokane River Basin (SRB).As in Lake CDA, surface sediments in the SRB are enriched in Pb, Zn, As, Cd, Sb and Hg relative to local background levels. Pb, Cd and Zn are the most elevated, with maximum enrichment occurring in the upper Spokane River in close proximity to Lake CDA. On average, enrichment decreases downstream, apparently reflecting both increased distance from the inferred source (the CDA River Basin), as well as increased dilution by locally derived but unenriched materials. Only Cd and Zn display marked enrichment throughout the SRB. Pb, Zn and Cd seem to be associated mainly with an operationally defined iron oxide phase, whereas the majority of the As and Sb seem to be matrix-held.Subsurface sediments also are enriched in Pb, Zn, As, Cd, Sb and Hg relative to background levels. Based on 137Cs and excess 210Pb dating, trace element enrichment began in the middle part of the SRB (Long Lake) between 1900 and 1920. This is contemporaneous with similar enrichments observed in Lake CDA, as well as the completion of Long Lake Dam (1913). In the most downstream part of the basin (Spokane River Arm of Lake Roosevelt), enrichment began substantially later, between 1930 and 1940. The temporal difference in enrichment between Long Lake and the River Arm may reflect the latter's greater distance from the presumed source of the enrichment (the CDA River Basin); however, the difference is more likely the result of the completion of Grand Coulee Dam (1934-1941), which formed Lake Roosevelt, backed up the Spokane River, and increased water levels in the River Arm by about 30 m.

  16. The effect of mining and related activities on the sediment-trace element geochemistry of Lake Coeur d'Alene, Idaho, USA. Part III. Downstream effects: The Spokane River Basin

    USGS Publications Warehouse

    Grosbois, C.A.; Horowitz, A.J.; Smith, J.J.; Elrick, K.A.

    2001-01-01

    During 1998/1999, surface and subsurface sediment samples were collected along the entire length of the Spokane River from its outlet at the northern end of Lake Coeur d'Alene (CDA), Idaho, to Lake Roosevelt on the Columbia River, Washington. The study was conducted to determine if the trace element enrichments observed in Lake CDA and on the floodplain and in the CDA River extend through the Spokane River Basin (SRB). As in Lake CDA, surface sediments in the SRB are enriched in Pb, Zn, As, Cd, Sb and Hg relative to local background levels. Pb, Cd and Zn are the most elevated, with maximum enrichment occurring in the upper Spokane River in close proximity to Lake CDA. On average, enrichment decreases downstream, apparently reflecting both increased distance from the inferred source (the CDA River Basin), as well as increased dilution by locally derived but unenriched materials. Only Cd and Zn display marked enrichment throughout the SRB. Pb, Zn and Cd seem to be associated mainly with an operationally defined iron oxide phase, whereas the majority of the As and Sb seem to be matrix-held. Subsurface sediments also are enriched in Pb, Zn, As, Cd, Sb and Hg relative to background levels. Based on 137Cs and excess 210Pb dating, trace element enrichment began in the middle part of the SRB (Long Lake) between 1900 and 1920. This is contemporaneous with similar enrichments observed in Lake CDA, as well as the completion of Long Lake Dam (1913). In the most downstream part of the basin (Spokane River Arm of Lake Roosevelt), enrichment began substantially later, between 1930 and 1940. The temporal difference in enrichment between Long Lake and the River Arm may reflect the latter's greater distance from the presumed source of the enrichment (the CDA River Basin); however, the difference is more likely the result of the completion of Grand Coulee Dam (1934-1941), which formed Lake Roosevelt, backed up the Spokane River, and increased water levels in the River Armby about 30

  17. Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug

    PubMed Central

    2012-01-01

    Background Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG. Methods The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9–11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided. Results FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC50 = 3.5nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206mm3, 95% CI = 0.068 to 0.344mm3; control: mean = 0.580mm3, 95% CI = 0.267 to 0.893mm3) and 0.24 (treated: mean = 0.131mm3, 95% CI = 0.09 to 0.180mm3; control

  18. Dipeptidyl Peptidase IV as a Potential Target for Selective Prodrug Activation and Chemotherapeutic Action in Cancers

    PubMed Central

    2015-01-01

    The efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells. Recently, the enzyme dipeptidyl peptidase IV (DPPIV) was considered as a potential target for the delivery of chemotherapeutic drugs. The purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to DPPIV as a strategy to enhance their specificity. The expression profile of DPPIV was obtained for seven cancer cell lines using DNA microarray data from the DTP database, and was validated by RT-PCR. A prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline-glycine dipeptide moiety, followed by hydrolysis studies in the seven cell lines with a standard substrate, as well as the glycyl-prolyl-melphalan (GP-Mel). Lastly, cell proliferation studies were carried out to demonstrate enzyme-dependent activation of the candidate prodrug. The relative RT-PCR expression levels of DPPIV in the cancer cell lines exhibited linear correlation with U95Av2 Affymetrix data (r2 = 0.94), and with specific activity of a standard substrate, glycine-proline-p-nitroanilide (r2 = 0.96). The significantly higher antiproliferative activity of GP-Mel in Caco-2 cells (GI50 = 261 μM) compared to that in SK-MEL-5 cells (GI50 = 807 μM) was consistent with the 9-fold higher specific activity of the prodrug in Caco-2 cells (5.14 pmol/min/μg protein) compared to SK-MEL-5 cells (0.68 pmol/min/μg protein) and with DPPIV expression levels in these cells. Our results demonstrate the great potential to exploit DPPIV as a prodrug activating enzyme for efficient chemotherapeutic drug targeting. PMID:25365774

  19. Further Evolution of Multifunctional Niosomes Based on Pluronic Surfactant: Dual Active Targeting and Drug Combination Properties.

    PubMed

    Tavano, Lorena; Mauro, Loredana; Naimo, Giuseppina Daniela; Bruno, Leonardo; Picci, Nevio; Andò, Sebastiano; Muzzalupo, Rita

    2016-09-01

    The loading of chemotherapics into smart nanocarriers that simultaneously possess more than one useful property for specifically targeting a tumor site improves their therapeutic effectiveness, reducing their side effects. Hence, we proposed a combined approach for the treatment of human breast cancer (BC) consisting of the co-encapsulation of doxorubicin and curcumin or doxorubicin and quercetin into multifunctional niosomes, which results in prolonged blood circulation and an ability to spontaneously accumulate at the tumor site (passive target) and to recognize and bind the tumor cells through dual ligand-receptor interactions (active target). The drug-loaded vesicles showed high stability and good capability of loading doxorubicin and antioxidants alone or in combination. Their diameter was around 400 nm. The drugs released from the vesicles were found to be controlled and sustained for over 24 h, with a strong dependence on the co-presence of the loaded molecules. Transferrin and/or folic acid were conjugated on the external surface of the niosomes as ligands, considerably improving the cellular uptake into MCF-7 and MDA-MB-231 malignant cells when compared with the uptake of nonconjugated samples. In vitro evaluation of anticancer activity demonstrated the strong potential of niosomes loaded with a doxorubicin/curcumin combination as useful devices in breast tumor treatment. These features hold great promise for the development of multifunctional devices that combine several advantages such as biocompatibility, stealth properties, loading capability, and active targeting, moving toward the development of more specific and efficient carriers for personalized tumoral therapy. PMID:27504856

  20. Meta-analysis of primary target genes of peroxisome proliferator-activated receptors

    PubMed Central

    Heinäniemi, Merja; Uski, J Oskari; Degenhardt, Tatjana; Carlberg, Carsten

    2007-01-01

    Background Peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the development of diseases, such as obesity, cardiovascular disease, type 2 diabetes and cancer. Here, an in silico screening method is presented, which incorporates experiment- and informatics-derived evidence, such as DNA-binding data of PPAR subtypes to a panel of PPAR response elements (PPREs), PPRE location relative to the transcription start site (TSS) and PPRE conservation across multiple species, for more reliable prediction of PPREs. Results In vitro binding and in vivo functionality evidence agrees with in silico predictions, validating the approach. The experimental analysis of 30 putative PPREs in eight validated PPAR target genes indicates that each gene contains at least one functional, strong PPRE that occurs without positional bias relative to the TSS. An extended analysis of the cross-species conservation of PPREs reveals limited conservation of PPRE patterns, although PPAR target genes typically contain strong or multiple medium strength PPREs. Human chromosome 19 was screened using this method, with validation of six novel PPAR target genes. Conclusion An in silico screening approach is presented, which allows increased sensitivity of PPAR binding site and target gene detection. PMID:17650321

  1. Cancer Nanotechnology: The impact of passive and active targeting in the era of modern cancer biology☆

    PubMed Central

    Bertrand, Nicolas; Wu, Jun; Xu, Xiaoyang; Kamaly, Nazila; Farokhzad, Omid C

    2014-01-01

    Cancer nanotherapeutics are progressing at a steady rate; research and development in the field has experienced an exponential growth since early 2000’s. The path to the commercialization of oncology drugs is long and carries significant risk; however, there is considerable excitement that nanoparticle technologies may contribute to the success of cancer drug development. The pace at which pharmaceutical companies have formed partnerships to use proprietary nanoparticle technologies has considerably accelerated. It is now recognized that by enhancing the efficacy and/or tolerability of new drug candidates, nanotechnology can meaningfully contribute to create differentiated products and improve clinical outcome. This review describes the lessons learned since the commercialization of the first-generation nanomedicines including DOXIL® and Abraxane®. It explores our current understanding of targeted and non-targeted nanoparticles that are under various stages of development, including BIND-014 and MM-398. It highlights the opportunities and challenges faced by nanomedicines in contemporary oncology, where personalized medicine is increasingly the mainstay of cancer therapy. We revisit the fundamental concepts of enhanced permeability and retention effect (EPR) and explore the mechanisms proposed to enhance preferential “retention” in the tumor, whether using active targeting of nanoparticles, binding of drugs to their tumoral targets or the presence of tumor associated macrophages. The overall objective of this review is to enhance our understanding in the design and development of therapeutic nanoparticles for treatment of cancers. PMID:24270007

  2. IRF1 marks activated genes in SLE and can induce target gene expression

    PubMed Central

    Zhang, Zhe; Shi, Lihua; Song, Li; Ephrem, Elshaddai; Petri, Michelle; Sullivan, Kathleen E.

    2014-01-01

    Objective IRF1 both mediates responses to type I interferons and the induction of interferons. It has been implicated in murine lupus models as a critical mediator of inflammation. A previous study of chromatin modifications in SLE patient monocytes implicated IRF1 as associated with increased histone acetylation in SLE patients. This study directly investigated IRF1 binding sites on chromatin using ChIP-seq. Methods Nine female SLE patients and seven female controls were examined. Monocytes were purified from peripheral blood and subjected to library preparation using a validated antibody to IRF1. The effect of IRF1 on target gene expression was confirmed using an overexpression system in cell lines and co-immunoprecipitation was used to define protein interactions. Results IRF1 binding around transcribed regions was increased in SLE patient monocytes but histone modifications at potential IRF1 binding sites without detectable IRF1 binding were also increased. IRF1 overexpression was sufficient to drive transcription of target genes. IRF1 overexpression was also able to alter histone modifications at a focus set of target genes and the use of an IRF1 inhibitor decreased both expression and histone modifications at target genes. IRF1 was found to interact with a select set of histone modifying enzymes and other transcription factors. Conclusions IRF1 is an important signaling protein in the interferon pathway. IRF1 not only activates gene expression as a transcription factor but may perpetuate disease by leading to a dysregulated epigenome. PMID:25418955

  3. Biodegradable Inorganic Nanovector: Passive versus Active Tumor Targeting in siRNA Transportation.

    PubMed

    Park, Dae-Hwan; Cho, Jaeyong; Kwon, Oh-Joon; Yun, Chae-Ok; Choy, Jin-Ho

    2016-03-24

    The biodegradable inorganic nanovector based on a layered double hydroxide (LDH) holds great promise for gene and drug delivery systems. However, in vivo targeted delivery of genes through LDH still remains a key challenge in the development of RNA interference therapeutics. Here, we describe in vivo and in vitro delivery system for Survivin siRNA (siSurvivin) assembled with passive LDH with a particle size of 100 nm or active LDH conjugated with a cancer overexpressing receptor targeting ligand, folic acid (LDHFA), conferring them an ability to target the tumor by either EPR-based clathrin-mediated or folate receptor-mediated endocytosis. When not only transfected into KB cells but also injected into xenograft mice, LDHFA/siSurvivin induced potent gene silencing at mRNA and protein levels in vitro, and consequently achieved a 3.0-fold higher suppression of tumor volume than LDH/siSurvivin in vivo. This anti-tumor effect was attributed to a selectively 1.2-fold higher accumulation of siSurvivin in tumor tissue compared with other organs. Targeting to the tumor with inorganic nanovector can guide and accelerate an evolution of next-generation theranosis system. PMID:26879376

  4. Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator.

    PubMed

    Jiang, Feng; Hua, Li-Ming; Jiao, Yun-Lu; Ye, Pin; Fu, Jin; Cheng, Zhi-Jun; Ding, Gang; Ji, Yong-Hua

    2014-02-01

    The mammalian target of rapamycin (mTOR) pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I (10 μg), induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in lumbar 5-6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4EBP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses (spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity). Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain. PMID:24132796

  5. Evidence of altered corticomotor excitability following targeted activation of gluteus maximus training in healthy individuals.

    PubMed

    Fisher, Beth E; Southam, Anna C; Kuo, Yi-Ling; Lee, Ya-Yun; Powers, Christopher M

    2016-04-13

    It has been proposed that strengthening and skill training of gluteus maximus (GM) may be beneficial in treating various knee injuries. Given the redundancy of the hip musculature and the small representational area of GM in the primary motor cortex (M1), learning to activate this muscle before prescribing strength exercises and modifying movement strategy would appear to be important. This study aimed to determine whether a short-term activation training program targeting the GM results in neuroplastic changes in M1. Using transcranial magnetic stimulation, motor evoked potentials (MEPs) were obtained in 12 healthy individuals at different stimulation intensities while they performed a double-leg bridge. Participants then completed a home exercise program for ∼1 h/day for 6 days that consisted of a single exercise designed to selectively target the GM. Baseline and post-training input-output curves (IOCs) were generated by graphing average MEP amplitudes and cortical silent period durations against corresponding stimulation intensities. Following the GM activation training, the linear slope of both the MEP IOC and cortical silent period IOC increased significantly. Short-term GM activation training resulted in a significant increase in corticomotor excitability as well as changes in inhibitory processes of the GM. We propose that the observed corticomotor plasticity will enable better utilization of the GM in the more advanced stages of a rehabilitation/training program. PMID:26981714

  6. The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

    PubMed Central

    Gao, Wei; Kim, Jin-Yong; Anderson, Jeffrey R.; Akopian, Tatos; Hong, Seungpyo; Jin, Ying-Yu; Kandror, Olga; Kim, Jong-Woo; Lee, In-Ae; Lee, Sun-Young; McAlpine, James B.; Mulugeta, Surafel; Sunoqrot, Suhair; Wang, Yuehong; Yang, Seung-Hwan; Yoon, Tae-Mi; Goldberg, Alfred L.; Pauli, Guido F.; Cho, Sanghyun

    2014-01-01

    Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development. PMID:25421483

  7. Thymosin beta4 targeting impairs tumorigenic activity of colon cancer stem cells.

    PubMed

    Ricci-Vitiani, Lucia; Mollinari, Cristiana; di Martino, Simona; Biffoni, Mauro; Pilozzi, Emanuela; Pagliuca, Alfredo; de Stefano, Maria Chiara; Circo, Rita; Merlo, Daniela; De Maria, Ruggero; Garaci, Enrico

    2010-11-01

    Thymosin β4 (Tβ4) is an actin-binding peptide overexpressed in several tumors, including colon carcinomas. The aim of this study was to investigate the role of Tβ4 in promoting the tumorigenic properties of colorectal cancer stem cells (CR-CSCs), which are responsible for tumor initiation and growth. We first found that CR-CSCs from different patients have higher Tβ4 levels than normal epithelial cells. Then, we used a lentiviral strategy to down-regulate Tβ4 expression in CR-CSCs and analyzed the effects of such modulation on proliferation, survival, and tumorigenic activity of CR-CSCs. Empty vector-transduced CR-CSCs were used as a control. Targeting of the Tβ4 produced CR-CSCs with a lower capacity to grow and migrate in culture and, interestingly, reduced tumor size and aggressiveness of CR-CSC-based xenografts in mice. Moreover, such loss in tumorigenic activity was accompanied by a significant increase of phosphatase and tensin homologue (PTEN) and a concomitant reduction of the integrin-linked kinase (ILK) expression, which resulted in a decreased activation of protein kinase B (Akt). Accordingly, exogenous expression of an active form of Akt rescued all the protumoral features lost after Tβ4 targeting in CR-CSCs. In conclusion, Tβ4 may have important implications for therapeutic intervention for treatment of human colon carcinoma. PMID:20566622

  8. Natural Killer Cell Immunomodulation: Targeting Activating, Inhibitory, and Co-stimulatory Receptor Signaling for Cancer Immunotherapy

    PubMed Central

    Chester, Cariad; Fritsch, Katherine; Kohrt, Holbrook E.

    2015-01-01

    There is compelling clinical and experimental evidence to suggest that natural killer (NK) cells play a critical role in the recognition and eradication of tumors. Efforts at using NK cells as antitumor agents began over two decades ago, but recent advances in elucidating NK cell biology have accelerated the development of NK cell-targeting therapeutics. NK cell activation and the triggering of effector functions is governed by a complex set of activating and inhibitory receptors. In the early phases of cancer immune surveillance, NK cells directly identify and lyse cancer cells. Nascent transformed cells elicit NK cell activation and are eliminated. However, as tumors progress, cancerous cells develop immunosuppressive mechanisms that circumvent NK cell-mediated killing, allowing for tumor escape and proliferation. Therapeutic intervention aims to reverse tumor-induced NK cell suppression and sustain NK cells’ tumorlytic capacities. Here, we review tumor–NK cell interactions, discuss the mechanisms by which NK cells generate an antitumor immune response, and discuss NK cell-based therapeutic strategies targeting activating, inhibitory, and co-stimulatory receptors. PMID:26697006

  9. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    PubMed Central

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background. PMID:23630316

  10. Target-Based Identification of Whole-Cell Active Inhibitors of Biotin Biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Park, Sae Woong; Casalena, Dominick; Wilson, Daniel; Dai, Ran; Nag, Partha; Liu, Feng; Boyce, Jim P.; Bittker, Joshua; Schreiber, Stuart; Finzel, Barry C.; Schnappinger, Dirk; Aldrich, Courtney C.

    2014-01-01

    SUMMARY Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counter-screen in either biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counter-screen proved crucial to filter out compounds whose whole-cell activity was off-target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were co-crystallized with BioA to provide a framework for future structure-based drug design efforts. PMID:25556942

  11. Ligand substitutions between ruthenium–cymene compounds can control protein versus DNA targeting and anticancer activity

    PubMed Central

    Adhireksan, Zenita; Davey, Gabriela E.; Campomanes, Pablo; Groessl, Michael; Clavel, Catherine M.; Yu, Haojie; Nazarov, Alexey A.; Yeo, Charmian Hui Fang; Ang, Wee Han; Dröge, Peter; Rothlisberger, Ursula; Dyson, Paul J.; Davey, Curt A.

    2014-01-01

    Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favourable toxicity and clearance properties. Nonetheless, their molecular targeting and mechanism of action are poorly understood. Here we study two prototypical ruthenium-arene agents—the cytotoxic antiprimary tumour compound [(η6-p-cymene)Ru(ethylene-diamine)Cl]PF6 and the relatively non-cytotoxic antimetastasis compound [(η6-p-cymene)Ru(1,3,5-triaza-7-phosphaadamantane)Cl2]—and discover that the former targets the DNA of chromatin, while the latter preferentially forms adducts on the histone proteins. Using a novel ‘atom-to-cell’ approach, we establish the basis for the surprisingly site-selective adduct formation behaviour and distinct cellular impact of these two chemically similar anticancer agents, which suggests that the cytotoxic effects arise largely from DNA lesions, whereas the protein adducts may be linked to the other therapeutic activities. Our study shows promise for developing new ruthenium drugs, via ligand-based modulation of DNA versus protein binding and thus cytotoxic potential, to target distinguishing epigenetic features of cancer cells. PMID:24637564

  12. Electrical properties of various gas mixtures for active target detector application

    NASA Astrophysics Data System (ADS)

    Yates, Daniel; Rogachev, Grigory; Koshchiy, Evgeniy; Uberseder, Ethan; Hooker, Josh

    2015-10-01

    Experiments with rare isotope beams (RIBs) open new opportunities to study properties of exotic nuclei and measure reaction cross sections relevant for nuclear astrophysics with radioactive ions. However, the low intensity of RIBs requires the development of new, more efficient detectors such as the Texas Active Target (TexAT) detector currently being developed at the Cyclotron Institute. With this detector, the target gas is also used as the active medium for tracking and energy loss measurements of charged recoils. Various gas mixtures will be used under different conditions and it is important that drift velocity and gas gain are well established. This study uses a time projection chamber with an applied electric field to measure drift velocity and electron gains of four gases to be used as targets in TexAT. The experimental values are then compared to simulation. Drift velocities of electrons were measured as a function of the electric field for each gas and pressure and then were compared to simulated values obtained from CERN's Garfield + + simulation package. The simulated and experimental drift velocities matched with root-mean-square deviations typically less than 10% for each pressure. These results provide important accuracy verification of the simulation programs and determine systematic uncertainties in track reconstructions with TexAT which rely on these simulations. Supported by NSF Grant No. 1263281.

  13. The search of the target of promotion: Phenylbenzoate esterase activities in hen peripheral nerve

    SciTech Connect

    Moretto, A. . E-mail: angelo.moretto@icps.it; Nicolli, A.; Lotti, M.

    2007-03-15

    Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crude homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC{sub 50} of about 0.08 mM (20 min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds.

  14. Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

    PubMed

    Loureiro, Ana; Nogueira, Eugénia; Azoia, Nuno G; Sárria, Marisa P; Abreu, Ana S; Shimanovich, Ulyana; Rollett, Alexandra; Härmark, Johan; Hebert, Hans; Guebitz, Georg; Bernardes, Gonçalo J L; Preto, Ana; Gomes, Andreia C; Cavaco-Paulo, Artur

    2015-11-01

    Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy. PMID:26241920

  15. Recent advances of cytotoxic chalconoids targeting tubulin polymerization: Synthesis and biological activity.

    PubMed

    Mirzaei, Hassan; Emami, Saeed

    2016-10-01

    Since microtubules have an important role in mitosis and other vital cellular functions, tubulin-targeting chemotherapy has been received growing attention in anticancer drug design and development. It was found that a number of naturally occurring compounds including distinct chalcones exert their effect by inhibition of tubulin polymerization. After the identification of tubulin polymerization as potential target for chalcone-type compounds, extensive researches have been made to design and synthesis of new anti-tubulin chalconoids. Although diverse chalcones have found to be potent anticancer agents but in the present review, we focused on the recently reported tubulin polymerization inhibitors from chalcone origin and related synthetic compounds, and their detailed synthetic methods and biological activities. PMID:27318983

  16. Targeting KIT on innate immune cells to enhance the antitumor activity of checkpoint inhibitors.

    PubMed

    Stahl, Maximilian; Gedrich, Richard; Peck, Ronald; LaVallee, Theresa; Eder, Joseph Paul

    2016-06-01

    Innate immune cells such as mast cells and myeloid-derived suppressor cells are key components of the tumor microenvironment. Recent evidence indicates that levels of myeloid-derived suppressor cells in melanoma patients are associated with poor survival to checkpoint inhibitors. This suggests that targeting both the innate and adaptive suppressive components of the immune system will maximize clinical benefit and elicit more durable responses in cancer patients. Preclinical data suggest that targeting signaling by the receptor tyrosine kinase KIT, particularly on mast cells, may modulate innate immune cell numbers and activity in tumors. Here, we review data highlighting the importance of the KIT signaling in regulating antitumor immune responses and the potential benefit of combining selective KIT inhibitors with immune checkpoint inhibitors. PMID:27349976

  17. Impact of high-risk conjunctions on Active Debris Removal target selection

    NASA Astrophysics Data System (ADS)

    Lidtke, Aleksander A.; Lewis, Hugh G.; Armellin, Roberto

    2015-10-01

    Space debris simulations show that if current space launches continue unchanged, spacecraft operations might become difficult in the congested space environment. It has been suggested that Active Debris Removal (ADR) might be necessary in order to prevent such a situation. Selection of objects to be targeted by ADR is considered important because removal of non-relevant objects will unnecessarily increase the cost of ADR. One of the factors to be used in this ADR target selection is the collision probability accumulated by every object. This paper shows the impact of high-probability conjunctions on the collision probability accumulated by individual objects as well as the probability of any collision occurring in orbit. Such conjunctions cannot be predicted far in advance and, consequently, not all the objects that will be involved in such dangerous conjunctions can be removed through ADR. Therefore, a debris remediation method that would address such events at short notice, and thus help prevent likely collisions, is suggested.

  18. Active Target detectors for studies with exotic beams: Present and next future

    NASA Astrophysics Data System (ADS)

    Mittig, W.; Beceiro-Novo, S.; Fritsch, A.; Abu-Nimeh, F.; Bazin, D.; Ahn, T.; Lynch, W. G.; Montes, F.; Shore, A.; Suzuki, D.; Usher, N.; Yurkon, J.; Kolata, J. J.; Howard, A.; Roberts, A. L.; Tang, X. D.; Becchetti, F. D.

    2015-06-01

    Reaccelerated radioactive beams near the Coulomb barrier, which are starting to be available from the ReA3 accelerator at NSCL and in next future at FRIB, will open up new opportunities for the study of nuclear structure near the drip lines. Efficient measurement techniques must be developed to compensate for the limited intensity of the most exotic beams. The Active-Target Time Projection Chamber (AT-TPC) constructed at MSU solves this problem by providing the increased luminosity of a thick target while maintaining a good energy resolution by tracking the reaction vertex over an essentially 4 π solid angle. The AT-TPC and similar detectors allow us to take full advantage of the radioactive ion beams at present and future nuclear physics facilities to explore the frontier of rare isotopes.

  19. Targeting macrophage activation for the prevention and treatment of S. aureus biofilm infections†

    PubMed Central

    Hanke, Mark L.; Heim, Cortney E.; Angle, Amanda; Sanderson, Sam D.; Kielian, Tammy

    2013-01-01

    Biofilm infections often lead to significant morbidity due to their chronicity and recalcitrance to antibiotics. We have demonstrated that methicillin-resistant Staphylococcus aureus (MRSA) biofilms can evade macrophage antibacterial effector mechanisms by skewing macrophages towards an alternatively activated M2 phenotype. To overcome this immune evasion, we have utilized two complementary approaches. In the first, a proinflammatory milieu was elicited by local administration of classically-activated M1 macrophages and second, by treatment with the C5a receptor (CD88) agonist EP67, which invokes macrophage proinflammatory activity. Early administration of M1-activated macrophages or EP67 significantly attenuated biofilm formation in a mouse model of MRSA catheter-associated infection. Several proinflammatory mediators were significantly elevated in biofilm infected tissues from macrophage- and EP67-treated animals, revealing effective reprogramming of the biofilm environment to a proinflammatory milieu. A requirement for macrophage proinflammatory activity was demonstrated by the fact that transfer of MyD88-deficient macrophages had minimal impact on biofilm growth. Likewise, neutrophil administration had no effect on biofilm formation. Treatment of established biofilm infections with M1-activated macrophages also significantly reduced catheter-associated biofilm burdens compared to antibiotic treatment. Collectively, these results demonstrate that targeting macrophage proinflammatory activity can overcome the local immune inhibitory environment created during biofilm infections and represents a novel therapeutic strategy. PMID:23365077

  20. A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity

    PubMed Central

    Peltonen, Karita; Colis, Laureen; Liu, Hester; Trivedi, Rishi; Moubarek, Michael S.; Moore, Henna M.; Bai, Bayoan; Rudek, Michelle A.; Bieberich, Charles J.; Laiho, Marikki

    2014-01-01

    SUMMARY We define here the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which is present at high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity. PMID:24434211

  1. Cucurbitacins: potential candidates targeting mitogen-activated protein kinase pathway for treatment of melanoma.

    PubMed

    Ahmed, Mahmoud S; Halaweish, Fathi T

    2014-04-01

    Cucurbitacins (Cucs) have been classified as signal transducer and activator of transcription 3 inhibitors. Kinase inhibition has been a validated drug target in multiple types of malignancies. B-RAF mutations are highly expressed in the melanoma. Our hypothesis is the Cucs can be a potential candidate to inhibit the signaling kinase pathway. The research presented is the evaluation of Cucs, as B-RAF and MEK1 kinase inhibitors. Virtual screening methods were employed to identify lead compounds. The hypothesis was tested on mutant B-RAF cell lines, A-375 and Sk-Mel-28 cell lines to determine the activity toward melanoma. A series of natural Cucs show an improved activity toward Sk-Mel-28 and A-375 cell lines. Cucs show potential inhibition for the total and phosphorylated ERK using ELISA kits. Cucs could be potential candidate for inhibiting cell growth. PMID:23368732

  2. Preclinical evaluation of a urokinase plasminogen activator receptor-targeted nanoprobe in rhesus monkeys

    PubMed Central

    Chen, Yushu; Gong, Li; Gao, Ning; Liao, Jichun; Sun, Jiayu; Wang, Yuqing; Wang, Lei; Zhu, Pengjin; Fan, Qing; Wang, Yongqiang Andrew; Zeng, Wen; Mao, Hui; Yang, Lily; Gao, Fabao

    2015-01-01

    Purpose To translate a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator receptor-targeted magnetic iron oxide (IO) nanoparticles (uPAR-targeted human ATF-IONPs) into clinical applications, we conducted a pilot study to evaluate the toxicity and pharmacokinetics of this nanoparticle in normal rhesus monkeys. Methods We assessed the changes in the following: magnetic resonance imaging (MRI) signals from pretreatment stage to 14 days posttreatment, serum iron concentrations from 5 minutes posttreatment to 12 weeks posttreatment, routine blood examination and serum chemistry analysis results from pretreatment stage to 12 weeks after administration, and results of staining of the liver with Perls’ Prussian Blue and hematoxylin–eosin at 24 hours and 3 months posttreatment in two rhesus monkeys following an intravenous administration of the targeted nanoparticles either with a polyethylene glycol (ATF-PEG-IONP) or without a PEG (ATF-IONP) coating. Results The levels of alkaline phosphatase, alanine transaminase, and direct bilirubin in the two monkeys increased immediately after the administration of the IONPs but returned to normal within 20 days and stayed within the normal reference range 3 months after the injection. The creatinine levels of the two monkeys stayed within the normal range during the study. In addition, red blood cells, white blood cells, hemoglobin level, and platelets remained normal during the 3 months of the study. Conclusion All of the results suggest that a transient injury in terms of normal organ functions, but no microscopic necrotic lesions, was observed at a systemic delivery dose of 5 mg/kg of iron equivalent concentration in the acute phase, and that no chronic toxicity was found 3 months after the injection. Therefore, we conclude that uPAR-targeted IONPs have the potential to be used as receptor-targeted MRI contrasts as well as theranostic agents for the detection and treatment of

  3. Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya.

    PubMed

    Ohto, H; Kamada, S; Tago, K; Tominaga, S I; Ozaki, H; Sato, S; Kawakami, K

    1999-10-01

    Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes. PMID:10490620

  4. Target-Cell Contact Activates a Highly Selective Capacitative Calcium Entry Pathway in Cytotoxic T Lymphocytes

    PubMed Central

    Zweifach, Adam

    2000-01-01

    Calcium influx is critical for T cell activation. Evidence has been presented that T cell receptor–stimulated calcium influx in helper T lymphocytes occurs via channels activated as a consequence of depletion of intracellular calcium stores, a mechanism known as capacitative Ca2+ entry (CCE). However, two key questions have not been addressed. First, the mechanism of calcium influx in cytotoxic T cells has not been examined. While the T cell receptor–mediated early signals in helper and cytotoxic T cells are similar, the physiology of the cells is strikingly different, raising the possibility that the mechanism of calcium influx is also different. Second, contact of T cells with antigen-presenting cells or targets involves a host of intercellular interactions in addition to those between antigen–MHC and the T cell receptor. The possibility that calcium influx pathways in addition to those activated via the T cell receptor may be activated by contact with relevant cells has not been addressed. We have used imaging techniques to show that target-cell–stimulated calcium influx in CTLs occurs primarily through CCE. We investigated the permeability of the CTL influx pathway for divalent cations, and compared it to the permeability of CCE in Jurkat human leukemic T cells. CCE in CTLs shows a similar ability to discriminate between calcium, barium, and strontium as CCE in Jurkat human leukemic T lymphocytes, where CCE is likely to mediated by Ca2+ release–activated Ca2+ current (CRAC) channels, suggesting that CRAC channels also underlie CCE in CTLs. These results are the first determination of the mechanism of calcium influx in cytotoxic T cells and the first demonstration that cell contact–mediated calcium signals in T cells occur via depletion-activated channels. PMID:10662784

  5. Transcriptional regulatory networks downstream of TAL1/SCL in T-cell acute lymphoblastic leukemia.

    PubMed

    Palomero, Teresa; Odom, Duncan T; O'Neil, Jennifer; Ferrando, Adolfo A; Margolin, Adam; Neuberg, Donna S; Winter, Stuart S; Larson, Richard S; Li, Wei; Liu, X Shirley; Young, Richard A; Look, A Thomas

    2006-08-01

    Aberrant expression of 1 or more transcription factor oncogenes is a critical component of the molecular pathogenesis of human T-cell acute lymphoblastic leukemia (T-ALL); however, oncogenic transcriptional programs downstream of T-ALL oncogenes are mostly unknown. TAL1/SCL is a basic helix-loop-helix (bHLH) transcription factor oncogene aberrantly expressed in 60% of human T-ALLs. We used chromatin immunoprecipitation (ChIP) on chip to identify 71 direct transcriptional targets of TAL1/SCL. Promoters occupied by TAL1 were also frequently bound by the class I bHLH proteins E2A and HEB, suggesting that TAL1/E2A as well as TAL1/HEB heterodimers play a role in transformation of T-cell precursors. Using RNA interference, we demonstrated that TAL1 is required for the maintenance of the leukemic phenotype in Jurkat cells and showed that TAL1 binding can be associated with either repression or activation of genes whose promoters occupied by TAL1, E2A, and HEB. In addition, oligonucleotide microarray analysis of RNA from 47 primary T-ALL samples showed specific expression signatures involving TAL1 targets in TAL1-expressing compared with -nonexpressing human T-ALLs. Our results indicate that TAL1 may act as a bifunctional transcriptional regulator (activator and repressor) at the top of a complex regulatory network that disrupts normal T-cell homeostasis and contributes to leukemogenesis. PMID:16621969

  6. Targeting Microglial Activation in Stroke Therapy: Pharmacological Tools and Gender Effects

    PubMed Central

    Chen, Y.; Won, S.J.; Xu, Y.; Swanson, R.A.

    2014-01-01

    Ischemic stroke is caused by critical reductions in blood flow to brain or spinal cord. Microglia are the resident immune cells of the central nervous system, and they respond to stroke by assuming an activated phenotype that releases cytotoxic cytokines, reactive oxygen species, proteases, and other factors. This acute, innate immune response may be teleologically adapted to limit infection, but in stroke this response can exacerbate injury by further damaging or killing nearby neurons and other cell types, and by recruiting infiltration of circulating cytotoxic immune cells. The microglial response requires hours to days to fully develop, and this time interval presents a clinically accessible time window for initiating therapy. Because of redundancy in cytotoxic microglial responses, the most effective therapeutic approach may be to target the global gene expression changes involved in microglial activation. Several classes of drugs can do this, including histone deacetylase inhibitors, minocycline and other PARP inhibitors, corticosteroids, and inhibitors of TNFα and scavenger receptor signaling. Here we review the pre-clinical studies in which these drugs have been used to suppress microglial activation after stroke. We also review recent advances in the understanding of sex differences in the CNS inflammatory response, as these differences are likely to influence the efficacy of drugs targeting post-stroke brain inflammation. PMID:24372213

  7. Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation.

    PubMed

    Li, Zhenfei; Nie, Fen; Wang, Sheng; Li, Lin

    2011-02-22

    Histone methylation has an important role in transcriptional regulation. However, unlike H3K4 and H3K9 methylation, the role of H4K20 monomethylation (H4K20me-1) in transcriptional regulation remains unclear. Here, we show that Wnt3a specifically stimulates H4K20 monomethylation at the T cell factor (TCF)-binding element through the histone methylase SET8. Additionally, SET8 is crucial for activation of the Wnt reporter gene and target genes in both mammalian cells and zebrafish. Furthermore, SET8 interacts with lymphoid enhancing factor-1 (LEF1)/TCF4 directly, and this interaction is regulated by Wnt3a. Therefore, we conclude that SET8 is a Wnt signaling mediator and is recruited by LEF1/TCF4 to regulate the transcription of Wnt-activated genes, possibly through H4K20 monomethylation at the target gene promoters. Our findings also indicate that H4K20me-1 is a marker for gene transcription activation, at least in canonical Wnt signaling. PMID:21282610

  8. Initial characterization of a dually radiolabeled peptide for simultaneous monitoring of protein targets and enzymatic activity

    PubMed Central

    Mebrahtu, Efrem; Zheleznyak, Alexander; Hur, Minjun A.; Laforest, Richard; Lapi, Suzanne E.

    2016-01-01

    Objective The goal of this study was to develop dually radiolabeled peptides for simultaneous imaging of cancer cell localization by targeting the αvβ3 integrin and their pathophysiology by targeting the activity of the proteolytic enzyme MMP2, involved in the metastatic process. Methods A hybrid peptide c(RGDfE)K(DOTA)PLGVRY containing a RGD motif for binding to the αvβ3 integrin, a metal chelator (DOTA) for radiolabeling with [64Cu], and the MMP2 substrate cleavage sequence PLGVRY with terminal tyrosine for labeling with [123I] was synthesized, labeled with [64Cu] and [123I], and evaluated in vitro as a potential imaging agent. Results The peptide was synthesized and labeled with [64Cu] and [123I] with 300 and 40 μCi/μg (542 and 72.2 mCi/μmol) specific activities, respectively, and radiochemical purity of>98%.c(RGDfE)K(DOTA)PLGVRY demonstrated high affinity for αvβ3 integrins(Kd = 83.4 ± 13.2 nM) in both substrate competition and cell binding assays. c(RGDfE)K(DOTA)PLGVRY peptide, but not the scrambled version, c(RGDfE)K(DOTA)GRPLVY was specifically cleaved by MMP2. Conclusions These results demonstrate the feasibility of developing dually radiolabeled peptides for the simultaneous imaging of cancer cells and their pathophysiologic activity. PMID:23154178

  9. Activation of wingless targets requires bipartite recognition of DNA by TCF.

    PubMed

    Chang, Mikyung V; Chang, Jinhee L; Gangopadhyay, Anu; Shearer, Andrew; Cadigan, Ken M

    2008-12-01

    Specific recognition of DNA by transcription factors is essential for precise gene regulation. In Wingless (Wg) signaling in Drosophila, target gene regulation is controlled by T cell factor (TCF), which binds to specific DNA sequences through a high mobility group (HMG) domain. However, there is considerable variability in TCF binding sites, raising the possibility that they are not sufficient for target location. Some isoforms of human TCF contain a domain, termed the C-clamp, that mediates binding to an extended sequence in vitro. However, the significance of this extended sequence for the function of Wnt response elements (WREs) is unclear. In this report, we identify a cis-regulatory element that, to our knowledge, was previously unpublished. The element, named the TCF Helper site (Helper site), is essential for the activation of several WREs. This motif greatly augments the ability of TCF binding sites to respond to Wg signaling. Drosophila TCF contains a C-clamp that enhances in vitro binding to TCF-Helper site pairs and is required for transcriptional activation of WREs containing Helper sites. A genome-wide search for clusters of TCF and Helper sites identified two new WREs. Our data suggest that DNA recognition by fly TCF occurs through a bipartite mechanism, involving both the HMG domain and the C-clamp, which enables TCF to locate and activate WREs in the nucleus. PMID:19062282

  10. Targeted recruitment of adults with type 2 diabetes for a physical activity intervention.

    PubMed

    Johnson, Elizabeth J; Niles, Barbara L; Mori, DeAnna L

    2015-05-01

    Recruiting sufficient numbers of participants for physical activity trials for individuals with diabetes can be difficult because there are often many behavioral demands for participants, and inclusion and exclusion criteria can be extensive. This study examined the recruitment strategies used for a randomized, controlled trial designed to investigate the efficacy of an automated telephone intervention to promote physical activity in adults with type 2 diabetes in an urban Veterans Administration health care system. Traditional recruitment approaches of posting flyers and obtaining referrals from clinicians did not yield sufficient numbers of interested patients. Using the electronic medical record system to identify patients with uncontrolled diabetes allowed staff to send targeted mailings to participants, and 77% of participants were recruited using this method. The targeted mailing approach elicited a positive response rate of 12% (328 of 2,764 potential participants identified) and appeared to produce a more representative and appropriate sample than other recruitment methods used. Lessons learned in this study may be helpful to researchers in future trials who attempt to recruit participants with diabetes for physical activity protocols. PMID:25987808

  11. The yeast Hot1 transcription factor is critical for activating a single target gene, STL1

    PubMed Central

    Bai, Chen; Tesker, Masha; Engelberg, David

    2015-01-01

    Transcription factors are commonly activated by signal transduction cascades and induce expression of many genes. They therefore play critical roles in determining the cell's fate. The yeast Hog1 MAP kinase pathway is believed to control the transcription of hundreds of genes via several transcription factors. To identify the bona fide target genes of Hog1, we inducibly expressed the spontaneously active variant Hog1D170A+F318L in cells lacking the Hog1 activator Pbs2. This system allowed monitoring the effects of Hog1 by itself. Expression of Hog1D170A+F318L in pbs2∆ cells imposed induction of just 105 and suppression of only 26 transcripts by at least twofold. We looked for the Hog1-responsive element within the promoter of the most highly induced gene, STL1 (88-fold). A novel Hog1 responsive element (HoRE) was identified and shown to be the direct target of the transcription factor Hot1. Unexpectedly, we could not find this HoRE in any other yeast promoter. In addition, the only gene whose expression was abolished in hot1∆ cells was STL1. Thus Hot1 is essential for transcription of just one gene, STL1. Hot1 may represent a class of transcription factors that are essential for transcription of a very few genes or even just one. PMID:25904326

  12. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2·− production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703–712. PMID:19761405

  13. Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

    PubMed Central

    Hui, Xin; Reither, Gregor; Kaestner, Lars

    2014-01-01

    Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of Gαs and Gαq, respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of Gαs and Gαq resulted in a differential translocation of the conventional PKCα to the plasma membrane while the novel PKCδ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKCδ translocation was driven by a novel Gαs-cyclic AMP-EPAC-RAP-PLCε pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKCδ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca2+ signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events. PMID:24732802

  14. Computational screening for active compounds targeting protein sequences: methodology and experimental validation.

    PubMed

    Wang, Fei; Liu, Dongxiang; Wang, Heyao; Luo, Cheng; Zheng, Mingyue; Liu, Hong; Zhu, Weiliang; Luo, Xiaomin; Zhang, Jian; Jiang, Hualiang

    2011-11-28

    The three-dimensional (3D) structures of most protein targets have not been determined so far, with many of them not even having a known ligand, a truly general method to predict ligand-protein interactions in the absence of three-dimensional information would be of great potential value in drug discovery. Using the support vector machine (SVM) approach, we constructed a model for predicting ligand-protein interaction based only on the primary sequence of proteins and the structural features of small molecules. The model, trained by using 15,000 ligand-protein interactions between 626 proteins and over 10,000 active compounds, was successfully used in discovering nine novel active compounds for four pharmacologically important targets (i.e., GPR40, SIRT1, p38, and GSK-3β). To our knowledge, this is the first example of a successful sequence-based virtual screening campaign, demonstrating that our approach has the potential to discover, with a single model, active ligands for any protein. PMID:21955088

  15. Novel epigallocatechin gallate (EGCG) analogs activate AMP-activated protein kinase pathway and target cancer stem cells

    PubMed Central

    Chen, Di; Pamu, Sreedhar; Cui, Qiuzhi; Chan, Tak Hang; Dou, Q. Ping

    2012-01-01

    AMP-activated protein kinase (AMPK) is a critical monitor of cellular energy status and also controls processes related to tumor development, including cell cycle progression, protein synthesis, cell growth and survival. Therefore AMPK as an anti-cancer target has received intensive attention recently. It has been reported that the anti-diabetic drug metformin and some natural compounds, such as quercetin, genistein, capsaicin and green tea polyphenol epigallocatechin gallate (EGCG), can activate AMPK and inhibit cancer cell growth. Indeed, natural products have been the most productive source of leads for the development of anti-cancer drugs but perceived disadvantages, such as low bioavailability and week potency, have limited their development and use in the clinic. In this study we demonstrated that synthetic EGCG analogs 4 and 6 were more potent AMPK activators than metformin and EGCG. Activation of AMPK by these EGCG analogs resulted in inhibition of cell proliferation, up-regulation of the cyclin-dependent kinase inhibitor p21, down-regulation of mTOR pathway, and suppression of stem cell population in human breast cancer cells. Our findings suggest that novel potent and specific AMPK activators can be discovered from natural and synthetic sources that have potential to be used for anti-cancer therapy in the clinic. PMID:22459208

  16. Molecular photoacoustic imaging of breast cancer using an actively targeted conjugated polymer

    PubMed Central

    Balasundaram, Ghayathri; Ho, Chris Jun Hui; Li, Kai; Driessen, Wouter; Dinish, US; Wong, Chi Lok; Ntziachristos, Vasilis; Liu, Bin; Olivo, Malini

    2015-01-01

    Conjugated polymers (CPs) are upcoming optical contrast agents in view of their unique optical properties and versatile synthetic chemistry. Biofunctionalization of these polymer-based nanoparticles enables molecular imaging of biological processes. In this work, we propose the concept of using a biofunctionalized CP for noninvasive photoacoustic (PA) molecular imaging of breast cancer. In particular, after verifying the PA activity of a CP nanoparticle (CP dots) in phantoms and the targeting efficacy of a folate-functionalized version of the same (folate-CP dots) in vitro, we systemically administered the probe into a folate receptor-positive (FR+ve) MCF-7 breast cancer xenograft model to demonstrate the possible application of folate-CP dots for imaging FR+ve breast cancers in comparison to CP dots with no folate moieties. We observed a strong PA signal at the tumor site of folate-CP dots-administered mice as early as 1 hour after administration as a result of the active targeting of the folate-CP dots to the FR+ve tumor cells but a weak PA signal at the tumor site of CP-dots-administered mice as a result of the passive accumulation of the probe by enhanced permeability and retention effect. We also observed that folate-CP dots produced ~4-fold enhancement in the PA signal in the tumor, when compared to CP dots. These observations demonstrate the great potential of this active-targeting CP to be used as a contrast agent for molecular PA diagnostic imaging in various biomedical applications. PMID:25609951

  17. Downstream processing of biopharmaceutical proteins produced in plants

    PubMed Central

    Buyel, Johannes Felix; Fischer, Rainer

    2014-01-01

    All biological platforms for the manufacture of biopharmaceutical proteins produce an initially turbid extract that must be clarified to avoid fouling sensitive media such as chromatography resins. Clarification is more challenging if the feed stream contains large amounts of dispersed particles, because these rapidly clog the filter media typically used to remove suspended solids. Charged polymers (flocculants) can increase the apparent size of the dispersed particles by aggregation, facilitating the separation of solids and liquids, and thus reducing process costs. However, many different factors can affect the behavior of flocculants, including the pH and conductivity of the medium, the size and charge distribution of the particulates, and the charge density and molecular mass of the polymer. Importantly, these properties can also affect the recovery of the target protein and the overall safety profile of the process. We therefore used a design of experiments approach to establish reliable predictive models that characterize the impact of flocculants during the downstream processing of biopharmaceutical proteins. We highlight strategies for the selection of flocculants during process optimization. These strategies will contribute to the quality by design aspects of process development and facilitate the development of safe and efficient downstream processes for plant-derived pharmaceutical proteins. PMID:24637706

  18. ROMA: Representation and Quantification of Module Activity from Target Expression Data

    PubMed Central

    Martignetti, Loredana; Calzone, Laurence; Bonnet, Eric; Barillot, Emmanuel; Zinovyev, Andrei

    2016-01-01

    In many analyses of high-throughput data in systems biology, there is a need to quantify the activity of a set of genes in individual samples. A typical example is the case where it is necessary to estimate the activity of a transcription factor (which is often not directly measurable) from the expression of its target genes. We present here ROMA (Representation and quantification Of Module Activities) Java software, designed for fast and robust computation of the activity of gene sets (or modules) with coordinated expression. ROMA activity quantification is based on the simplest uni-factor linear model of gene regulation that approximates the expression data of a gene set by its first principal component. The proposed algorithm implements novel functionalities: it provides several method modifications for principal components computation, including weighted, robust and centered methods; it distinguishes overdispersed modules (based on the variance explained by the first principal component) and coordinated modules (based on the significance of the spectral gap); finally, it computes statistical significance of the estimated module overdispersion or coordination. ROMA can be applied in many contexts, from estimating differential activities of transcriptional factors to finding overdispersed pathways in single-cell transcriptomics data. We describe here the principles of ROMA providing several practical examples of its use. ROMA source code is available at https://github.com/sysbio-curie/Roma. PMID:26925094

  19. Single-molecule study of protein-DNA target search mechanisms for dimer-active protein complexes

    NASA Astrophysics Data System (ADS)

    Landry, Markita; Huang, Wai Mun; Chemla, Yann

    2012-02-01

    Protein-DNA interactions are essential to cellular processes, many of which require proteins to recognize a specific DNA target-site. This search process is well-documented for monomeric proteins, but not as well understood for systems that require dimerization at the target site for activity. We present a single-molecule study of the target-search mechanism of Protelomerase TelK, a recombinase-like protein that is only active as a dimer. We observe that TelK undergoes 1D diffusion on non-target DNA as a monomer, as expected, but becomes immobile on DNA as a dimer or oligomer despite the absence of its target site. We further show that TelK condenses non-target DNA upon dimerization, forming a tightly bound nucleo-protein complex. Together with simulations, our results suggest a search model whereby monomers diffuse along DNA, and subsequently dimerize to form an active complex on target DNA. These results show that target-finding occurs faster than nonspecific dimerization at biologically relevant protein concentrations. This model may provide insights into the search mechanisms of proteins that are active as multimeric complexes for a more accurate and comprehensive model for the target-search process by sequence specific proteins.

  20. Rapid, Specific, No-wash, Far-red Fluorogen Activation in Subcellular Compartments by Targeted Fluorogen Activating Proteins

    PubMed Central

    2015-01-01

    Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20–30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments. PMID:25650487

  1. Development of CNS Active Target for Deuteron Induced Reactions with High Intensity Exotic Beam

    NASA Astrophysics Data System (ADS)

    Ota, Shinsuke; Tokieda, H.; Lee, C. S.; Kojima, R.; Watanabe, Y. N.; Corsi, A.; Dozono, M.; Gibelin, J.; Hashimoto, T.; Kawabata, T.; Kawase, S.; Kubono, S.; Kubota, Y.; Maeda, Y.; Matsubara, H.; Matsuda, Y.; Michimasa, S.; Nakao, T.; Nishi, T.; Obertelli, A.; Otsu, H.; Santamaria, C.; Sasano, M.; Takaki, M.; Tanaka, Y.; Leung, T.; Uesaka, T.; Yako, K.; Yamaguchi, H.; Zenihiro, J.; Takada, E.

    An active target system called CAT, has been developed aiming at the measurement of deuteron induced reactions with high intensity beams in inverse kinematics. The CAT consists of a time projection chamber using THGEM and an array of Si detectors or NaI scintilators. The effective gain for the recoil particle is deisgned to be 5 - 10 × 103, while one for the beam is reduced by 102 using mesh grid to match the amplified signal to the dynamic range same as the one for recoil particle. The structure of CAT and the effect of the mesh grid are reported.

  2. Parasite Mitogen-Activated Protein Kinases as Drug Discovery Targets to Treat Human Protozoan Pathogens

    PubMed Central

    Brumlik, Michael J.; Pandeswara, Srilakshmi; Ludwig, Sara M.; Murthy, Kruthi; Curiel, Tyler J.

    2011-01-01

    Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known. PMID:21637385

  3. Therapeutic Targets for Neurodevelopmental Disorders Emerging from Animal Models with Perinatal Immune Activation

    PubMed Central

    Ibi, Daisuke; Yamada, Kiyofumi

    2015-01-01

    Increasing epidemiological evidence indicates that perinatal infection with various viral pathogens enhances the risk for several psychiatric disorders. The pathophysiological significance of astrocyte interactions with neurons and/or gut microbiomes has been reported in neurodevelopmental disorders triggered by pre- and postnatal immune insults. Recent studies with the maternal immune activation or neonatal polyriboinosinic polyribocytidylic acid models of neurodevelopmental disorders have identified various candidate molecules that could be responsible for brain dysfunction. Here, we review the functions of several candidate molecules in neurodevelopment and brain function and discuss their potential as therapeutic targets for psychiatric disorders. PMID:26633355

  4. History of the bubble chamber and related active- and internal-target nuclear tracking detectors

    NASA Astrophysics Data System (ADS)

    Becchetti, F. D.

    2015-06-01

    Donald Glaser, 1960 Nobel laureate in Physics, recently passed away (2013), as have many of his colleagues who were involved with the early development of bubble chambers at the University of Michigan. In this paper I will review those early years and the subsequent wide-spread application of active-target (AT) bubble chambers that dominated high-energy physics (HEP) research for over thirty years. Some of the related, but more modern nuclear tracking detectors being used in HEP, neutrino astrophysics and dark-matter searches also will be discussed.

  5. Active Helium 3/4 Target for Use in MAMI Crystal Ball

    NASA Astrophysics Data System (ADS)

    Demell, Jennifer; A2 Collaboration

    2014-09-01

    By using a Helium 3 Active target (AT) with the A2 photon beam in MAMI, the polarizability of the neutron, a value that has not yet been well defined, can be determined. In order to be used in the MAMI Crystal Ball, the size of the 3He Active Target needed to be decreased. For our experiment we tested new, compact photomultiplier tubes (PMTs) by comparing their response to changes in nitrogen admixture concentration to those of the original, larger PMTs. We also examined the contribution of NINO discriminators, to be attached to the new PMTs to decrease noise effects. We found that the new PMTs and NINO discriminator functioned well and will be used in the future experiment, though a decrease in voltage detection was experienced. Additionally, using AT Geant4, simulations of the upcoming experiment were performed and background and resolution studies conducted. We specifically examined mass loss due to quasi free Compton Scattering, π0 production and the breakup of the 3He nucleus. By using a Helium 3 Active target (AT) with the A2 photon beam in MAMI, the polarizability of the neutron, a value that has not yet been well defined, can be determined. In order to be used in the MAMI Crystal Ball, the size of the 3He Active Target needed to be decreased. For our experiment we tested new, compact photomultiplier tubes (PMTs) by comparing their response to changes in nitrogen admixture concentration to those of the original, larger PMTs. We also examined the contribution of NINO discriminators, to be attached to the new PMTs to decrease noise effects. We found that the new PMTs and NINO discriminator functioned well and will be used in the future experiment, though a decrease in voltage detection was experienced. Additionally, using AT Geant4, simulations of the upcoming experiment were performed and background and resolution studies conducted. We specifically examined mass loss due to quasi free Compton Scattering, π0 production and the breakup of the 3He nucleus

  6. Oxaliplatin immuno hybrid nanoparticles for active targeting: an approach for enhanced apoptotic activity and drug delivery to colorectal tumors.

    PubMed

    Tummala, Shashank; Gowthamarajan, K; Satish Kumar, M N; Wadhwani, Ashish

    2016-06-01

    Tumor necrosis factor related apoptosis inducing ligand (TRAIL) proved to be a promising new target for colorectal cancer treatment. Elevated expression of TRAIL protein in tumor cells distinguishes it from healthy cells, thereby delivering the drug at the specific site. Here, we formulated oxaliplatin immunohybrid nanoparticles (OIHNPs) to deliver oxaliplatin and anti-TRAIL for colorectal cancer treatment in xenograft tumor models. The polymeric chitosan layer binds to the lipid film with the mixture of phospholipids by an ultra sound method followed by conjugating with thiolated antibody using DSPE-PEG-mal3400, resulting in the formation of OIHNPs. The polymer layer helps in more encapsulation of the drug (71 ± 0.09%) with appreciable particle size (95 ± 0.01 nm), and lipid layer prevents degradation of the drug in serum by preventing nanoparticle aggregation. OIHNPs have shown a 4-fold decrease in the IC50 value compared to oxaliplatin in HT-29 cells by the MTT assay. These immuno-nanoparticles represent the successful uptake and internalization of oxaliplatin in HT-29 cells rather than in MCF-7 cells determined by triple fluorescence method. Apoptotic activity in vitro of OIHNPs was determined by the change in the mitochondria membrane potential that further elevates its anti-tumor property. Furthermore, the conjugated nanoparticles can effectively deliver the drug to the tumor sites, which can be attributed to its ability in reducing tumor mass and tumor volume in xenograft tumor models in vivo along with sustaining its release in vitro. These findings indicated that the oxaliplatin immuno-hybrid nanoparticles would be a promising nano-sized active targeted formulation for colorectal-tumor targeted therapy. PMID:26377238

  7. HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment

    SciTech Connect

    Weigelt, Britta; Lo, Alvin T; Park, Catherine C; Gray, Joe W; Bissell, Mina J

    2009-07-27

    Development of effective and durable breast cancer treatment strategies requires a mechanistic understanding of the influence of the microenvironment on response. Previous work has shown that cellular signaling pathways and cell morphology are dramatically influenced by three-dimensional (3D) cultures as opposed to traditional two-dimensional (2D) monolayers. Here, we compared 2D and 3D culture models to determine the impact of 3D architecture and extracellular matrix (ECM) on HER2 signaling and on the response of HER2-amplified breast cancer cell lines to the HER2-targeting agents Trastuzumab, Pertuzumab and Lapatinib. We show that the response of the HER2-amplified AU565, SKBR3 and HCC1569 cells to these anti-HER2 agents was highly dependent on whether the cells were cultured in 2D monolayer or 3D laminin-rich ECM gels. Inhibition of {beta}1 integrin, a major cell-ECM receptor subunit, significantly increased the sensitivity of the HER2-amplified breast cancer cell lines to the humanized monoclonal antibodies Trastuzumab and Pertuzumab when grown in a 3D environment. Finally, in the absence of inhibitors, 3D cultures had substantial impact on HER2 downstream signaling and induced a switch between PI3K-AKT- and RAS-MAPKpathway activation in all cell lines studied, including cells lacking HER2 amplification and overexpression. Our data provide direct evidence that breast cancer cells are able to rapidly adapt to different environments and signaling cues by activating alternative pathways that regulate proliferation and cell survival, events that may play a significant role in the acquisition of resistance to targeted therapies.

  8. Peroxisome Proliferator Activated Receptor A Ligands as Anticancer Drugs Targeting Mitochondrial Metabolism