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Sample records for activated ras alters

  1. Activated Ras alters lens and corneal development through induction of distinct downstream targets

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    Reneker Lixing

    2010-01-01

    Full Text Available Abstract Background Mammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas. Results Ras transgenic lenses and corneal epithelial cells showed increased proliferation with concomitant increases in cyclin D1 and D2 expression. This initial increase in proliferation is sustained in the cornea but not in the lens epithelial cells. Coincidentally, cdk inhibitors p27Kip1 and p57Kip2 were upregulated in the Ras transgenic lenses but not in the corneas. Phospho-Erk1 and Erk2 levels were elevated in the lens but not in the cornea and Spry 1 and Spry 2, negative regulators of Ras-Raf-Erk signaling, were upregulated more in the corneal than in the lens epithelial cells. Both lens and corneal differentiation programs were sensitive to Ras activation. Ras transgenic embryos showed a distinctive alteration in the architecture of the lens pit. Ras activation, though sufficient for upregulation of Prox1, a transcription factor critical for cell cycle exit and initiation of fiber differentiation, is not sufficient for induction of terminal fiber differentiation. Expression of Keratin 12, a marker of corneal epithelial differentiation, was reduced in the Ras transgenic corneas. Conclusions Collectively, these

  2. Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

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    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M; Abel, Steven M; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S; Hansen, Scott D; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K; Kuriyan, John; Groves, Jay T

    2014-07-01

    Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.

  3. Ras activation by SOS

    DEFF Research Database (Denmark)

    Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen;

    2014-01-01

    Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual ...

  4. Raft protein clustering alters N-Ras membrane interactions and activation pattern

    NARCIS (Netherlands)

    Eisenberg, Sharon; Beckett, Alison J; Prior, Ian A; Dekker, Frank J; Hedberg, Christian; Waldmann, Herbert; Ehrlich, Marcelo; Henis, Yoav I; Dekker, Frank

    2011-01-01

    The trafficking, membrane localization, and lipid raft association of Ras proteins, which are crucial oncogenic mediators, dictate their isoform-specific biological responses. Accordingly, their spatiotemporal dynamics are tightly regulated. While extensively studied for H- and K-Ras, such informati

  5. Inhibition of Ras oncogenic activity by Ras protooncogenes.

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    Diaz, Roberto; Lue, Jeffrey; Mathews, Jeremy; Yoon, Andrew; Ahn, Daniel; Garcia-España, Antonio; Leonardi, Peter; Vargas, Marcelo P; Pellicer, Angel

    2005-01-10

    Point mutations in ras genes have been found in a large number and wide variety of human tumors. These oncogenic Ras mutants are locked in an active GTP-bound state that leads to a constitutive and deregulated activation of Ras function. The dogma that ras oncogenes are dominant, whereby the mutation of a single allele in a cell will predispose the host cell to transformation regardless of the presence of the normal allele, is being challenged. We have seen that increasing amounts of Ras protooncogenes are able to inhibit the activity of the N-Ras oncogene in the activation of Elk in NIH 3T3 cells and in the formation of foci. We have been able to determine that the inhibitory effect is by competition between Ras protooncogenes and the N-Ras oncogene that occurs first at the effector level at the membranes, then at the processing level and lastly at the effector level in the cytosol. In addition, coexpression of the N-Ras protooncogene in thymic lymphomas induced by the N-Ras oncogene is associated with increased levels of p107, p130 and cyclin A and decreased levels of Rb. In the present report, we have shown that the N-Ras oncogene is not truly dominant over Ras protooncogenes and their competing activities might be depending on cellular context.

  6. Regulation of p21ras activity

    DEFF Research Database (Denmark)

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

    1992-01-01

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

  7. ras activation in human tumors and in animal model systems

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    Corominas, M.; Sloan, S.R.; Leon, J.; Kamino, Hideko; Newcomb, E.W.; Pellicer, A. (New York Univ. Medical Center, New York (United States))

    1991-06-01

    Environmental agents such as radiation and chemicals are known to cause genetic damage. Alterations in a limited set of cellular genes called proto-oncogenes lead to unregulated proliferation and differentiation. The authors have studied the role of the ras gene family in carcinogenesis using two different animal models. In one case, thymic lymphomas were induced in mice by either gamma or neutron radiation, and in the other, keratoacanthomas were induced in rabbit skin with dimethylbenzanthracene. Human keratoacanthomas similar to the ones induced in rabbits were also analyzed. They found that different types of radiation such as gamma rays and neutrons, induced different point mutations in ras genes. A novel K-ras mutation in codon 146 has been found in thymic lymphomas induced by neutrons. Keratoacanthomas induced in rabbit skin by dimethylbenzanthracene show a high frequency of H-ras-activated genes carrying a mutation in codon 61. The same is observed in human keratoacanthomas, although mutations are in both the 12th and the 61st codons of the H-ras gene. H-ras activation is less frequent in human squamous cell carcinomas than in keratoacanthomas, suggesting that ras genes could play a role in vivo in differentiation as well as in proliferation.

  8. Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

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    Hurwitz, N; Segal, M; Marbach, I; Levitzki, A

    1995-11-21

    Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements. PMID:7479926

  9. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells

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    Jesse E. Jun

    2013-09-01

    Full Text Available The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and SOS-family GEFs.Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood.One large group of biomolecules critically involved in the control of Ras-GEFs´functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells.

  10. Altered expression of Bcl-2, c-Myc, H-Ras, K-Ras, and N-Ras does not influence the course of mycosis fungoides

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    Maj, Joanna; Jankowska-Konsur, Alina; Plomer-Niezgoda, Ewa; Sadakierska-Chudy, Anna

    2013-01-01

    Introduction Data about genetic alterations in mycosis fungoides (MF) are limited and their significance not fully elucidated. The aim of the study was to explore the expression of various oncogenes in MF and to assess their influence on the disease course. Material and methods Skin biopsies from 27 MF patients (14 with early MF and 13 with advanced disease) and 8 healthy volunteers were analyzed by real-time polymerase chain reaction (PCR) to detect Bcl-2, c-Myc, H-Ras, K-Ras and N-Ras expression. All PCR reactions were performed using an Applied Biosystems 7900HT Fast Real-Time PCR System and interpreted using Sequence Detection Systems software which utilizes the comparative delta Ct method. The level of mRNA was normalized to GAPDH expression. All data were analyzed statistically. Results All evaluated oncogenes were found to be expressed in the skin from healthy controls and MF patients. Bcl-2 (–4.2 ±2.2 vs. –2.2 ±1.1; p = 0.01), H-Ras (–3.0 ±3.3 vs. 0.6 ±2.6; p = 0.01) and N-Ras (–3.6 ±2.0 vs. –1.1 ±2.4; p = 0.03) were expressed at significantly lower levels in MF. No relationships between oncogene expression and disease stage, presence of distant metastases and survival were observed (p > 0.05 for all comparisons). Conclusions The pathogenic role and prognostic significance of analyzed oncogenes in MF seem to be limited and further studies are needed to establish better prognostic factors for patients suffering from MF. PMID:24273576

  11. Gamma band activity in the reticular activating system (RAS

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    Francisco J Urbano

    2012-01-01

    Full Text Available This review considers recent evidence showing that cells in three regions of the reticular activating system (RAS exhibit gamma band activity, and describes the mechanisms behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN, intralaminar parafascicular nucleus (Pf, and pontine Subcoeruleus nucleus dorsalis (SubCD all fire in the beta/gamma band range when maximally activated, but no higher. The mechanisms behind this ceiling effect have been recently elucidated. We describe recent findings showing that every cell in the PPN have high threshold, voltage-dependent P/Q-type calcium channels that are essential, while N-type calcium channels are permissive, to gamma band activity. Every cell in the Pf also showed that P/Q-type and N-type calcium channels are responsible for this activity. On the other hand, every SubCD cell exhibited sodium-dependent subthreshold oscillations. A novel mechanism for sleep-wake control based on well-known transmitter interactions, electrical coupling, and gamma band activity is described. The data presented here on inherent gamma band activity demonstrates the global nature of sleep-wake oscillation that is orchestrated by brainstem-thalamic mechanism, and questions the undue importance given to the hypothalamus for regulation of sleep-wakefulness. The discovery of gamma band activity in the RAS follows recent reports of such activity in other subcortical regions like the hippocampus and cerebellum. We hypothesize that, rather than participating in the temporal binding of sensory events as seen in the cortex, gamma band activity manifested in the RAS may help stabilize coherence related to arousal, providing a stable activation state during waking and paradoxical sleep. Most of our thoughts and actions are driven by preconscious processes. We speculate that continuous sensory input will induce gamma band activity in the RAS that could participate in the

  12. Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

    Institute of Scientific and Technical Information of China (English)

    Qing Wang; Zhi Ying Lin; Xiao Li Feng

    2001-01-01

    AIM To demonstrate the relationship betweenH-ras oncogene and hepatocellular carcinoma(HCC) metastasis.METHODS Activated H-ras oncogene wastransfected into SMMC 7721, a cell line derivedfrom human HCC, by calcium phosphatetransfection method. Some metastasis-relatedparameters were detected in vitro, includingadhesion assay, migration assay, expression ofcollagenase ⅣV (c ⅣV ase) and epidermal growthfactor receptor (EGFR).RESULTS The abilities of H-ras-transfected cellclones in adhesion to laminin (LN) or fibronectin(FN), migration, c Ⅳ ase secretion increasedmarkedly, and the expression of EGFR elevatedmoderately. More importantly, these alterationswere consistent positively with the expressionof p21, the protein product of H-ras oncogene.CONCLUSION H-ras oncogene could inducethe metastatic phenotype of HCC cell in vitro toraise its metastatic potential.

  13. A Genetic Screen Identifies PITX1 as a Suppressor of RAS Activity and Tumorigenicity

    NARCIS (Netherlands)

    Kolfschoten, I.G.M.; Leeuwen, Bart van; Berns, K.; Mullenders, J.; Beijersbergen, R.L.; Bernards, R.A.; Voorhoeve, P.M.; Agami, R.

    2005-01-01

    Activating mutations of RAS frequently occur in subsets of human cancers, indicating that RAS activation is important for tumorigenesis. However, a large proportion of these cancers still retain wild-type RAS alleles, suggesting that either the RAS pathway is activated in a distinct manner or anothe

  14. Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

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    Winter, Jon J G; Anderson, Malcolm; Blades, Kevin; Brassington, Claire; Breeze, Alexander L; Chresta, Christine; Embrey, Kevin; Fairley, Gary; Faulder, Paul; Finlay, M Raymond V; Kettle, Jason G; Nowak, Thorsten; Overman, Ross; Patel, S Joe; Perkins, Paula; Spadola, Loredana; Tart, Jonathan; Tucker, Julie A; Wrigley, Gail

    2015-03-12

    Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.

  15. R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

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    Sawada, Junko; Li, Fangfei; Komatsu, Masanobu

    2015-01-01

    R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling. PMID:27029009

  16. Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes

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    Olschwang Sylviane

    2008-10-01

    Full Text Available Abstract Background Chronic myelomonocytic leukemia (CMML is a hematological disease close to, but separate from both myeloproliferative disorders (MPD and myelodysplastic syndromes and may show either myeloproliferative (MP-CMML or myelodysplastic (MD-CMML features. Not much is known about the molecular biology of this disease. Methods We studied a series of 30 CMML samples (13 MP- and 11 MD-CMMLs, and 6 acutely transformed cases from 29 patients by using Agilent high density array-comparative genomic hybridization (aCGH and sequencing of 12 candidate genes. Results Two-thirds of samples did not show any obvious alteration of aCGH profiles. In one-third we observed chromosome abnormalities (e.g. trisomy 8, del20q and gain or loss of genes (e.g. NF1, RB1 and CDK6. RAS mutations were detected in 4 cases (including an uncommon codon 146 mutation in KRAS and PTPN11 mutations in 3 cases. We detected 11 RUNX1 alterations (9 mutations and 2 rearrangements. The rearrangements were a new, cryptic inversion of chromosomal region 21q21-22 leading to break and fusion of RUNX1 to USP16. RAS and RUNX1 alterations were not mutually exclusive. RAS pathway mutations occurred in MP-CMMLs (~46% but not in MD-CMMLs. RUNX1 alterations (mutations and cryptic rearrangement occurred in both MP and MD classes (~38%. Conclusion We detected RAS pathway mutations and RUNX1 alterations. The latter included a new cryptic USP16-RUNX1 fusion. In some samples, two alterations coexisted already at this early chronic stage.

  17. Inactivation of RASA1 promotes melanoma tumorigenesis via R-Ras activation

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    Sung, Hyeran; Kanchi, Krishna L.; Wang, Xue; Hill, Kristen S.; Messina, Jane L.; Lee, Ji-Hyun; Kim, Youngchul; Dees, Nathan D.; Ding, Li; Teer, Jamie K.; Yang, Shengyu; Sarnaik, Amod A.; Sondak, Vernon K.; Mulé, James J.; Wilson, Richard K.; Weber, Jeffrey S.; Kim, Minjung

    2016-01-01

    Inactivation of Ras GTPase activating proteins (RasGAPs) can activate Ras, increasing the risk for tumor development. Utilizing a melanoma whole genome sequencing (WGS) data from 13 patients, we identified two novel, clustered somatic missense mutations (Y472H and L481F) in RASA1 (RAS p21 protein activator 1, also called p120RasGAP). We have shown that wild type RASA1, but not identified mutants, suppresses soft agar colony formation and tumor growth of BRAF mutated melanoma cell lines via its RasGAP activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform. Moreover, R-Ras increased and RASA1 suppressed Ral-A activation among Ras downstream effectors. In addition to mutations, loss of RASA1 expression was frequently observed in metastatic melanoma samples on melanoma tissue microarray (TMA) and a low level of RASA1 mRNA expression was associated with decreased overall survival in melanoma patients with BRAF mutations. Thus, these data support that RASA1 is inactivated by mutation or by suppressed expression in melanoma and that RASA1 plays a tumor suppressive role by inhibiting R-Ras, a previously less appreciated member of the Ras small GTPases. PMID:26993606

  18. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

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    Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))

    1992-08-01

    Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

  19. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

    DEFF Research Database (Denmark)

    Adari, H; Lowy, D R; Willumsen, B M;

    1988-01-01

    A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras...... as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl....... Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair...

  20. An Active RFID Accountability System (RAS) for Constrained Wireless Environments

    Energy Technology Data Exchange (ETDEWEB)

    Barker, Alan M [ORNL; Hanson, Gregory R [ORNL; Sexton, Angela Kay [ORNL; Jones Jr, J P [ORNL; Freer, Eva B [ORNL; Sjoreen, Andrea L [ORNL

    2011-01-01

    A team from Oak Ridge National Laboratory (ORNL) has developed an RFID Accountability System (RAS) that allows items with active RFID tags to be tracked in environments where tags may not be able to transmit their location continuously. The system uses activators that transmit a short range signal. Active RFID tags are in a sleep state until they encounter an activator. Then they transmit a signal that is picked up by the antennas installed throughout the building. This paper presents the theory of operation, application areas, lessons learned, and key features developed over the course of seven years of development and use.

  1. Ras-mutant cancer cells display B-Raf binding to Ras that activates extracellular signal-regulated kinase and is inhibited by protein kinase A phosphorylation.

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    Li, Yanping; Takahashi, Maho; Stork, Philip J S

    2013-09-20

    The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

  2. Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

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    Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

    2014-03-01

    Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

  3. Formation of the Ras dimer is essential for Raf-1 activation.

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    Inouye, K; Mizutani, S; Koide, H; Kaziro, Y

    2000-02-11

    Although it is well established that Ras requires membrane localization for activation of its target molecule, Raf-1, the reason for this requirement is not fully understood. In this study, we found that modified Ras, which is purified from Sf9 cells, could activate Raf-1 in a cell-free system, when incorporated into liposome. Using a bifunctional cross-linker and a protein-fragmentation complementation assay, we detected dimer formation of Ras in the liposome and in the intact cells, respectively. These results suggest that dimerization of Ras in the lipid membrane is essential for activation of Raf-1. To support this, we found that, when fused to glutathione S-transferase (GST), unprocessed Ras expressed in Escherichia coli could bypass the requirement for liposome. A Ras-dependent Raf-1 activator, which we previously reported (Mizutani, S., Koide, H., and Kaziro, Y. (1998) Oncogene 16, 2781-2786), was still required for Raf-1 activation by GST-Ras. Furthermore, an enforced dimerization of unmodified oncogenic Ras mutant in human embryonic kidney (HEK) 293 cells, using a portion of gyrase B or estrogen receptor, also resulted in activation of Raf-1. From these results, we conclude that membrane localization allows Ras to form a dimer, which is essential, although not sufficient, for Raf-1 activation.

  4. Study Illuminates K-Ras4B Activation, Which May Help Predict Drug Resistance | Poster

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    Until recently, researchers studying RAS, a family of proteins involved in transmitting signals within cells, believed that the exchange of guanosine 5’-diphosphate (GDP) by guanosine triphosphate (GTP) was sufficient to activate the protein. Once activated, RAS can cause unintended and overactive signaling in cells, which can lead to cell division and, ultimately, cancer.

  5. RasGRP1, but not RasGRP3, is required for efficient thymic β-selection and ERK activation downstream of CXCR4.

    Directory of Open Access Journals (Sweden)

    Dominic P Golec

    Full Text Available T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4(-CD8(- 'double negative' (DN thymocytes, pass through a checkpoint termed "β-selection" before maturing into CD4(+CD8(+ 'double positive' (DP thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ(+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

  6. The Ras mutant D119N is both dominant negative and activated

    NARCIS (Netherlands)

    Cool, RH; Schmidt, G; Lenzen, CU; Prinz, H; Vogt, D; Wittinghofer, A

    1999-01-01

    The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentrat

  7. Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.

    OpenAIRE

    Hofer, F.; Fields, S; Schneider, C; Martin, G S

    1994-01-01

    The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The...

  8. CARD9 mediates Dectin-1-induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity.

    Science.gov (United States)

    Jia, Xin-Ming; Tang, Bing; Zhu, Le-Le; Liu, Yan-Hui; Zhao, Xue-Qiang; Gorjestani, Sara; Hsu, Yen-Michael S; Yang, Long; Guan, Jian-Hong; Xu, Guo-Tong; Lin, Xin

    2014-10-20

    Dectin-1 functions as a pattern recognition receptor for sensing fungal infection. It has been well-established that Dectin-1 induces innate immune responses through caspase recruitment domain-containing protein 9 (CARD9)-mediated NF-κB activation. In this study, we find that CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands, such as curdlan or Candida albicans yeast. In contrast, we find that CARD9 regulates H-Ras activation by linking Ras-GRF1 to H-Ras, which mediates Dectin-1-induced extracellular signal-regulated protein kinase (ERK) activation and proinflammatory responses when stimulated by their ligands. Mechanistically, Dectin-1 engagement initiates spleen tyrosine kinase (Syk)-dependent Ras-GRF1 phosphorylation, and the phosphorylated Ras-GRF1 recruits and activates H-Ras through forming a complex with CARD9, which leads to activation of ERK downstream. Finally, we show that inhibiting ERK activation significantly accelerates the death of C. albicans-infected mice, and this inhibitory effect is dependent on CARD9. Together, our studies reveal a molecular mechanism by which Dectin-1 induces H-Ras activation that leads to ERK activation for host innate immune responses against fungal infection. PMID:25267792

  9. Identification of H-Ras-Specific Motif for the Activation of Invasive Signaling Program in Human Breast Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Hae-Young Yong

    2011-02-01

    Full Text Available Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR, consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

  10. CARD9 mediates Dectin-1–induced ERK activation by linking Ras-GRF1 to H-Ras for antifungal immunity

    OpenAIRE

    Jia, Xin-ming; Tang, Bing; Zhu, Le-Le; Liu, Yan-Hui; Zhao, Xue-Qiang; Gorjestani, Sara; Hsu, Yen-Michael S.; Yang, Long; Guan, Jian-Hong; Xu, Guo-Tong; Lin, Xin

    2014-01-01

    Dectin-1 functions as a pattern recognition receptor for sensing fungal infection. It has been well-established that Dectin-1 induces innate immune responses through caspase recruitment domain-containing protein 9 (CARD9)–mediated NF-κB activation. In this study, we find that CARD9 is dispensable for NF-κB activation induced by Dectin-1 ligands, such as curdlan or Candida albicans yeast. In contrast, we find that CARD9 regulates H-Ras activation by linking Ras-GRF1 to H-Ras, which mediates De...

  11. Active Erk Regulates Microtubule Stability in H-ras-Transformed Cells

    Directory of Open Access Journals (Sweden)

    Rene E. Harrison

    2001-01-01

    Full Text Available Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs and mitotic spindles in both parental and mutant active rastransfected 10T1 /2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

  12. Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

    Directory of Open Access Journals (Sweden)

    Jennifer S. Liu

    2012-11-01

    Full Text Available Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

  13. Molecular alterations of Ras-Raf-mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt signaling pathways in colorectal cancers from a tertiary hospital at Kuala Lumpur, Malaysia.

    Science.gov (United States)

    Yip, Wai Kien; Choo, Chee Wei; Leong, Vincent Ching-Shian; Leong, Pooi Pooi; Jabar, Mohd Faisal; Seow, Heng Fong

    2013-10-01

    Molecular alterations in KRAS, BRAF, PIK3CA, and PTEN have been implicated in designing targeted therapy for colorectal cancer (CRC). The present study aimed to determine the status of these molecular alterations in Malaysian CRCs as such data are not available in the literature. We investigated the mutations of KRAS, BRAF, and PTEN, the gene amplification of PIK3CA, and the protein expression of PTEN and phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110α) by direct DNA sequencing, quantitative real-time PCR, and immunohistochemistry, respectively, in 49 CRC samples. The frequency of KRAS (codons 12, 13, and 61), BRAF (V600E), and PTEN mutations, and PIK3CA amplification was 25.0% (11/44), 2.3% (1/43), 0.0% (0/43), and 76.7% (33/43), respectively. Immunohistochemical staining demonstrated loss of PTEN protein in 54.5% (24/44) of CRCs and no significant difference in PI3K p110α expression between CRCs and the adjacent normal colonic mucosa (p = 0.380). PIK3CA amplification was not associated with PI3K p110α expression level, but associated with male cases (100% of male cases vs 56% of female cases harbored amplified PIK3CA, p = 0.002). PI3K p110α expression was significantly higher (p = 0.041) in poorly/moderately differentiated carcinoma compared with well-differentiated carcinoma. KRAS mutation, PIK3CA amplification, PTEN loss, and PI3K p110α expression did not correlate with Akt phosphorylation or Ki-67 expression. KRAS mutation, PIK3CA amplification, and PTEN loss were not mutually exclusive. This is the first report on CRC in Malaysia showing comparable frequency of KRAS mutation and PTEN loss, lower BRAF mutation rate, higher PIK3CA amplification frequency, and rare PTEN mutation, as compared with published reports.

  14. NMR characterization of full-length farnesylated and non-farnesylated H-Ras and its implications for Raf activation.

    Science.gov (United States)

    Thapar, Roopa; Williams, Jason G; Campbell, Sharon L

    2004-11-01

    The C terminus, also known as the hypervariable region (residues 166-189), of H-, N-, and K-Ras proteins has sequence determinants necessary for full activation of downstream effectors such as Raf kinase and PI-3 kinase as well as for the correct targeting of Ras proteins to lipid rafts and non-raft membranes. There is considerable interest in understanding how residues in the extreme C terminus of the different Ras proteins and farnesylation of the CaaX box cysteine affect Ras membrane localization and allosteric activation of Raf kinase. To provide insights into the structural and dynamic changes that occur in Ras upon farnesylation, we have used NMR spectroscopy to compare the properties of truncated H-Ras (1-166), to non-processed full-length H-Ras (residues 1-185) and full-length (1-189) farnesylated H-Ras. We report that the C-terminal helix alpha-5 extends to residue N172, and the remaining 17 amino acid residues in the C terminus are conformationally averaged in solution. Removal of either 23 or 18 amino acid residues from the C terminus of full length H-Ras generates truncated H-Ras (1-166) and H-Ras (1-171) proteins, respectively, that have been structurally characterized and are biochemical active. Here we report that C-terminal truncation of H-Ras results in minor structural and dynamic perturbations that are propagated throughout the H-Ras protein including increased flexibility of the central beta-sheet and the C-terminal helix alpha-5. Ordering of residues in loop-2, which is involved in Raf CRD binding is also observed. Farnesylation of full-length H-Ras at C186 does not result in detectable conformational changes in H-Ras. Chemical shift mapping studies of farnesylated and non-farnesylated forms of H-Ras with the Raf-CRD show that the farnesyl moiety, the extreme H-Ras C terminus and residues 23-30, contribute to H-Ras:Raf-CRD interactions, thereby increasing the affinity of H-Ras for the Raf-CRD.

  15. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover.

    Science.gov (United States)

    Hong, Xin; Nguyen, Hung Thanh; Chen, Qingfeng; Zhang, Rui; Hagman, Zandra; Voorhoeve, P Mathijs; Cohen, Stephen M

    2014-11-01

    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the βTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1. PMID:25180228

  16. Role of neuronal Ras activity in adult hippocampal neurogenesis and cognition

    Directory of Open Access Journals (Sweden)

    Martina eManns

    2011-02-01

    Full Text Available Hippocampal neurogenesis in the adult mammalian brain is modulated by various signals like growth factors, hormones, neuropeptides, and neurotransmitters. All of these factors can (but not necessarily do converge on the activation of the G protein p21Ras. We used a transgenic mouse model (synRas mice expressing constitutively activated G12V-Harvey Ras selectively in differentiated neurons to investigate the possible effects onto neurogenesis. Ras activation in neurons attenuates hippocampal precursor cell generation at an early stage of the proliferative cascade before neuronal lineage determination occurs. Therefore it is unlikely that the transgenically activated Ras in neurons mediates this effect by a direct, intracellular signaling mechanism. Voluntary exercise restores neurogenesis up to wild type level presumably mediated by brain derived neurotrophic factor. Reduced neurogenesis is linked to impairments in spatial short-term memory and object recognition, the latter can be rescued by voluntary exercise, as well. These data support the view that new cells significantly increase complexity that can be processed by the hippocampal network when experience requires high demands to associate stimuli over time and/or space.

  17. Dual Activation of Phospholipase C-ε by Rho and Ras GTPases*

    OpenAIRE

    Seifert, Jason P.; Zhou, Yixing; Hicks, Stephanie N.; Sondek, John; Harden, T. Kendall

    2008-01-01

    Phospholipase C-ε (PLC-ε) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-ε to stimulate its phospholipase activity. Purified PLC-ε was activated in a guanine nucleotide- and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P2-containing ph...

  18. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    Science.gov (United States)

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

    2016-09-01

    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis. PMID:27501536

  19. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    Science.gov (United States)

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

    2016-09-01

    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

  20. A WXW Motif Is Required for the Anticancer Activity of the TAT-RasGAP317–326 Peptide*

    Science.gov (United States)

    Barras, David; Chevalier, Nadja; Zoete, Vincent; Dempsey, Rosemary; Lapouge, Karine; Olayioye, Monilola A.; Michielin, Olivier; Widmann, Christian

    2014-01-01

    TAT-RasGAP317–326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317–326 sequence for the anticancer activities of TAT-RasGAP317–326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317–326. PMID:25008324

  1. A WXW motif is required for the anticancer activity of the TAT-RasGAP317-326 peptide.

    Science.gov (United States)

    Barras, David; Chevalier, Nadja; Zoete, Vincent; Dempsey, Rosemary; Lapouge, Karine; Olayioye, Monilola A; Michielin, Olivier; Widmann, Christian

    2014-08-22

    TAT-RasGAP317-326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317-326 sequence for the anticancer activities of TAT-RasGAP317-326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317-326.

  2. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    Science.gov (United States)

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  3. K-Ras mediated murine epidermal tumorigenesis is dependent upon and associated with elevated Rac1 activity.

    Directory of Open Access Journals (Sweden)

    Michael S Samuel

    Full Text Available A common goal for potential cancer therapies is the identification of differences in protein expression or activity that would allow for the selective targeting of tumor vs. normal cells. The Ras proto-oncogene family (K-Ras, H-Ras and N-Ras are amongst the most frequently mutated genes in human cancers. As a result, there has been substantial effort dedicated to determining which pathways are activated by Ras signaling and, more importantly, which of these contribute to cancer. Although the most widely studied Ras-regulated signaling pathway is the Raf/mitogen-activated protein kinase cascade, previous research in model systems has revealed that the Rac1 GTP-binding protein is also required for Ras-induced biological responses. However, what have been lacking are rigorous in vivo Rac1 target validation data and a clear demonstration that in Ras-driven hyperplastic lesions, Rac1 activity is increased. Using a combination of genetically-modified mouse models that allow for the tissue-selective activation or deletion of signaling molecules and an activation-state sensitive Rac1 antibody that detects GTP-bound Rac1, we found that Rac1 contributes to K-Ras induced epidermal papilloma initiation and growth and that Rac1 activity is elevated by oncogenic K-Ras in vivo. Previously, it was not practical to assess Rac1 activation status in the most commonly used format for clinical tumor specimens, formalin-fixed paraffin embedded (FFPE tissues samples. However, this study clearly demonstrates that Rac1 is essential for K-Ras driven epithelial cell hyperproliferation and that Rac1 activity is elevated in tissues expressing mutant oncogenic K-Ras, while also characterizing the activation-state specific Rac1-GTP antibody as a probe to examine Rac1 activation status in FFPE samples. Our findings will facilitate further research on the status of Rac1 activity in human tumors and will help to define the tumor types of the patient population that could

  4. An orthosteric inhibitor of the RAS-SOS interaction.

    Science.gov (United States)

    Nickerson, Seth; Joy, Stephen T; Arora, Paramjit S; Bar-Sagi, Dafna

    2013-01-01

    Rat sarcoma (RAS) proteins are signaling nodes that transduce extracellular cues into precise alterations in cellular physiology by engaging effector pathways. RAS signaling thus regulates diverse cell processes including proliferation, migration, differentiation, and survival. Owing to this central role in governing mitogenic signals, RAS pathway components are often dysregulated in human diseases. Targeted therapy of RAS pathways has generally not been successful, largely because of the robust biochemistry of the targets and their multifaceted network of molecular regulators. The rate-limiting step of RAS activation is Son of Sevenless (SOS)-mediated nucleotide exchange involving a single evolutionarily conserved catalytic helix from SOS. Structure function data of this mechanism provided a strong platform to design an SOS-derived, helically constrained peptide mimic as an inhibitor of the RAS-SOS interaction. In this chapter, we review RAS-SOS signaling dynamics and present evidence supporting the novel paradigm of inhibiting their interaction as a therapeutic strategy. We then describe a method of generating helically constrained peptide mimics of protein surfaces, which we have employed to inhibit the RAS-SOS active site interaction. The biochemical and functional properties of this SOS mimic support the premise that inhibition of RAS-nucleotide exchange can effectively block RAS activation and downstream signaling.

  5. Combined Rational Design and a High Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras-activating Enzyme*

    Science.gov (United States)

    Evelyn, Chris R.; Biesiada, Jacek; Duan, Xin; Tang, Hong; Shang, Xun; Papoian, Ruben; Seibel, William L.; Nelson, Sandra; Meller, Jaroslaw; Zheng, Yi

    2015-01-01

    The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions. PMID:25825487

  6. Ras-activated Dsor1 promotes Wnt signaling in Drosophila development.

    Science.gov (United States)

    Hall, Eric T; Verheyen, Esther M

    2015-12-15

    Wnt/Wingless (Wg) and Ras-MAPK signaling both play fundamental roles in growth and cell fate determination, and when dysregulated, can lead to tumorigenesis. Several conflicting modes of interaction between Ras-MAPK and Wnt signaling have been identified in specific cellular contexts, causing synergistic or antagonistic effects on target genes. We find novel evidence that the Drosophila homolog of the dual specificity kinases MEK1/2 (also known as MAP2K1/2), Downstream of Raf1 (Dsor1), is required for Wnt signaling. Knockdown of Dsor1 results in loss of Wg target gene expression, as well as reductions in stabilized Armadillo (Arm; Drosophila β-catenin). We identify a close physical interaction between Dsor1 and Arm, and find that catalytically inactive Dsor1 causes a reduction in active Arm. These results suggest that Dsor1 normally counteracts the Axin-mediated destruction of Arm. We find that Ras-Dsor1 activity is independent of upstream activation by EGFR, and instead it appears to be activated by the insulin-like growth factor receptor to promote Wg signaling. Taken together, our results suggest that there is a new crosstalk pathway between insulin and Wg signaling that is mediated by Dsor1. PMID:26542023

  7. Activation of c-Ki-ras gene in human pancreatic cancer.

    Science.gov (United States)

    Prassolov, V S; Sakamoto, H; Nishimura, S; Terada, M; Sugimura, T

    1985-09-01

    DNA isolated from a lymph node with metastasis from pancreatic adenocarcinoma in a Japanese male patient transformed NIH3T3 cells upon transfection by the calcium-phosphate precipitation technique. Analysis of DNA from the transformant revealed the presence of an activated human c-Ki-ras gene, which is considered to be responsible for the transformation of the NIH3T3 cells.

  8. Constitutive CCND1/CDK2 activity substitutes for p53 loss, or MYC or oncogenic RAS expression in the transformation of human mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Damian J Junk

    Full Text Available Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1 occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.

  9. Structural basis for the interaction of the adaptor protein grb14 with activated ras.

    Directory of Open Access Journals (Sweden)

    Rohini Qamra

    Full Text Available Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA domain, a pleckstrin-homology (PH domain, a family-specific BPS (between PH and SH2 region, and a C-terminal Src-homology-2 (SH2 domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V. The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity.

  10. INHIBITION OF FARNESYL PROTEIN TRANSFERASE AND P21RAS MEMEBRANE ASSOCIATION BY D-LIMONENE IN HUMAN PANCREAS TUMOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ The monoterpene d-limonene inhibit the plasma-membrane associated P21ras expression and the posttranslational isoprenylation of P21ras, a mechanism that may contribute to its efficacy in the chemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study,the relative abilities of d-limonene to inhibit membrane associated P21ras expression in pancreas tumor cell(PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FTPase) activity during the Ras protein isoprenylation and cell proliferation were determined.Concomitantly,the effects of d-limonene on P21ras localization by immunohistochemistry and H-ras oncogene expression in PaCa tumor cell line by Northern blotting were observed. The results showed that d-limonene inhibited FPTase activity, thus to reduce P21H-ras isoprenylation. d-limonene could decrease P21ras membrane association and increase cytosolic accumulation of P21ras. This phenomenon was also noted when d-limonene-treated PaCa cells were stained immunohistochemically with anti-P21ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21ras membrane association and the alteration of P21ras localization. Inhibition of farnesylation of P21ras altered their intracellular localization and, hence, disrupted their biological activity,but no relationship with H-ras oncogene expression was found.

  11. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    Directory of Open Access Journals (Sweden)

    Jianhua Gong

    Full Text Available Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency, hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  12. Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation

    Directory of Open Access Journals (Sweden)

    Ze-Yi Zheng

    2015-07-01

    Full Text Available Basal-like breast cancers (BLBCs are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

  13. Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation.

    Science.gov (United States)

    Zheng, Ze-Yi; Tian, Lin; Bu, Wen; Fan, Cheng; Gao, Xia; Wang, Hai; Liao, Yi-Hua; Li, Yi; Lewis, Michael T; Edwards, Dean; Zwaka, Thomas P; Hilsenbeck, Susan G; Medina, Daniel; Perou, Charles M; Creighton, Chad J; Zhang, Xiang H-F; Chang, Eric C

    2015-07-21

    Basal-like breast cancers (BLBCs) are aggressive, and their drivers are unclear. We have found that wild-type N-RAS is overexpressed in BLBCs but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, whereas overexpression enhances these processes even in preinvasive BLBC cells. We identified N-Ras-responsive genes, most of which encode chemokines; e.g., IL8. Expression levels of these chemokines and N-RAS in tumors correlate with outcome. N-Ras, but not K-Ras, induces IL-8 by binding and activating the cytoplasmic pool of JAK2; IL-8 then acts on both the cancer cells and stromal fibroblasts. Thus, BLBC progression is promoted by increasing activities of wild-type N-Ras, which mediates autocrine/paracrine signaling that can influence both cancer and stroma cells.

  14. Activating Ras mutations fail to ensure efficient replication of adenovirus mutants lacking VA-RNA

    DEFF Research Database (Denmark)

    Schümann, Michael; Dobbelstein, Matthias

    2006-01-01

    Adenoviruses lacking their PKR-antagonizing VA RNAs replicate poorly in primary cells. It has been suggested that these virus recombinants still replicate efficiently in tumor cells with Ras mutations and might therefore be useful in tumor therapy. The ability of interferon-sensitive viruses...... to grow in Ras-mutant tumor cells is generally ascribed to a postulated inhibitory effect of mutant Ras on PKR. We have constructed a set of isogenic adenoviruses that lack either or both VA RNA species, and tested virus replication in a variety of cell species with different Ras status. In tendency, VA......-less viruses replicated with higher efficiency in Ras-mutant cells, as compared to cell lines without Ras mutation. However, several exceptions to this rule were observed, arguing against a direct inhibition of PKR by mutant Ras. Phosphorylation of the PKR-substrate eIF2alpha was observed regardless of the Ras...

  15. In Drosophila, RhoGEF2 cooperates with activated Ras in tumorigenesis through a pathway involving Rho1–Rok–Myosin-II and JNK signalling

    Directory of Open Access Journals (Sweden)

    Peytee Khoo

    2013-05-01

    The Ras oncogene contributes to ∼30% of human cancers, but alone is not sufficient for tumorigenesis. In a Drosophila screen for oncogenes that cooperate with an activated allele of Ras (RasACT to promote tissue overgrowth and invasion, we identified the GTP exchange factor RhoGEF2, an activator of Rho-family signalling. Here, we show that RhoGEF2 also cooperates with an activated allele of a downstream effector of Ras, Raf (RafGOF. We dissect the downstream pathways through which RhoGEF2 cooperates with RasACT (and RafGOF, and show that RhoGEF2 requires Rho1, but not Rac, for tumorigenesis. Furthermore, of the Rho1 effectors, we show that RhoGEF2 + Ras (Raf-mediated tumorigenesis requires the Rho kinase (Rok–Myosin-II pathway, but not Diaphanous, Lim kinase or protein kinase N. The Rho1–Rok–Myosin-II pathway leads to the activation of Jun kinase (JNK, in cooperation with RasACT. Moreover, we show that activation of Rok or Myosin II, using constitutively active transgenes, is sufficient for cooperative tumorigenesis with RasACT, and together with RasACT leads to strong activation of JNK. Our results show that Rok–Myosin-II activity is necessary and sufficient for Ras-mediated tumorigenesis. Our observation that activation of Myosin II, which regulates Filamentous actin (F-actin contractility without affecting F-actin levels, cooperates with RasACT to promote JNK activation and tumorigenesis, suggests that increased cell contractility is a key factor in tumorigenesis. Furthermore, we show that signalling via the Tumour necrosis factor (TNF; also known as Egr-ligand–JNK pathway is most likely the predominant pathway that activates JNK upon Rok activation. Overall, our analysis highlights the need for further analysis of the Rok–Myosin-II pathway in cooperation with Ras in human cancers.

  16. INHIBITION OF FARNESYL PROTEIN TRANSFERASE AND P21RAS MEMEBRANE ASSOCIATION BY D-LIMONENE IN HUMAN PANCREAS TUMOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    陈晓光; YoshihisaYano; TadayoshiHasuma; ToshikoYoshimata; WangYinna; ShuzoOtani

    1999-01-01

    The monoterpene d-limonene inhibit the plasma membrane associated P21ras expresion and the post-translational isoprenylatlon of P21ras, a mechanism that may contribute to its efficacy in the ehemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study, the relative abilities of d-limonene to inhibit membrane associated P21ras expression in Imncreas tumorcell (PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FT-Pase ) activity during the Rns p~otebi isoprenylation and cell proliferation were determined. Concomitantly,the effects of d-limonene on P21ras localization hy immunohimcchemistry and H-ras oncogene expression in PaCe tutor cell line by Northern blotting were observed. The results showed that ddimonene inhibited FPTase activity, thus to reduce P21H-ras isoprenylation, d limonene couM decrease P21ras meanhrane asso-ciation and increase cytosolie accumulation of P21ras This phenomenon was also noted when d-llmmaene-treated PaCe cells were stained immunohistcchemieally with anti-P21ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21ras membrane assoclaticon aad the alteration of P21ras localization. Inhibition ot farnesylation of P21ras altered their intraeellulsr localization and, hence, disrupted their biological activity, hut no relationship with H ras oneogene expression was found.

  17. Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ai-Guo, E-mail: wangaiguotl@hotmail.com; Song, Ya-Nan; Chen, Jun; Li, Hui-Ling; Dong, Jian-Yi; Cui, Hai-Peng; Yao, Liang; Li, Xue-Feng; Gao, Wen-Ting; Qiu, Ze-Wen; Wang, Fu-Jin; Wang, Jing-Yu, E-mail: wangjingyus@163.com

    2014-09-26

    Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12V oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5-week

  18. ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially

    Directory of Open Access Journals (Sweden)

    Bonini Chiara

    2006-06-01

    Full Text Available Abstract Background The mitogen-activated protein (MAP kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. Results Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. Conclusion These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity.

  19. Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

    2012-04-15

    Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

  20. INHIBITION OF ACTIVATED K-RAS GENE BY SIRNA EXPRESSION CASSETTES IN HUMAN PANCREATIC CARCINOMA CELL LINE MIAPACA-2

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; WANG Chun-you; DONG Ji-hua; CHEN Xiong; ZHANG Min; ZHAO Gang

    2005-01-01

    Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194,491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR,immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR,immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

  1. Activated ras Prevents Downregulation of Bcl-XL Triggered by Detachment from the Extracellular Matrix

    OpenAIRE

    Rosen, Kirill; RAK, Janusz; Leung, Thomas; Dean, Nicholas M.; Kerbel, Robert S.; Filmus, Jorge

    2000-01-01

    Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptoti...

  2. Detection of K-ras point mutation and telomerase activity during endoscopic retrograde cholangiopancreatography in diagnosis of pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Guo-Xiong Zhou; Jie-Fei Huang; Zhao-Shen Li; Guo-Ming Xu; Feng Liu; Hong Zhang

    2004-01-01

    AIM: To study the value of monitoring K-ras point mutation at codon 12 and telomerase activity in exfoliated cells obtained from pancreatic duct brushings during endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis of pancreatic cancer.METHODS: Exfoliated cells obtained from pancreatic duct brushings during ERCP were examined in 27 patients: 23with pancreatic cancers, 4 with chronic pancreatitis. K-fas point mutation was detected with the polymerase chain reaction and restriction fragment-length polymorphism (PCR-RFLP). Telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAPELISA).RESULTS: The telomerase activities in 27 patients were measured in 21 exfoliated cell samples obtained from pancreatic duct brushings. D450 value of telomerase activities in pancreatic cancer and chronic pancreatitis were 0.446±0.27and 0.041±0.0111, respectively. Seventy-seven point eight percent (14/18) of patients with pancreatic cancer and none of the patients with chronic pancreatitis showed telomerase activity in cells collected from pancreatic duct brushings when cutoff value of telomerase activity was set at 2.0. The K-ras gene mutation rate (72.2%) in pancreatic cancer was higher than that in chronic pancreatitis (33.3%)(P<0.05). In considering of both telomerase activities and K-ras point mutation, the total positive rate was 83.3%(15/18), and the specificity was 100%.CONCLUSION: Changes of telomerase activities and K-ras point mutation at codon 12 may be an early event of malignant progression in pancreatic cancer. Detection of telomerase activity and K-ras point mutation at codon 12may be complementary to each other, and is useful in diagnosis of pancreatic cancer.

  3. The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

    Institute of Scientific and Technical Information of China (English)

    Chiara Sandri; Guido Serini; Francesca Caccavari; Donatella Valdembri; Chiara Camillo; Stefan Veltel; Martina Santambrogio; Letizia Lanzetti; Fedenco Bussolino; Johanna Ivaska

    2012-01-01

    During developmental and tumor angiogenesis,semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases.R-Ras is mainly expressed in vascular cells,where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms.We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and-angiogenic activity of R-Ras.Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia.Upon binding,GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF)to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes.Here,the R-Ras/RIN2/Rab5 signaling module activates Racl-dependent cell adhesion via TIAM1,a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate.In conclusion,the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Racl.

  4. H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo.

    Science.gov (United States)

    Best, C J; Tanzer, L R; Phelps, P C; Merriman, R L; Boder, G G; Trump, B F; Elliget, K A

    1999-04-01

    We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells. H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.

  5. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    International Nuclear Information System (INIS)

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)–transfected cells enhanced activation of the phosphatidylinositol-3-kinase–Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)–neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor α was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR–phosphatidylinositol-3-kinase–Akt cascade.

  6. OSBP-related protein 3 (ORP3) coupling with VAMP-associated protein A regulates R-Ras activity.

    Science.gov (United States)

    Weber-Boyvat, Marion; Kentala, Henriikka; Lilja, Johanna; Vihervaara, Terhi; Hanninen, Raisa; Zhou, You; Peränen, Johan; Nyman, Tuula A; Ivaska, Johanna; Olkkonen, Vesa M

    2015-02-15

    ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3-VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and β1-integrin activity were enhanced by ORP3-VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3-VAPA complexes stimulate R-Ras signaling.

  7. Role of 239Pu-induced chromosome alterations and mutated Ki-v-ras oncogene during liver-cancer induction in Chinese hamsters and mice

    International Nuclear Information System (INIS)

    Chromosome aberrations and mutated oncogenes can cause important changes during carcinogenesis. Model systems are being studied in which defined cellular and molecular changes can be quantitated and altered, and tumor frequency, type, and time of appearance can be evaluated. Dose-response relationships for Pu Citrate-induced chromosome aberrations and liver cancer were measured in Chinese hamsters. Chromosome aberrations increased linearly according to dose, with a slope of 4.8 x 10-1 aberrations/cell/Gy; liver-tumor incidence was 1.1 x 10-1 tumors/animal/Gy. The dose was calculated at the 50% survival time. The interaction between Pu and Ki-v-ras, an altered, dominant-acting oncogene, on the induction of liver cancer was measured in B6C3F1 mice. The neo oncogene was used as a negative control in these studies. The Ki-v-ras oncogene was inserted into a viral vector and incorporated into the livers of mice either 30 days before or after the incorporation of 239Pu. Compared with both the controls and the mice injected with a single insult, mortality increased in groups of animals that received combined exposure to oncogenes, CCl4, and 239Pu. The relationships between molecular and cellular damage and the induction of cancer is being defined in both mice and Chinese hamsters

  8. The RafC1 cysteine-rich domain contains multiple distinct regulatory epitopes which control Ras-dependent Raf activation.

    Science.gov (United States)

    Daub, M; Jöckel, J; Quack, T; Weber, C K; Schmitz, F; Rapp, U R; Wittinghofer, A; Block, C

    1998-11-01

    Activation of c-Raf-1 (referred to as Raf) by Ras is a pivotal step in mitogenic signaling. Raf activation is initiated by binding of Ras to the regulatory N terminus of Raf. While Ras binding to residues 51 to 131 is well understood, the role of the RafC1 cysteine-rich domain comprising residues 139 to 184 has remained elusive. To resolve the function of the RafC1 domain, we have performed an exhaustive surface scanning mutagenesis. In our study, we defined a high-resolution map of multiple distinct functional epitopes within RafC1 that are required for both negative control of the kinase and the positive function of the protein. Activating mutations in three different epitopes enhanced Ras-dependent Raf activation, while only some of these mutations markedly increased Raf basal activity. One contiguous inhibitory epitope consisting of S177, T182, and M183 clearly contributed to Ras-Raf binding energy and represents the putative Ras binding site of the RafC1 domain. The effects of all RafC1 mutations on Ras binding and Raf activation were independent of Ras lipid modification. The inhibitory mutation L160A is localized to a position analogous to the phorbol ester binding site in the protein kinase C C1 domain, suggesting a function in cofactor binding. Complete inhibition of Ras-dependent Raf activation was achieved by combining mutations K144A and L160A, which clearly demonstrates an absolute requirement for correct RafC1 function in Ras-dependent Raf activation.

  9. Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

    Directory of Open Access Journals (Sweden)

    Nadja Chevalier

    Full Text Available Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes. Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin. All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

  10. Transforming activity of the c-Ha-ras oncogene having two point mutations in codons 12 and 61.

    Science.gov (United States)

    Sekiya, T; Prassolov, V S; Fushimi, M; Nishimura, S

    1985-09-01

    A recombinant plasmid carrying the human c-Ha-ras gene with two point mutations in codons 12 and 61 was constructed and its transforming activity on mouse NIH 3T3 cells was compared with those of genes with a single mutation in either codon 12 or 61. Quantitative analyses revealed that the gene with two mutations had essentially the same transforming activity as the genes with single mutations. These results indicate that a single mutation of the c-Ha-ras gene in either codon 12 or 61 is sufficient to activate the gene and that neither of the two mutation sites involved in activation of the gene needs to be intact for transforming activity.

  11. Renoprotective effect of renal liver-type fatty acid binding protein and angiotensin II type 1a receptor loss in renal injury caused by RAS activation.

    Science.gov (United States)

    Ichikawa, Daisuke; Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Shibagaki, Yugo; Yasuda, Takashi; Katayama, Kimie; Hoshino, Seiko; Igarashi-Migitaka, Junko; Hirata, Kazuaki; Kimura, Kenjiro

    2014-03-15

    The aim of this study was to assess the renoprotective effect of renal human liver-type fatty acid binding protein (hL-FABP) and angiotensin II (ANG II) type 1A receptor (AT1a) loss in renal injury caused by renin-angiotensin system (RAS) activation. We established hL-FABP chromosomal transgenic mice (L-FABP(+/-)AT1a(+/+)), crossed the L-FABP(+/-)AT1a(+/+) with AT1a knockdown homo mice (L-FABP(-/-)AT1a(-/-)), and generated L-FABP(+/-)AT1a hetero mice (L-FABP(+/-)AT1a(+/-)). After the back-cross of these cubs, L-FABP(+/-)AT1a(-/-) were obtained. To activate the renal RAS, wild-type mice (L-FABP(-/-)AT1a(+/+)), L-FABP(+/-)AT1a(+/+), L-FABP(-/-)AT1a(+/-), L-FABP(+/-)AT1a(+/-), L-FABP(-/-)AT1a(-/-), and L-FABP(+/-)AT1a(-/-) were administered high-dose systemic ANG II infusion plus a high-salt diet for 28 days. In the L-FABP(-/-)AT1a(+/+), RAS activation (L-FABP(-/-)AT1a(+/+)RAS) caused hypertension and tubulointerstitial damage. In the L-FABP(+/-)AT1a(+/+)RAS, tubulointerstitial damage was significantly attenuated compared with L-FABP(-/-)AT1a(+/+)RAS. In the AT1a partial knockout (AT1a(+/-)) or complete knockout (AT1a(-/-)) mice, reduction of AT1a expression led to a significantly lower degree of renal injury compared with L-FABP(-/-)AT1a(+/+)RAS or L-FABP(+/-)AT1a(+/+)RAS mice. Renal injury in L-FABP(+/-)AT1a(+/-)RAS mice was significantly attenuated compared with L-FABP(-/-)AT1a(+/-)RAS mice. In both L-FABP(-/-)AT1a(-/-)RAS and L-FABP(+/-)AT1a(-/-)RAS mice, renal damage was rarely found. The degrees of renal hL-FABP expression and urinary hL-FABP levels increased by RAS activation and gradually decreased along with reduction of AT1a expression levels. In conclusion, in this mouse model, renal hL-FABP expression and a decrease in AT1a expression attenuated tubulointerstitial damage due to RAS activation.

  12. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations

    OpenAIRE

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc’h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the del...

  13. Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells.

    Science.gov (United States)

    Koh, Min-Soo; Moon, Aree

    2011-03-01

    There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.

  14. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes.

    Science.gov (United States)

    Lerner, E C; Qian, Y; Blaskovich, M A; Fossum, R D; Vogt, A; Sun, J; Cox, A D; Der, C J; Hamilton, A D; Sebti, S M

    1995-11-10

    Ras-induced malignant transformation requires Ras farnesylation, a lipid posttranslational modification catalyzed by farnesyltransferase (FTase). Inhibitors of this enzyme have been shown to block Ras-dependent transformation, but the mechanism by which this occurs remains largely unknown. We have designed FTI-276, a peptide mimetic of the COOH-terminal Cys-Val-Ile-Met of K-Ras4B that inhibited potently FTase in vitro (IC50 = 500 pM) and was highly selective for FTase over geranylgeranyltransferase I (GGTase I) (IC50 = 50 nM). FTI-277, the methyl ester derivative of FTI-276, was extremely potent (IC50 = 100 nM) at inhibiting H-Ras, but not the geranylgeranylated Rap1A processing in whole cells. Treatment of H-Ras oncogene-transformed NIH 3T3 cells with FTI-277 blocked recruitment to the plasma membrane and subsequent activation of the serine/threonine kinase c-Raf-1 in cells transformed by farnesylated Ras (H-RasF), but not geranylgeranylated, Ras (H-RasGG). FTI-277 induced accumulation of cytoplasmic non-farnesylated H-Ras that was able to bind Raf and form cytoplasmic Ras/Raf complexes in which Raf kinase was not activated. Furthermore, FTI-277 blocked constitutive activation of mitogen-activated protein kinase (MAPK) in H-RasF, but not H-RasGG, or Raf-transformed cells. FTI-277 also inhibited oncogenic K-Ras4B processing and constitutive activation of MAPK, but the concentrations required were 100-fold higher than those needed for H-Ras inhibition. The results demonstrate that FTI-277 blocks Ras oncogenic signaling by accumulating inactive Ras/Raf complexes in the cytoplasm, hence preventing constitutive activation of the MAPK cascade.

  15. Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

    Directory of Open Access Journals (Sweden)

    Sujit V Janardhan

    Full Text Available Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

  16. RLIP76 regulates Arf6-dependent cell spreading and migration by linking ARNO with activated R-Ras at recycling endosomes.

    Science.gov (United States)

    Wurtzel, Jeremy G T; Lee, Seunghyung; Singhal, Sharad S; Awasthi, Sanjay; Ginsberg, Mark H; Goldfinger, Lawrence E

    2015-11-27

    R-Ras small GTPase enhances cell spreading and motility via RalBP1/RLIP76, an R-Ras effector that links GTP-R-Ras to activation of Arf6 and Rac1 GTPases. Here, we report that RLIP76 performs these functions by binding cytohesin-2/ARNO, an Arf GTPase guanine exchange factor, and connecting it to R-Ras at recycling endosomes. RLIP76 formed a complex with R-Ras and ARNO by binding ARNO via its N-terminus (residues 1-180) and R-Ras via residues 180-192. This complex was present in Rab11-positive recycling endosomes and the presence of ARNO in recycling endosomes required RLIP76, and was not supported by RLIP76(Δ1-180) or RLIP76(Δ180-192). Spreading and migration required RLIP76(1-180), and RLIP76(Δ1-180) blocked ARNO recruitment to recycling endosomes, and spreading. Arf6 activation with an ArfGAP inhibitor overcame the spreading defects in RLIP76-depleted cells or cells expressing RLIP76(Δ1-180). Similarly, RLIP76(Δ1-180) or RLIP76(Δ180-192) suppressed Arf6 activation. Together these results demonstrate that RLIP76 acts as a scaffold at recycling endosomes by binding activated R-Ras, recruiting ARNO to activate Arf6, thereby contributing to cell spreading and migration.

  17. Rck1 up-regulates pseudohyphal growth by activating the Ras2 and MAP kinase pathways independently in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chang, Miwha; Kang, Chang-Min; Park, Yong-Sung; Yun, Cheol-Won

    2014-02-21

    Previously, we reported that Rck1 regulates Hog1 and Slt2 activities and affects MAP kinase activity in Saccharomyces cerevisiae. Recently, we found that Rck1 up-regulates phospho-Kss1 and phospho-Fus3. Kss1 has been known as a component in the pseudohyphal growth pathway, and we attempted to identify the function of Rck1 in pseudohyphal growth. Rck1 up-regulated Ras2 at the protein level, not the transcriptional level. Additionally, FLO11 transcription was up-regulated by RCK1 over-expression. RCK1 expression was up-regulated during growth on SLAD+1% butanol medium. On nitrogen starvation agar plates, RCK1 over-expression induced pseudohyphal growth of colonies, and cells over-expressing RCK1 showed a filamentous morphology when grown in SLAD medium. Furthermore, 1-butanol greatly induced filamentous growth when RCK1 was over-expressed. Moreover, invasive growth was activated in haploid cells when RCK1 was over-expressed. The growth defect of cells observed on 1-butanol medium was recovered when RCK1 was over-expressed. Interestingly, Ras2 and phospho-Kss1 were up-regulated by Rck1 independently. Together, these results suggest that Rck1 promotes pseudohyphal growth by activating Ras2 and Kss1 via independent pathways in S. cerevisiae. PMID:24491552

  18. Impeded Nedd4-1-Mediated Ras Degradation Underlies Ras-Driven Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Taoling Zeng

    2014-05-01

    Full Text Available RAS genes are among the most frequently mutated proto-oncogenes in cancer. However, how Ras stability is regulated remains largely unknown. Here, we report a regulatory loop involving the E3 ligase Nedd4-1, Ras, and PTEN. We found that Ras signaling stimulates the expression of Nedd4-1, which in turn acts as an E3 ubiquitin ligase that regulates Ras levels. Importantly, Ras activation, either by oncogenic mutations or by epidermal growth factor (EGF signaling, prevents Nedd4-1-mediated Ras ubiquitination. This leads to Ras-induced Nedd4-1 overexpression, and subsequent degradation of the tumor suppressor PTEN in both human cancer samples and cancer cells. Our study thus unravels the molecular mechanisms underlying the interplay of Ras, Nedd4-1, and PTEN and suggests a basis for the high prevalence of Ras-activating mutations and EGF hypersignaling in cancer.

  19. Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells.

    OpenAIRE

    Owen, R D; Ostrowski, M C

    1987-01-01

    Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-r...

  20. Biological aspects and tumorigenic activity of the Ras proto-oncogenic family Aspectos biológicos e atividade tumorigênica da família proto-oncogênica Ras

    Directory of Open Access Journals (Sweden)

    Juliano André Boquett

    2010-12-01

    Full Text Available Proto-oncogenes play an important role in the regulation of the cellular cycle, being critical to the tumorigenesis. In this category we can find the RAS family. Due to the high transformation potential of these genes, this family is the best described and most studied one. It is formed by the H-, K- and the N-RAS genes, that codify highly related proteins expressed in several types of cells, denominated p21.These proteins act in the sign transduction from the membrane to the nucleus, as well as in the control of proliferation, differentiation and cellular death, and they are regulated by the interaction with GDP (inactive and GTP (active. These proteins show variation in only 10 - 15% of the primary structure, in the C-terminal portion denominated hyper-variant region. When in the oncogenic form, the p21 proteins remain active, providing continuous stimuli to the cellular proliferation. Among the RAS genes, K-RAS ones have been the most studied for presenting more frequent mutations and for being present in more aggressive tumors, implying the patients’ shorter survival time. Due to these facts and relative bibliography lack in the Portuguese language on this family, we presented in this work a systematized and updated review on the RAS genes. Os proto-oncogenes desempenham importante papel na regulação do ciclo celular, e são críticos à tumorigênese. Nessa categoria se encontra a família RAS, que, devido ao elevado potencial transformante dos genes que a compõem, é uma das mais bem descritas e estudadas. É formada pelos genes H-, K- e N-RAS, que codificam proteínas altamente relacionadas expressas em vários tipos de células, denominadas p21. Estas atuam na transdução de sinal da membrana ao núcleo, estão envolvidas no controle da proliferação, diferenciação e morte celular e são reguladas pela interação com GDP (inativa e GTP (ativa. As proteínas p21 diferem em apenas 10-15% da sua estrutura primária, na porção C

  1. High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27Kip Cdk-complexes

    DEFF Research Database (Denmark)

    Groth, Anja; Willumsen, Berthe Marie

    2005-01-01

    The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells...... ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27Kip1 levels rose and the inhibitor accumulated in both Cdk4......-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27Kip1 induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal...

  2. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Gennaro Altamura

    2013-01-01

    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  3. Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

    Science.gov (United States)

    Ayllón, Verónica; Martínez-A, Carlos; García, Alphonse; Cayla, Xavier; Rebollo, Angelita

    2000-01-01

    Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras. PMID:10811615

  4. Grb2 Is a Negative Modulator of the Intrinsic Ras-GEF Activity of hSos1

    OpenAIRE

    Zarich, Natasha; Oliva, José Luis; Martínez, Natalia; Jorge, Rocío; Ballester, Alicia; Gutiérrez-Eisman, Silvia; García-Vargas, Susana; Rojas, José M

    2006-01-01

    hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modu...

  5. A New View of Ras Isoforms in Cancers.

    Science.gov (United States)

    Nussinov, Ruth; Tsai, Chung-Jung; Chakrabarti, Mayukh; Jang, Hyunbum

    2016-01-01

    Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics. PMID:26659836

  6. The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the ApcMin/+ mouse small intestine

    NARCIS (Netherlands)

    C.E. Patek (Charles); M.J. Arends (Mark); L. Rose (Lorraine); F. Luo (Feijun); M.R. Walker (Marion); P.S. Devenney (Paul); R.L. Berry (Rachel); N.J. Lawrence (Nicola); R.A. Ridgway (Rachel); O.J. Sansom (Owen); M.L. Hooper (Martin)

    2008-01-01

    textabstractBackground: Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, Kras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC.

  7. The RAS Initiative

    Science.gov (United States)

    NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

  8. RAS Laboratory Groups

    Science.gov (United States)

    The RAS Initiative uses multiple technologies to attack RAS-driven cancers. The resources of the Frederick National Lab allocated to the RAS Hub are organized into seven laboratory groups, each contributing to the collaborative effort.

  9. Oncogenic Ras pushes (and pulls) cell cycle progression through ERK activation.

    Science.gov (United States)

    Campbell, Paul M

    2014-01-01

    The Ras-Raf-MEK-ERK signaling cascade is capable of channeling a wide variety of extracellular signals into control of cell proliferation, differentiation, senescence, and death. Because aberrant regulation at all steps of this signaling axis is observed in cancer, it remains an area of great interest in the field of tumor biology. Here we present evidence of the intricate and delicate levels of control of this pathway as it pertains to cell cycle regulation and illustrate how this control is not simply a rheostat.

  10. Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype

    Directory of Open Access Journals (Sweden)

    Pluk Helma

    2009-07-01

    Full Text Available Abstract Background The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. Results Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. Conclusion The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.

  11. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    Science.gov (United States)

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

  12. Inhibitors of Ras-SOS Interactions.

    Science.gov (United States)

    Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

    2016-04-19

    Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal.

  13. Ras does not contribute to the facilitation of hippocampal synaptic plasticity enabled by environmental enrichment.

    Science.gov (United States)

    Novkovic, T; Heumann, R; Manahan-Vaughan, D

    2015-11-19

    Environmental enrichment (EE), which mimics the wealth of sensory, motor and cognitive stimuli that arise through intense interactions with the ambient environment, results in enhanced hippocampal long-term potentiation (LTP) and spatial learning. A key molecular factor in the mediation of these changes is the brain-derived neurotrophic factor (BDNF). One of the downstream cascades that is activated by BDNF is the cascade linked to the small GTPase, Ras, that triggers mitogen-activated protein kinase (MAPK) activity and is part of the cAMP response element-binding protein (CREB) pathway that can lead to synaptic restructuring to support LTP. Here, we explored whether persistent activation of Ras in neurons further enhances LTP following EE of rodents. Immediately following weaning, transgenic mice that expressed constitutively activated neuronal Ras, or their wildtype (Wt) littermates, underwent 3weeks of constant EE. In the absence of EE, theta burst stimulation (TBS) evoked LTP in the CA1 region of transgenic mice that was not significantly different from LTP in Wts. After 3weeks of EE, hippocampal LTP was improved in Wt mice. Enriched transgenic mice showed an equivalent level of LTP to enriched Wts, but it was not significantly different from non-enriched synRas controls. Western blot analysis performed after a pull-down assay showed that non-enriched transgenic mice expressed higher Ras activity compared to non-enriched Wts. Following EE, Ras activity was reduced in transgenics to levels detected in Wts. These results show that constitutive activation of Ras does not mimic the effects of EE on LTP. In addition, EE results in an equivalent enhancement of LTP transgenics and Wts, coupled with a decrease in Ras activity to Wt levels. This suggests that permanent activation of Ras in neurons of synRas animals following EE results in an altered feedback regulation of endogenous Ras activity that is not a key factor in LTP enhancements. The maintenance of Ras within

  14. Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Fiorella Guadagni

    2012-01-01

    Full Text Available The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine at codon 57. In addition, we found in the same patient’s sample a silent polymorphism at codon 11 (Ala11Ala of exon 1. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 729–733

  15. New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution

    Directory of Open Access Journals (Sweden)

    Klink Björn U

    2010-10-01

    Full Text Available Abstract Background In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes. Results X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido-triphosphate, a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom. Conclusion For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.

  16. The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization.

    Science.gov (United States)

    Licata, Stephanie C; Pierce, R Christopher

    2003-04-01

    Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization. PMID:12641723

  17. Neural cell adhesion molecule-stimulated neurite outgrowth depends on activation of protein kinase C and the Ras-mitogen-activated protein kinase pathway

    DEFF Research Database (Denmark)

    Kolkova, K; Novitskaya, V; Pedersen, N;

    2000-01-01

    , inhibitors of the nonreceptor tyrosine kinase p59(fyn), PLC, PKC and MEK and an activator of PKC, phorbol-12-myristate-13-acetate (PMA). MEK2 transfection rescued cells treated with all inhibitors. The same was found for PMA treatment, except when cells concomitantly were treated with the MEK inhibitor....... Arachidonic acid rescued cells treated with antibodies to the FGF receptor or the PLC inhibitor, but not cells in which the activity of PKC, p59(fyn), FAK, Ras, or MEK was inhibited. Interaction of NCAM with a synthetic NCAM peptide ligand, known to induce neurite outgrowth, was shown to stimulate...

  18. Loss of RASAL1:a new mechanism of ras activation in cancers%RASAL1蛋白丢失——恶性肿瘤中ras活化的新机制

    Institute of Scientific and Technical Information of China (English)

    杨晴; 钱程佳

    2009-01-01

    ras 是一类与恶性肿瘤密切相关的蛋白,其持续活化可促进肿瘤的发生发展.RASAL1蛋白是一种钙离子介导的 ras GTP 酶活化蛋白,其表达下降或缺失,可使 ras 活化,从而导致肿瘤的发生发展.本文综述 ras 活性调节机制、RASAL1 与恶性肿瘤的关系以及RASAL1的表达下调的相关机制.%Ras is a kind of protein closely related to neoplasms,the persistent activation of which would induce tumorigenesis and progression.RASAL1 is a Ca~(2+) regulated ras GTPase-activating-like protein, the loss or decreased expression of RASAL1 induces ras activation, then results in tumongenesis and progression.In this review we sum up the mechanism of ras activation, the relationship between neoplasms and RASAL1 and the mechanism of RASAL1 expression decreasing.

  19. A novel quinoline, MT477: suppresses cell signaling through Ras molecular pathway, inhibits PKC activity, and demonstrates in vivo anti-tumor activity against human carcinoma cell lines.

    Science.gov (United States)

    Jasinski, Piotr; Welsh, Brandon; Galvez, Jorge; Land, David; Zwolak, Pawel; Ghandi, Lori; Terai, Kaoru; Dudek, Arkadiusz Z

    2008-06-01

    MT477 is a novel thiopyrano[2,3-c]quinoline that has been identified using molecular topology screening as a potential anticancer drug with a high activity against protein kinase C (PKC) isoforms. The objective of the present study was to determine the mechanism of action of MT477 and its activity against human cancer cell lines. MT477 interfered with PKC activity as well as phosphorylation of Ras and ERK1/2 in H226 human lung carcinoma cells. It also induced poly-caspase-dependent apoptosis. MT477 had a dose-dependent (0.006 to 0.2 mM) inhibitory effect on cellular proliferation of H226, MCF-7, U87, LNCaP, A431 and A549 cancer cell lines as determined by in vitro proliferation assays. Two murine xenograft models of human A431 and H226 lung carcinoma were used to evaluate tumor response to intraperitoneal administration of MT477 (33 microg/kg, 100 microg/kg, and 1 mg/kg). Tumor growth was inhibited by 24.5% in A431 and 43.67% in H226 xenografts following MT477 treatment, compared to vehicle controls (p < 0.05). In conclusion, our empirical findings are consistent with molecular modeling of MT477's activity against PKC. We also found, however, that its mechanism of action occurs through suppressing Ras signaling, indicating that its effects on apoptosis and tumor growth in vivo may be mediated by Ras as well as PKC. We propose, therefore, that MT477 warrants further development as an anticancer drug. PMID:17957339

  20. The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine

    Directory of Open Access Journals (Sweden)

    Ahmad EI

    2011-07-01

    Full Text Available Ebtesam I Ahmad, Heba H Gawish, Nashwa MA Al Azizi, Ashraf M ElhefniClinical Pathology Department, Hematology and Oncology Unit of Internal Medicine Department, Faculty of Medicine, Zagazig University, Sharkia, EgyptBackground: Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML. However, little is known about its clinical relevance in the treatment outcome for this leukemia.Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C used in postinduction consolidation chemotherapy in adult AML patients.Patients and methods: The study comprised of 71 de novo AML patients with male/female ratio 1.4:1; their ages ranged from 21–59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G-banding (Giemsa staining, and K-RAS mutation detection using real-time polymerase chain reaction. The patients were randomized into two groups according to the ara-C dose used in consolidation treatment, the high the dose ara-C (HDAC group receiving 400 mg ara-C and-low-dose ara-C (LDAC group receiving 100 mg ara-C; they were followed over a period of five years.Results: Mutations in the K-RAS gene (mutRAS were detected in 23 patients (32% with the remaining 48 patients (68% having wild-type RAS (wtRAS. The percent of blast cells was significantly lower in mutRAS compared to wtRAS patients (P ≤ 0.001 while M4 subtype of AML and Inv(16 frequencies were significantly higher in mutRAS compared to wtRAS patients (P = 0.015 and (P = 0.003, respectively. The patients were followed up for a median of 43 months (range 11–57 months. There was no significant difference in overall survival (OS between mutRAS and wtRAS (P = 0.326. Within the mutRAS

  1. Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS.

    Science.gov (United States)

    Huang, William Y C; Yan, Qingrong; Lin, Wan-Chen; Chung, Jean K; Hansen, Scott D; Christensen, Sune M; Tu, Hsiung-Lin; Kuriyan, John; Groves, Jay T

    2016-07-19

    The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

  2. 肿瘤中k-ras基因异常激活及其临床意义%The active mechanism of k -ras in cancers and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    黄晓宇; 贾晓艳

    2015-01-01

    k -ras 作为一种原癌基因,在调控细胞增殖方面起着重要作用。k -ras 基因被多种因素激活后,可产生一系列生物学效应,导致多种肿瘤的发生与演进。k -ras 异常激活还可影响肿瘤分子靶向治疗的疗效。本文对肿瘤中 k -ras 基因的激活机制异常激活及其对临床药物疗效的影响作一综述。%k -ras,which was known as an important proto -oncogene,plays an important role in cell proliferation. The active k -ras could lead series of biological effects,which can cause the progression of cancer.Activated k -ras also has influence on the targeted therapy of cancer.The active mechanism of k -ras and how does it affect clinical medication were summarized.

  3. RAS Initiative - Events

    Science.gov (United States)

    The NCI RAS Initiative has organized multiple events with outside experts to discuss how the latest scientific and technological breakthroughs can be applied to discover vulnerabilities in RAS-driven cancers.

  4. RasGRP1 and RasGRP3 Are Required for Efficient Generation of Early Thymic Progenitors.

    Science.gov (United States)

    Golec, Dominic P; Henao Caviedes, Laura M; Baldwin, Troy A

    2016-09-01

    T cell development is dependent on the migration of progenitor cells from the bone marrow to the thymus. Upon reaching the thymus, progenitors undergo a complex developmental program that requires inputs from various highly conserved signaling pathways including the Notch and Wnt pathways. To date, Ras signaling has not been implicated in the very earliest stages of T cell differentiation, but members of a family of Ras activators called RasGRPs have been shown to be involved at multiple stages of T cell development. We examined early T cell development in mice lacking RasGRP1, RasGRP3, and RasGRPs 1 and 3. We report that RasGRP1- and RasGRP3-deficient thymi show significantly reduced numbers of early thymic progenitors (ETPs) relative to wild type thymi. Furthermore, RasGRP1/3 double-deficient thymi show significant reductions in ETP numbers compared with either RasGRP1 or RasGRP3 single-deficient thymi, suggesting that both RasGRP1 and RasGRP3 regulate the generation of ETPs. In addition, competitive bone marrow chimera experiments reveal that RasGRP1/3 double-deficient progenitors intrinsically generate ETPs less efficiently than wild type progenitors. Finally, RasGRP1/3-deficient progenitors show impaired migration toward the CCR9 ligand, CCL25, suggesting that RasGRP1 and RasGRP3 may regulate progenitor entry into the thymus through a CCR9-dependent mechanism. These data demonstrate that, in addition to Notch and Wnt, the highly conserved Ras pathway is critical for the earliest stages of T cell development and further highlight the importance of Ras signaling during thymocyte maturation. PMID:27465532

  5. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

    Science.gov (United States)

    Cheng, Yougan; Othmer, Hans

    2016-01-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty

  6. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network.

    Science.gov (United States)

    Cheng, Yougan; Othmer, Hans

    2016-05-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that [Formula: see text] cycling modulated by Ric8, a nonreceptor guanine exchange factor for [Formula: see text] in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both [Formula: see text] and Gβγ are essential for direction sensing, in that membrane-localized [Formula: see text], the activated GTP-bearing form of [Formula: see text], leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient 'memory' to eliminate the 'back-of-the wave' problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since

  7. Activated Ras signaling pathways and reovirus oncolysis: an update on the mechanism of preferential reovirus replication in cancer cells

    Directory of Open Access Journals (Sweden)

    Jun eGong

    2014-06-01

    Full Text Available The development of wild-type, unmodified Type 3 Dearing (T3D strain reovirus as an anticancer agent has currently expanded to 32 clinical trials (both completed and ongoing involving reovirus in the treatment of cancer. It has been more than 30 years since the potential of reovirus as an anticancer agent was first identified in studies that demonstrated the preferential replication of reovirus in transformed cell lines but not in normal cells. Later investigations have revealed the involvement of activated Ras signaling pathways (both upstream and downstream and key steps of the reovirus infectious cycle in promoting preferential replication in cancer cells with reovirus-induced cancer cell death occurring through necrotic, apoptotic, and autophagic pathways. There is increasing evidence that reovirus-induced antitumor immunity involving both innate and adaptive responses also contributes to therapeutic efficacy though this discussion is beyond the scope of this article. Here we review our current understanding of the mechanism of oncolysis contributing to the broad anticancer activity of reovirus. Further understanding of reovirus oncolysis is critical in enhancing the clinical development and efficacy of reovirus.

  8. cis-Active Ras G2-like sequence implicated in the heterotropic activation of the deoxyadenosine kinase of Lactobacillus acidophilus R-26.

    Science.gov (United States)

    Guo, S; Ma, N; Ives, D H

    1997-03-14

    Deoxyadenosine kinase (dAK) forms a heterodimer with either deoxyguanosine kinase (dGK) or deoxycytidine kinase (dCK), and is heterotropically activated 3-5 times by dGuo or dCyd. Expressed alone, dAK is inactive and exhibits no response to dGuo or dCyd; activity and heterotropic response are fully restored upon reassociation with dGK or dCK. However, turnover of independently expressed dGK or dCK is nearly maximal, being further activated only 50-100% upon reassociation with dAK. In neither case is the heterotropic activation due to ligand-induced heterodimer formation. A proline/alanine substitution within a dAK segment homologous to loop G2 of Ras proteins yielded a heterodimer with dAK permanently cis-activated 2-fold, with a corresponding reduction in heterotropic activation by dGuo. A chimeric dAK, with 25% of its C terminus substituted by the homologous sequence from dGK, was inactive alone, and its characteristics were unchanged in the reconstituted heterodimer. Superimposing the Pro/Ala substitution on this chimera also reduced heterotropic activation by half. Cross-linking the dimer by 1,5-difluoro-2,4-dinitrobenzene was inhibited by ATP, dATP, dGTP, and dAdo, suggesting the proximity of the active site(s) to the interface. These data suggest that dAK depends on dGK or dCK in a manner resembling the reliance of Ras upon GTPase activating protein.

  9. Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane.

    Science.gov (United States)

    Chiu, C-F; Ho, M-Y; Peng, J-M; Hung, S-W; Lee, W-H; Liang, C-M; Liang, S-M

    2013-02-01

    Prohibitin (PHB) is indispensable for Ras-induced Raf-1 activation, cell migration and growth; however, the exact role of PHB in the molecular pathogenesis of cancer metastasis remains largely unexamined. Here, we found a positive correlation between plasma membrane-associated PHB and the clinical stages of cancer. The level of PHB phosphorylated at threonine 258 (T258) and tyrosine 259 (Y259) in human cancer-cell membranes correlated with the invasiveness of cancer cells. Overexpression of phosphorylated PHB (phospho-PHB) in the lipid-raft domain of the cell membrane enhanced cell migration/invasion through PI3K/Akt and Raf-1/ERK activation. It also enhanced epithelial-mesenchymal transition, matrix metalloproteinase-2 activity and invasiveness of cancer cells in vitro. Immunoprecipitation analysis demonstrated that phospho-PHB associated with Raf-1, Akt and Ras in the membrane and was essential for the activation of Raf-1 signaling by Ras. Mice implanted with cancer cells stably overexpressing PHB in the plasma membrane showed enlarged cervical tumors, enhanced metastasis and shorter survival time compared with mice implanted with cancer cells without PHB overexpression. Dephosphorylation of PHB at T258 by site-directed mutagenesis diminished the in vitro and in vivo effects of PHB. These results suggest that increase in phospho-PHB T258 in the raft domain of the plasma membrane has a role in the Ras-driven activation of PI3K/Akt and Raf-1/ERK-signaling cascades and results in the promotion of cancer metastasis.

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

    Science.gov (United States)

    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

  11. Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover

    DEFF Research Database (Denmark)

    Hong, Xin; Nguyen, Thanh Hung; Chen, Qingfeng;

    2014-01-01

    Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of ...

  12. Cardiac remodelling and RAS inhibition.

    Science.gov (United States)

    Ferrario, Carlos M

    2016-06-01

    Risk factors such as hypertension and diabetes are known to augment the activity and tissue expression of angiotensin II (Ang II), the major effector peptide of the renin-angiotensin system (RAS). Overstimulation of the RAS has been implicated in a chain of events that contribute to the pathogenesis of cardiovascular (CV) disease, including the development of cardiac remodelling. This chain of events has been termed the CV continuum. The concept of CV disease existing as a continuum was first proposed in 1991 and it is believed that intervention at any point within the continuum can modify disease progression. Treatment with antihypertensive agents may result in regression of left ventricular hypertrophy, with different drug classes exhibiting different degrees of efficacy. The greatest decrease in left ventricular mass is observed following treatment with angiotensin converting enzyme inhibitors (ACE-Is), which inhibit Ang II formation. Although ACE-Is and angiotensin receptor blockers (ARBs) provide significant benefits in terms of CV events and stroke, mortality remains high. This is partly due to a failure to completely suppress the RAS, and, as our knowledge has increased, an escape phenomenon has been proposed whereby the human sequence of the 12 amino acid substrate angiotensin-(1-12) is converted to Ang II by the mast cell protease, chymase. Angiotensin-(1-12) is abundant in a wide range of organs and has been shown to increase blood pressure in animal models, an effect abolished by the presence of ACE-Is or ARBs. This review explores the CV continuum, in addition to examining the influence of the RAS. We also consider novel pathways within the RAS and how new therapeutic approaches that target this are required to further reduce Ang II formation, and so provide patients with additional benefits from a more complete blockade of the RAS. PMID:27105891

  13. Activation of RAS family members confers resistance to ROS1 targeting drugs.

    OpenAIRE

    Cargnelutti, Marilisa; Corso, Simona; Pergolizzi, Margherita; Mévellec, Laurence; Aisner, Dara L.; Dziadziuszko, Rafal; Varella-Garcia, Marileila; Comoglio, Paolo M.; Doebele, Robert C.; Vialard, Jorge; Giordano, Silvia

    2014-01-01

    The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse. To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activatio...

  14. ASPP2 Is a Novel Pan-Ras Nanocluster Scaffold

    Science.gov (United States)

    Posada, Itziar M. D.; Serulla, Marc; Zhou, Yong; Oetken-Lindholm, Christina

    2016-01-01

    Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2’s interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2’s ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events. PMID:27437940

  15. RASAL1 expression and ras activity with gastrointestinal tumors: a review%RASAL1表达及ras活性与胃肠道肿瘤临床关系的研究进展

    Institute of Scientific and Technical Information of China (English)

    吕成余; 谢洪虎; 樊红

    2011-01-01

    Approximately 20% of human tumors are associated with mutations of ras genes. Ras proteins control cellular signaling pathways responsible for cell proliferation, differentiation, survival and apoptosis. Ras proteins belong to the large family of small GTPases, which are activated in response-to various extracellular stimuli. RASAL1, as a ras GTPase activating protein, inhibits ras activation. Regulation of ras and RASAL1 activities may lead the direction of target treatment of tumors. Detection of ras and RASAL1 activities can provide more relevant information about diagnosis and prognosis of gastrointestinal tumors. In this paper, we reviewed the structure, the biochemical properties of ras proteins, and the role of its mutations in tumors. We further discussed the inhibitive effects of RASAL1 on ras activities. Finally, we commented the correlation of ras and RASAL1 with gastrointestinal tumors.%大约有20%的人类肿瘤与ras的突变有关.ras蛋白调控细胞的一系列生物学行为,包括细胞增殖、分化、生存及凋亡等.ras蛋白属于小GTP结合蛋白,通过响应细胞外信号产生活性.RASAL1是一种ras GTP酶激活蛋白,可以抑制ras的活性.调节ras以及RASAL1的活性,可能是肿瘤靶向治疗的方向之一.检测ras以及RASAL1的活性有助于胃肠道肿瘤的早期诊断以及预后的判断.本文从ras的结构、功能及突变与肿瘤的关系,RASALl作为ras的效应分子抑制ras的活性,ras、RASAL1与胃肠道肿瘤的关系三方面来综述了RASAL1表达及ras活性与胃肠道肿瘤临床关系的研究进展.

  16. RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines.

    Science.gov (United States)

    Ksionda, O; Melton, A A; Bache, J; Tenhagen, M; Bakker, J; Harvey, R; Winter, S S; Rubio, I; Roose, J P

    2016-07-14

    Ras GTPases are activated by RasGEFs and inactivated by RasGAPs, which stimulate the hydrolysis of RasGTP to inactive RasGDP. GTPase-impairing somatic mutations in RAS genes, such as KRAS(G12D), are among the most common oncogenic events in metastatic cancer. A different type of cancer Ras signal, driven by overexpression of the RasGEF RasGRP1 (Ras guanine nucleotide-releasing protein 1), was recently implicated in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and murine models, in which RasGRP1 T-ALLs expand in response to treatment with interleukins (ILs) 2, 7 and 9. Here, we demonstrate that IL-2/7/9 stimulation activates Erk and Akt pathways downstream of Ras in RasGRP1 T-ALL but not in normal thymocytes. In normal lymphocytes, RasGRP1 is recruited to the membrane by diacylglycerol (DAG) in a phospholipase C-γ (PLCγ)-dependent manner. Surprisingly, we find that leukemic RasGRP1-triggered Ras-Akt signals do not depend on acute activation of PLCγ to generate DAG but rely on baseline DAG levels instead. In agreement, using three distinct assays that measure different aspects of the RasGTP/GDP cycle, we established that overexpression of RasGRP1 in T-ALLs results in a constitutively high GTP-loading rate of Ras, which is constantly counterbalanced by hydrolysis of RasGTP. KRAS(G12D) T-ALLs do not show constitutive GTP loading of Ras. Thus, we reveal an entirely novel type of leukemogenic Ras signals that is based on a RasGRP1-driven increased in flux through the RasGTP/GDP cycle, which is mechanistically very different from KRAS(G12D) signals. Our studies highlight the dynamic balance between RasGEF and RasGAP in these T-ALLs and put forth a new model in which IL-2/7/9 decrease RasGAP activity. PMID:26549032

  17. Mutational analysis of a ras catalytic domain

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Kung, H F;

    1986-01-01

    We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal...... transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic...

  18. Interferon-induced revertants of ras-transformed cells: resistance to transformation by specific oncogenes and retransformation by 5-azacytidine.

    Science.gov (United States)

    Samid, D; Flessate, D M; Friedman, R M

    1987-01-01

    Prolonged alpha/beta interferon (IFN-alpha/beta) treatment of NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintained a nontransformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3, cells. The PR retransformants reverted again with IFN, suggesting that DNA methylation is involved in IFN-induced persistent reversion. Images PMID:2439904

  19. Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and ras stimulation of rac activity in immortalized gonadotropin-releasing hormone neuronal cells.

    Science.gov (United States)

    Nielsen-Preiss, Sheila M; Allen, Melissa P; Xu, Mei; Linseman, Daniel A; Pawlowski, John E; Bouchard, R J; Varnum, Brian C; Heidenreich, Kim A; Wierman, Margaret E

    2007-06-01

    GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration. PMID:17332061

  20. NSD2 contributes to oncogenic RAS-driven transcription in lung cancer cells through long-range epigenetic activation.

    Science.gov (United States)

    García-Carpizo, Verónica; Sarmentero, Jacinto; Han, Bomie; Graña, Osvaldo; Ruiz-Llorente, Sergio; Pisano, David G; Serrano, Manuel; Brooks, Harold B; Campbell, Robert M; Barrero, Maria J

    2016-01-01

    The histone methyltransferase NSD2/WHSC1/MMSET is overexpressed in a number of solid tumors but its contribution to the biology of these tumors is not well understood. Here, we describe that NSD2 contributes to the proliferation of a subset of lung cancer cell lines by supporting oncogenic RAS transcriptional responses. NSD2 knock down combined with MEK or BRD4 inhibitors causes co-operative inhibitory responses on cell growth. However, while MEK and BRD4 inhibitors converge in the downregulation of genes associated with cancer-acquired super-enhancers, NSD2 inhibition affects the expression of clusters of genes embedded in megabase-scale regions marked with H3K36me2 and that contribute to the RAS transcription program. Thus, combinatorial therapies using MEK or BRD4 inhibitors together with NSD2 inhibition are likely to be needed to ensure a more comprehensive inhibition of oncogenic RAS-driven transcription programs in lung cancers with NSD2 overexpression. PMID:27604143

  1. Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

    Science.gov (United States)

    Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A

    2013-07-26

    RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

  2. K-Ras(V14I) -induced Noonan syndrome predisposes to tumour development in mice.

    Science.gov (United States)

    Hernández-Porras, Isabel; Schuhmacher, Alberto J; Garcia-Medina, Raquel; Jiménez, Beatriz; Cañamero, Marta; de Martino, Alba; Guerra, Carmen

    2016-06-01

    The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock-in mouse model that carries one of the most frequent KRAS-NS-related mutations, the K-Ras(V14I) substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K-Ras(V14I) mutation is a mild activating K-Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K-Ras(G12V) oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K-Ras(V14I) mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K-Ras(V14I) activating protein is able to induce cancer, although at a much lower level than the classical K-Ras(G12V) oncogene, and that it can be significantly modulated by both genetic and non-genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  3. VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking.

    Science.gov (United States)

    Zhou, Mo; Wiener, Heidi; Su, Wenjuan; Zhou, Yong; Liot, Caroline; Ahearn, Ian; Hancock, John F; Philips, Mark R

    2016-08-15

    Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells. PMID:27502489

  4. XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice

    Science.gov (United States)

    Cheung, Henry; Tourrette, Yves; Maas, Peter; Schalken, Jack A; van der Pluijm, Gabri

    2016-01-01

    Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy. PMID:27427904

  5. XRP44X, an Inhibitor of Ras/Erk Activation of the Transcription Factor Elk3, Inhibits Tumour Growth and Metastasis in Mice.

    Science.gov (United States)

    Semenchenko, Kostyantyn; Wasylyk, Christine; Cheung, Henry; Tourrette, Yves; Maas, Peter; Schalken, Jack A; van der Pluijm, Gabri; Wasylyk, Bohdan

    2016-01-01

    Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy. PMID:27427904

  6. Chemical biology tools for regulating RAS signaling complexity in space and time.

    Science.gov (United States)

    van Hattum, Hilde; Waldmann, Herbert

    2014-09-18

    Rat sarcoma (RAS) family members are small GTPases that control a number of signaling pathways important for normal cellular proliferation. Therefore, it is no surprise that a significant portion of human tumors express constitutively active mutated RAS proteins, which leads to deregulation of RAS signaling pathways, resulting in pathological perturbations of cell growth and death. Although the molecular details of RAS signaling cascades are well understood, there is still a largely unmet need for small molecule probes to control RAS signaling in space and time. More broadly, given the prevalence of mutated RAS in cancer, the need to translate the insights obtained from using small molecule probes into clinically useful drugs is also significant. In this review, we introduce RAS proteins and the signaling pathways they are involved in, and discuss some of the innovative chemical biology approaches to regulate RAS signaling, which include the exploitation of newly identified binding pockets, covalent inhibitors for mutated RAS, and RAS localization impairment.

  7. The activity of Mblk-1, a mushroom body-selective transcription factor from the honeybee, is modulated by the ras/MAPK pathway.

    Science.gov (United States)

    Park, Jung-Min; Kunieda, Takekazu; Kubo, Takeo

    2003-05-16

    We previously identified a gene, termed Mblk-1, that encodes a putative transcription factor with two DNA-binding motifs expressed preferentially in the mushroom body of the honeybee brain, and its preferred binding sequence, termed Mblk-1-binding element (MBE) (Takeuchi, H., Kage, E., Sawata, M., Kamikouchi, A., Ohashi, K., Ohara, M., Fujiyuki, T., Kunieda, T., Sekimizu, K., Natori, S., and Kubo, T. (2001) Insect Mol Biol 10, 487-494; Park, J.-M., Kunieda. T., Takeuchi, H., and Kubo, T. (2002) Biochem. Biophys. Res. Commun. 291, 23-28). In the present study, the effect of Mblk-1 on transcription of genes containing MBE in Drosophila Schneider's Line 2 cells was examined using a luciferase assay. Mblk-1 expression transactivated promoters containing MBEs approximately 2-7-fold. Deletion experiments revealed that RHF2, the second DNA-binding domain of Mblk-1, was necessary for the transcriptional activity. Furthermore, mitogen-activated protein kinase (MAPK) phosphorylated Mblk-1 at Ser-444 in vitro, and the Mblk-1-induced transactivation was stimulated by phosphorylation of Ser-444 by the Ras/MAPK pathway in the luciferase assay. These results suggest that Mblk-1 is a transcription factor that might function in the mushroom body neuronal circuits downstream of the Ras/MAPK pathway in the honeybee brain.

  8. Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko

    2015-05-26

    K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

  9. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

    Science.gov (United States)

    Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

    1995-03-01

    Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

  10. Ras-activated RSK1 phosphorylates EBP50 to regulate its nuclear localization and promote cell proliferation.

    Science.gov (United States)

    Lim, Hooi Cheng; Jou, Tzuu-Shuh

    2016-03-01

    Differential subcellular localization of EBP50 leads to its controversial role in cancer biology either as a tumor suppressor when it resides at the membrane periphery, or a tumor facilitator at the nucleus. However, the mechanism behind nuclear localization of EBP50 remains unclear. A RNA interference screening identified the downstream effector of the Ras-ERK cascade, RSK1, as the molecule unique for nuclear transport of EBP50. RSK1 binds to EBP50 and phosphorylates it at a conserved threonine residue at position 156 (T156) under the regulation of growth factor. Mutagenesis experiments confirmed the significance of T156 residue in nuclear localization of EBP50, cellular proliferation, and oncogenic transformation. Our study sheds light on a possible therapeutic strategy targeting at this aberrant nuclear expression of EBP50 without affecting the normal physiological function of EBP50 at other subcellular localization.

  11. Ras Conformational Ensembles, Allostery, and Signaling.

    Science.gov (United States)

    Lu, Shaoyong; Jang, Hyunbum; Muratcioglu, Serena; Gursoy, Attila; Keskin, Ozlem; Nussinov, Ruth; Zhang, Jian

    2016-06-01

    Ras proteins are classical members of small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and inactivation is terminated by GTPase-activating proteins that accelerate the intrinsic GTP hydrolysis rate by orders of magnitude. In this review, we focus on data that have accumulated over the past few years pertaining to the conformational ensembles and the allosteric regulation of Ras proteins and their interpretation from our conformational landscape standpoint. The Ras ensemble embodies all states, including the ligand-bound conformations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, transition states, and nonfunctional states serving as a reservoir for emerging functions. The ensemble is shifted by distinct mutational events, cofactors, post-translational modifications, and different membrane compositions. A better understanding of Ras biology can contribute to therapeutic strategies. PMID:26815308

  12. Mammalian target of rapamycin complex I (mTORC1 activity in ras homologue enriched in brain (Rheb-deficient mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Marlous J Groenewoud

    Full Text Available The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs, we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.

  13. Anti-tumor effect of CTLs activated by dendritic cells pulsed with K-ras mutant peptide and whole tumor antigen on pancreatic cancer%K-ras突变多肽与全细胞抗原致敏DCs诱导CTLs对胰腺癌的杀伤活性研究

    Institute of Scientific and Technical Information of China (English)

    Guang Tan; Zhongyu Wang; Xin Zhang; Zhengang Cai; Junkai Zhang

    2010-01-01

    Objective:We studied the role of specific cytotoxic T lymphocytes(CTLs)activated by dendritic cells(DCs)presenting cationic nanoparticles with the K-ras(12-Val)mutant peptide and whole tumor antigen in the killing of different pancreatic cancer cell lines in vitro and in vitro.Methods:Peripheral blood DCs were induced by rhGM-CSF and IL-4 and cultured.DCs were sensitized by whole antigen of a pancreatic cancer cell line(PANC-1)with expression of K-ras mutant,K-ras mutant peptide(K-ras+peptide)and cationic nanoparticles with K-ras mutant peptide(K-ras+peptide-CNP),respectively.Cell surface markers were measured by flow cytometry.Lymphocyte proliferation was detected by the 3H-TdR test,and ELISA was performed to detect IFN-γ secretion.125I-UdR was used to measure the killing effect of CTLs.We also evaluated the antitumor activity of CTLs in vivo in a tumor-bearing nude mouse model prepared with the PANC-1(K-ras+)and SW1990(K-ras-)cell lines.Results:Compared with K-ras+peptide,low concentration K-ras+peptide-CNP can be effectively presented by DCs(P0.05)on SW1990 cell lines(P>0.05).Conclusion:Cationic nanoparticles with K-res(12-Val)mutant peptide can be effectively presented by DCs at a low concentration in a short time.CTLs induced by K-ras+peptide-CNP had specific killing activity for the pancreatic cancer cell line with the K-ras(12-Val)mutant and could significantly inhibit tumor growth and increase the survival time of tumor-bearing nude mice.

  14. Pharmacological modulation of oncogenic Ras by natural products and their derivatives: Renewed hope in the discovery of novel anti-Ras drugs.

    Science.gov (United States)

    Quah, Shun Ying; Tan, Michelle Siying; Teh, Yuan Han; Stanslas, Johnson

    2016-06-01

    Oncogenic rat sarcoma (Ras) is linked to the most fatal cancers such as those of the pancreas, colon, and lung. Decades of research to discover an efficacious drug that can block oncogenic Ras signaling have yielded disappointing results; thus, Ras was considered "undruggable" until recently. Inhibitors that directly target Ras by binding to previously undiscovered pockets have been recently identified. Some of these molecules are either isolated from natural products or derived from natural compounds. In this review, we described the potential of these compounds and other inhibitors of Ras signaling in drugging Ras. We highlighted the modes of action of these compounds in suppressing signaling pathways activated by oncogenic Ras, such as mitogen-activated protein kinase (MAPK) signaling and the phosphoinositide-3-kinase (PI3K) pathways. The anti-Ras strategy of these compounds can be categorized into four main types: inhibition of Ras-effector interaction, interference of Ras membrane association, prevention of Ras-guanosine triphosphate (GTP) formation, and downregulation of Ras proteins. Another promising strategy that must be validated experimentally is enhancement of the intrinsic Ras-guanosine triphosphatase (GTPase) activity by small chemical entities. Among the inhibitors of Ras signaling that were reported thus far, salirasib and TLN-4601 have been tested for their clinical efficacy. Although both compounds passed phase I trials, they failed in their respective phase II trials. Therefore, new compounds of natural origin with relevant clinical activity against Ras-driven malignancies are urgently needed. Apart from salirasib and TLN-4601, some other compounds with a proven inhibitory effect on Ras signaling include derivatives of salirasib, sulindac, polyamine, andrographolide, lipstatin, levoglucosenone, rasfonin, and quercetin. PMID:27016467

  15. Regulation of H-Ras-driven MAPK signaling, transformation and tumorigenesis, but not PI3K signaling and tumor progression, by plasma membrane microdomains.

    Science.gov (United States)

    Michael, J V; Wurtzel, J G T; Goldfinger, L E

    2016-05-30

    In this study, we assessed the contributions of plasma membrane (PM) microdomain targeting to the functions of H-Ras and R-Ras. These paralogs have identical effector-binding regions, but variant C-terminal targeting domains (tDs) which are responsible for lateral microdomain distribution: activated H-Ras targets to lipid ordered/disordered (Lo/Ld) domain borders, and R-Ras to Lo domains (rafts). We hypothesized that PM distribution regulates Ras-effector interactions and downstream signaling. We used tD swap mutants, and assessed effects on signal transduction, cell proliferation, transformation and tumorigenesis. R-Ras harboring the H-Ras tD (R-Ras-tH) interacted with Raf, and induced Raf and ERK phosphorylation similar to H-Ras. R-Ras-tH stimulated proliferation and transformation in vitro, and these effects were blocked by both MEK and PI3K inhibition. Conversely, the R-Ras tD suppressed H-Ras-mediated Raf activation and ERK phosphorylation, proliferation and transformation. Thus, Ras access to Raf at the PM is sufficient for MAPK activation and is a principal component of Ras mitogenesis and transformation. Fusion of the R-Ras extended N-terminal domain to H-Ras had no effect on proliferation, but inhibited transformation and tumor progression, indicating that the R-Ras N-terminus also contributes negative regulation to these Ras functions. PI3K activation was tD independent; however, H-Ras was a stronger activator of PI3K than R-Ras, with either tD. PI3K inhibition nearly ablated transformation by R-Ras-tH, H-Ras and H-Ras-tR, whereas MEK inhibition had a modest effect on Ras-tH-driven transformation but no effect on H-Ras-tR transformation. R-Ras-tH supported tumor initiation, but not tumor progression. While H-Ras-tR-induced transformation was reduced relative to H-Ras, tumor progression was robust and similar to H-Ras. H-Ras tumor growth was moderately suppressed by MEK inhibition, which had no effect on H-Ras-tR tumor growth. In contrast, PI3K inhibition

  16. Signal integration by lipid-mediated spatial cross talk between Ras nanoclusters.

    Science.gov (United States)

    Zhou, Yong; Liang, Hong; Rodkey, Travis; Ariotti, Nicholas; Parton, Robert G; Hancock, John F

    2014-03-01

    Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing.

  17. Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

    Science.gov (United States)

    Pierret, P; Vallée, A; Mechawar, N; Dower, N A; Stone, J C; Richardson, P M; Dunn, R J

    2001-01-01

    Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function. Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

  18. Ras and Rheb Signaling in Survival and Cell Death

    International Nuclear Information System (INIS)

    One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb, the cellular context is likely to modulate the whole Rheb-mTor pathway towards cellular death or survival, respectively

  19. The human c-Kirsten ras gene is activated by a novel mutation in codon 13 in the breast carcinoma cell line MDA-MB231.

    OpenAIRE

    Kozma, S C; Bogaard, M E; Buser, K; Saurer, S M; Bos, J. L.; Groner, B; Hynes, N E

    1987-01-01

    We have detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231. Hybridization of synthetic oligonucleotides specific for human Ki-ras sequences showed a mutation at codon 13. The polymerase chain reaction with Ki-ras specific amplimers revealed a guanosine to adenosine transition at the second position of codon 13, resulting in a substitution of glycine by aspartic acid. The codon 13 mutation is a...

  20. Altered myoelectric activity in the experimental blind loop syndrome.

    Science.gov (United States)

    Justus, P G; Fernandez, A; Martin, J L; King, C E; Toskes, P P; Mathias, J R

    1983-09-01

    Nutrient malabsorption and diarrhea are characteristic of the blind loop syndrome. Alterations in motility have been implicated as a cause of bacterial overgrowth, but the possibility that altered motility may result from alterations in the flora has not been explored. The purpose of this study was to characterize the myoelectric activity of the small intestine in the blind loop rat model. Eight groups of rats were studied: rats with self-filling blind loops, which develop bacterial overgrowth; rats with self-emptying blind loops, which are surgical controls that do not develop overgrowth; unoperated litter mates; rats with self-filling blind loops and unoperated controls treated with chloramphenicol, 200 mg/d i.p.; rats with surgically removed self-filling blind loops; operated control rats; and gnotobiotic rats with self-filling blind loops. In the untreated rats with self-filling blind loops, there was altered myoelectric activity characterized by an increased percentage of slow waves occupied by action potentials and by organized activity similar to the migrating action potential complex. Migrating action potential complex activity and percentage of slow waves occupied by action potentials were significantly decreased with chloramphenicol therapy; that decrease correlated with a decrease in aerobes and anaerobes. Migrating action potential complex activity was abolished in rats with surgically removed self-filling blind loops; they also showed a significant decrease in percentage of slow waves occupied by action potentials. Gnotobiotic rats with self-filling blind loops showed no alteration in myoelectric activity. These data indicate: (a) bacterial overgrowth is associated with a significant increase in percentage of slow waves occupied by action potentials and migrating action potential complex activity; (b) chloramphenicol significantly reduced both percentage of slow waves occupied by action potentials and migrating action potential complex activity; and (c

  1. Silencing of SPRY1 Triggers Complete Regression of Rhabdomyosarcoma Tumors Carrying a Mutated RAS Gene

    NARCIS (Netherlands)

    G. Schaaf; M. Hamdi; D. Zwijnenburg; A. Lakeman; D. Geerts; R. Versteeg; M. Kool

    2010-01-01

    RAS oncogenes are among the most frequently mutated genes in human cancer, but effective strategies for therapeutic inhibition of the RAS pathway have been elusive. Sprouty1 (SPRY1) is an upstream antagonist of RAS that is activated by extracellular signal-related kinase (ERK), providing a negative

  2. Design, synthesis and biological evaluation of sugar-derived Ras inhibitors.

    Science.gov (United States)

    Peri, Francesco; Airoldi, Cristina; Colombo, Sonia; Martegani, Enzo; van Neuren, Anske Stephanie; Stein, Matthias; Marinzi, Chiara; Nicotra, Francesco

    2005-10-01

    The design and synthesis of novel Ras inhibitors with a bicyclic scaffold derived from the natural sugar D-arabinose are presented. Molecular modelling showed that these ligands can bind Ras by accommodating the aromatic moieties and the phenylhydroxylamino group in a cavity near the Switch II region of the protein. All the synthetic compounds were active in inhibiting nucleotide exchange on p21 human Ras in vitro, and two of them selectively inhibited Ras-dependent cell growth in vivo.

  3. Neoplastic transformation of a human prostate epithelial cell line by the v-Ki-ras oncogene.

    Science.gov (United States)

    Parda, D S; Thraves, P J; Kuettel, M R; Lee, M S; Arnstein, P; Kaighn, M E; Rhim, J S; Dritschilo, A

    1993-01-01

    Investigations of mechanisms of human prostate carcinogenesis are limited by the unavailability of a suitable in vitro model system. We have demonstrated that an immortal, but nontumorigenic, human epithelial cell line (267B1) established from fetal prostate tissue can be malignantly transformed by a biological carcinogen, and can serve as a useful model for investigations of the progression steps of carcinogenesis. Activated Ki-ras was introduced into 267B1 cells by infection with the Kirsten murine sarcoma virus. Morphological alterations and anchorage-independent growth were observed; when cells were injected into nude mice, poorly differentiated adenocarcinomas developed. These findings represent the first evidence of malignant transformation of human prostate epithelial cells in culture, and support a role for Ki-ras activation in a multistep process for prostate neoplastic transformation.

  4. Methionine adenosyltransferase 2B-GIT1 complex serves as a scaffold to regulate Ras/Raf/MEK1/2 activity in human liver and colon cancer cells.

    Science.gov (United States)

    Peng, Hui; Li, Tony W H; Yang, Heping; Moyer, Mary P; Mato, Jose M; Lu, Shelly C

    2015-04-01

    Methionine adenosyltransferase 2B (MAT2B) encodes for variant proteins V1 and V2 that interact with GIT1 to increase ERK activity and growth in human liver and colon cancer cells. MAT2B or GIT1 overexpression activates MEK. This study explores the mechanism for MEK activation. We examined protein-protein interactions by co-immunoprecipitation and verified by confocal microscopy and pull-down assay using recombinant or in vitro translated proteins. Results were confirmed in an orthotopic liver cancer model. We found that MAT2B and GIT1-mediated MEK1/2 activation was not mediated by PAK1 or Src in HepG2 or RKO cells. Instead, MAT2B and GIT1 interact with B-Raf and c-Raf and enhance recruitment of Raf proteins to MEK1/2. MAT2B-GIT1 activates c-Raf, which is the key mediator for MEK/12 activation, because this still occurred in RKO cells that express constitutively active B-Raf mutant. The mechanism lies with the ability of MAT2B-GIT1 to activate Ras and promote B-Raf/c-Raf heterodimerization. Interestingly, MAT2B but not GIT1 can directly interact with Ras, which increases protein stability. Finally, increased Ras-Raf-MEK signaling occurred in phenotypically more aggressive liver cancers overexpressing MAT2B variants and GIT1. In conclusion, interaction between MAT2B and GIT1 serves as a scaffold and facilitates signaling in multiple steps of the Ras/Raf/MEK/ERK pathway, further emphasizing the importance of MAT2B/GIT1 interaction in cancer growth.

  5. Endogenous K-ras signaling in erythroid differentiation.

    Science.gov (United States)

    Zhang, Jing; Lodish, Harvey F

    2007-08-15

    K-ras is one of the most frequently mutated genes in virtually all types of human cancers. Using mouse fetal liver erythroid progenitors as a model system, we studied the role of endogenous K-ras signaling in erythroid differentiation. When oncogenic K-ras is expressed from its endogenous promoter, it hyperactivates cytokine-dependent signaling pathways and results in a partial block in erythroid differentiation. In erythroid progenitors deficient in K-ras, cytokine-dependent Akt activation is greatly reduced, leading to delays in erythroid differentiation. Thus, both loss- and gain-of-Kras functions affect erythroid differentiation through modulation of cytokine signaling. These results support the notion that in human cancer patients oncogenic Ras signaling might be controlled by antagonizing essential cytokines.

  6. Myelin alters the inflammatory phenotype of macrophages by activating PPARs

    OpenAIRE

    Bogie, Jeroen; Jorissen, Winde; Mailleux, Jo; Vanmierlo, Tim; van Horssen, Jack; Hellings, Niels; Stinissen, Piet; Hendriks, J. J. A.; Nijland, Philip G.; Zelcer, Noam

    2013-01-01

    Background Foamy macrophages, containing myelin degradation products, are abundantly found in active multiple sclerosis (MS) lesions. Recent studies have described an altered phenotype of macrophages after myelin internalization. However, mechanisms by which myelin affects the phenotype of macrophages and how this phenotype influences lesion progression remain unclear. Results We demonstrate that myelin as well as phosphatidylserine (PS), a phospholipid found in myelin, reduce nitri...

  7. Opposing effects of a ras oncogene on growth factor-stimulated phosphoinositide hydrolysis: desensitization to platelet-derived growth factor and enhanced sensitivity to bradykinin

    International Nuclear Information System (INIS)

    Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradyknin to activate phospholipase C-mediated phosphoinositide hydrolysis. In [3H]inositol-labeled rat-1 fibroblasts, PDGF resulted in a 2-fold increase in the level of [3H]inositol trisphosphate (InsP3) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of [3H]inositol monophosphate (InsP1) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either [3H]InsP3 or [3H]InsP1. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21/sup ras/ and the loss of PDGF-stimulated [3H]InsP1 accumulation. The authors propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors

  8. Spectropolarimetric monitoring of active galaxy 3C390.3 with 6m telescope SAO RAS in the period 2009-2014

    CERN Document Server

    Afanasiev, V L; Popovic, L C; Borisov, N V

    2015-01-01

    Here we present the spectropolarimetric observations of the radio loud active galaxy 3C 390.3 in the period 2009-2014 (24 epochs). The galaxy has been observed with the 6-meter telescope of SAO RAS using the SCORPIO spectropolarimeter. We explore the variability and lags in the polarized light of the continuum and broad H$\\alpha$ line. We give the Stokes parameters $Q, U$, degree of linear polarization $P$ and the position angle of the polarization plane, $\\varphi$, for 24 epochs. We find a small lag~(10-40 days) between the unpolarized and polarized continuum that is significantly smaller than the estimated lags for the unpolarized broad emission lines (lag(H$\\alpha$)$\\sim$138-186 and lag(H$\\beta$)$\\sim$60-79 days). This shows that the region of the variable polarized continuum is significantly smaller than the broad line region, indicating that a part of the polarized continuum is coming from the jet. The lag of the polarized light in the H$\\alpha$ line (89-156 days) indicates an additional component to the...

  9. Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors

    International Nuclear Information System (INIS)

    The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, the authors have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 104 foci per μg of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, they found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which the authors had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment

  10. The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription

    DEFF Research Database (Denmark)

    Nielsen, O; Davey, William John; Egel, R;

    1992-01-01

    Loss of ras1+ function renders fission yeast cells unable to undergo morphological changes in response to mating pheromones, whereas cells carrying activated mutations in ras1 are hyper-responsive. This has led to the suggestion that the ras1 gene product plays a role in mating pheromone signal...... transduction. Using partially purified M factor we demonstrate that the mat1-Pm gene, which controls entry into meiosis, is transcribed in response to a pheromone signal. Strains mutated in the ras1 gene or in ste6, the fission yeast homologue of Ras protein GDP/GTP exchange factor, are unable to induce...... transcription of mat1-Pm in response to M factor. Furthermore, an activated ras1val17 mutant exhibits a stronger induction of the mat1-Pm transcript. However, transcription still depends on nitrogen deprivation as well as on the presence of pheromone, showing that activation of the Ras1 protein alone does...

  11. Alterations in HIV-1 LTR promoter activity during AIDS progression

    International Nuclear Information System (INIS)

    HIV-1 variants evolving in AIDS patients frequently show increased replicative capacity compared to those present during early asymptomatic infection. It is known that late stage HIV-1 variants often show an expanded coreceptor tropism and altered Nef function. In the present study we investigated whether enhanced HIV-1 LTR promoter activity might also evolve during disease progression. Our results demonstrate increased LTR promoter activity after AIDS progression in 3 of 12 HIV-1-infected individuals studied. Further analysis revealed that multiple alterations in the U3 core-enhancer and in the transactivation-response (TAR) region seem to be responsible for the enhanced functional activity. Our findings show that in a subset of HIV-1-infected individuals enhanced LTR transcription contributes to the increased replicative potential of late stage virus isolates and might accelerate disease progression

  12. Human activities change marine ecosystems by altering predation risk.

    Science.gov (United States)

    Madin, Elizabeth M P; Dill, Lawrence M; Ridlon, April D; Heithaus, Michael R; Warner, Robert R

    2016-01-01

    In ocean ecosystems, many of the changes in predation risk - both increases and decreases - are human-induced. These changes are occurring at scales ranging from global to local and across variable temporal scales. Indirect, risk-based effects of human activity are known to be important in structuring some terrestrial ecosystems, but these impacts have largely been neglected in oceans. Here, we synthesize existing literature and data to explore multiple lines of evidence that collectively suggest diverse human activities are changing marine ecosystems, including carbon storage capacity, in myriad ways by altering predation risk. We provide novel, compelling evidence that at least one key human activity, overfishing, can lead to distinct, cascading risk effects in natural ecosystems whose magnitude exceeds that of presumed lethal effects and may account for previously unexplained findings. We further discuss the conservation implications of human-caused indirect risk effects. Finally, we provide a predictive framework for when human alterations of risk in oceans should lead to cascading effects and outline a prospectus for future research. Given the speed and extent with which human activities are altering marine risk landscapes, it is crucial that conservation and management policy considers the indirect effects of these activities in order to increase the likelihood of success and avoid unfortunate surprises. PMID:26448058

  13. Activated oxygen alters cerebral microvascular responses in newborn pigs

    Energy Technology Data Exchange (ETDEWEB)

    Leffler, C.W.; Busiia, D.W.; Armstead, W.M.; Mirro, R.; Thelin, O. (Univ. of Tennessee, Memphis (United States))

    1990-02-26

    In piglets, cerebral ischemia/reperfusion blocks prostanoid dependent cerebral vasodilation to hypercapnia (CO{sub 2}) and hypotension but not prostanoid independent dilation to isoproterenol (Isu) or constriction to norepinephrine (NE). Ischemia/reperfusion increases activated-O{sub 2} production by piglet brains. Using cranial windows in piglets, the authors investigated the hypothesis that activated oxygen can block prostanoid dependent cerebral vasodilator responses to CO{sub 2} and hypotension without altering responses to Isu and NE. Exposure to an activated oxygen generating system of xanthine oxidase, hypoxanthine, and Fe that made about 3 times the activated-O{sub 2} on the brain surface as ischemia/reperfusion caused reversible pial arteriolar dilation. After exposure, pial arteriolar dilation was reduced to CO{sub 2} and hypotension but not to Isu. NE constrictor responses were also unaltered. H{sub 2}O{sub 2} or H{sub 2}O{sub 2} + Fe caused constriction followed by reversible dilation. After exposure, pial arteriolar dilation in response to CO{sub 2} and hypotension was not altered. However, addition of xanthine oxidase and hypoxanthine with H{sub 2}O{sub 2} and Fe totally eliminated pial arteriolar dilator responses to CO{sub 2} and hypotension but did not decrease dilation caused by Isu or constriction caused by NE. The authors conclude that activated oxygen could produce the altered prostanoid dependent pial arteriolar responses observed following ischemia in piglets.

  14. Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling.

    Science.gov (United States)

    Pazman, C; Mayes, C A; Fanto, M; Haynes, S R; Mlodzik, M

    2000-04-01

    The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.

  15. EMT-induced stemness and tumorigenicity are fueled by the EGFR/Ras pathway.

    Directory of Open Access Journals (Sweden)

    Dominic Chih-Cheng Voon

    Full Text Available Recent studies have revealed that differentiated epithelial cells would acquire stem cell-like and tumorigenic properties following an Epithelial-Mesenchymal Transition (EMT. However, the signaling pathways that participate in this novel mechanism of tumorigenesis have not been fully characterized. In Runx3 (-/- p53 (-/- murine gastric epithelial (GIF-14 cells, EMT-induced plasticity is reflected in the expression of the embryonal proto-oncogene Hmga2 and Lgr5, an exclusive gastrointestinal stem cell marker. Here, we report the concurrent activation of an EGFR/Ras gene expression signature during TGF-β1-induced EMT in GIF-14 cells. Amongst the altered genes was the induction of Egfr, which corresponded with a delayed sensitization to EGF treatment in GIF-14. Co-treatment with TGF-β1 and EGF or the expression of exogenous KRas led to increased Hmga2 or Lgr5 expression, sphere initiation and colony formation in soft agar assay. Interestingly, the gain in cellular plasticity/tumorigenicity was not accompanied by increased EMT. This uncoupling of EMT and the induction of plasticity reveals an involvement of distinct signaling cues, whereby the EGFR/Ras pathway specifically promotes stemness and tumorigenicity in EMT-altered GIF-14 cells. These data show that the EGFR/Ras pathway requisite for the sustenance of gastric stem cells in vivo and in vitro is involved in the genesis and promotion of EMT-induced tumor-initiating cells.

  16. Ras signalling regulates differentiation and UCP1 expression in models of brown adipogenesis

    DEFF Research Database (Denmark)

    Murholm, Maria; Dixen, Karen; Hansen, Jacob B

    2010-01-01

    BACKGROUND: The Ras/Raf/MEK/ERK pathway has been recognised as an important signalling module in adipogenesis and adipocyte function, but whether it promotes or inhibits the formation of fat cells has not been reconciled. METHODS: Here we investigate the significance of Ras signalling intensity...... on two unrelated models of mouse brown adipocyte differentiation. RESULTS: A constitutively active H-Ras mutant (Ras V12) caused a complete block of adipose conversion, as manifested by a lack of both lipid accumulation and induction of adipocyte gene expression. The Ras V12-mediated impediment...

  17. Performance of McRAS-AC in the GEOS-5 AGCM: Part 1, Aerosol-Activated Cloud Microphysics, Precipitation, Radiative Effects, and Circulation

    Science.gov (United States)

    Sud, Y. C.; Lee, D.; Oreopoulos, L.; Barahona, D.; Nenes, A.; Suarez, M. J.

    2012-01-01

    A revised version of the Microphysics of clouds with Relaxed Arakawa-Schubert and Aerosol-Cloud interaction (McRAS-AC), including, among others, the Barahona and Nenes ice nucleation parameterization, is implemented in the GEOS-5 AGCM. Various fields from a 10-year long integration of the AGCM with McRAS-AC were compared with their counterparts from an integration of the baseline GEOS-5 AGCM, and with satellite data as observations. Generally using McRAS-AC reduced biases in cloud fields and cloud radiative effects are much better over most of the regions of the Earth. Two weaknesses are identified in the McRAS-AC runs, namely, too few cloud particles around 40S-60S, and too high cloud water path during northern hemisphere summer over the Gulf Stream and North Pacific. Sensitivity analyses showed that these biases potentially originated from biases in the aerosol input. The first bias is largely eliminated in a sensitivity test using 50% smaller aerosol particles, while the second bias is much reduced when interactive aerosol chemistry was turned on. The main drawback of McRAS-AC is dearth of low-level marine stratus clouds, probably due to lack of dry-convection, not yet implemented into the cloud scheme. Despite these biases, McRAS-AC does simulate realistic clouds and their optical properties that can improve with better aerosol-input and thereby has the potential to be a valuable tool for climate modeling research because of its aerosol indirect effect simulation capabilities involving prediction of cloud particle number concentration and effective particle size for both convective and stratiform clouds is quite realistic.

  18. 1H, 15N and 13C backbone assignments of GDP-bound human H-Ras mutant G12V.

    Science.gov (United States)

    Amin, Nader; Chiarparin, Elisabetta; Coyle, Joe; Nietlispach, Daniel; Williams, Glyn

    2016-04-01

    Harvey Ras (H-Ras) is a membrane-associated GTPase with critical functions in cell proliferation and differentiation. The G12V mutant of H-Ras is one of the most commonly encountered oncoproteins in human cancer. This mutation disrupts the GTPase activity of H-Ras, leading to constitutive activation and aberrant downstream signalling. Here we report the backbone resonance assignments of human H-Ras mutant G12V lacking the C-terminal membrane attachment domain.

  19. RAS/RAF/MEK/ERK and PI3K/PTEN/AKT Signaling in Malignant Melanoma Progression and Therapy

    Directory of Open Access Journals (Sweden)

    Ichiro Yajima

    2012-01-01

    Full Text Available Cutaneous malignant melanoma is one of the most serious skin cancers and is highly invasive and markedly resistant to conventional therapy. Melanomagenesis is initially triggered by environmental agents including ultraviolet (UV, which induces genetic/epigenetic alterations in the chromosomes of melanocytes. In human melanomas, the RAS/RAF/MEK/ERK (MAPK and the PI3K/PTEN/AKT (AKT signaling pathways are two major signaling pathways and are constitutively activated through genetic alterations. Mutations of RAF, RAS, and PTEN contribute to antiapoptosis, abnormal proliferation, angiogenesis, and invasion for melanoma development and progression. To find better approaches to therapies for patients, understanding these MAPK and AKT signaling mechanisms of melanoma development and progression is important. Here, we review MAPK and AKT signaling networks associated with melanoma development and progression.

  20. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    NARCIS (Netherlands)

    O. Kauko; T.D. Laajala; M. Jumppanen; P. Hintsanen; V. Suni; P. Haapaniemi; G. Corthals; T. Aittokallio; J. Westermarck; S.Y. Imanishi

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how

  1. RAS AND p53 EXPRESSION IN HUMAN THYROID CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the possible interaction between the ras and p53 genes over-expression in thyroid carcinoma, and whether there is a correlation between the ras and p53 over-expression and clinicopathological criteria. Methods: Eighty patients with thyroid lesions were examined for expression of ras and p53 genes by the labeled streptavidin biotin peroxidase (LSAB) method. Of these patients, 54 were diagnosed (average age: 39.9± 15.9 years) with malignant lesions. Of those included in the study, 31 has papillary carcinoma, 13 had follicular carcinoma, 7 had medullary carcinoma, 3 had undifferentiated carcinoma and 19 were stratified to stage I, 28 to stage II, 2 to stage III and 5 to stage IV according to TNM staging system. Twenty-six benign nodular thyroid disorders were studied as control. Results: Positive immunostain results for ras and p53 genes were statistically significant between thyroid carcinomas and benign disorders (90.7% vs 23%, 55.5% vs 30.7%, P<0.05). Both p53 and ras overexpressions coexisted in 30 thyroid carcinomas, and of these, 3 died and 5 had recurrences within 4 years. Conclusions: Activation of ras gene and inactivation of p53 gene were cooperatively associated in thyroid tumorigenesis. The concurrent overexpressions of ras and p53 could result in a poor prognosis.

  2. Analysis of beta-catenin, Ki-ras, and microsatellite stability in azoxymethane-induced colon tumors of BDIX/Orl Ico rats

    DEFF Research Database (Denmark)

    Sørensen, Nanna Møller; Kobaek-Larsen, Morten; Bonne, Anita;

    2003-01-01

    The aim of the study reported here was to investigate whether the azoxymethane (AOM)-induced colon cancer rat model mimics the human situation with regard to microsatellite stability, changes in expression of beta-catenin, and/or changes in the sequence of the proto-oncogene Ki-ras. Colon cancer...... result is in accordance with those of similar studies in other rat models and with most human colorectal cancers. Immunohistochemical analyses of beta-catenin did not reveal loss of gene activity, nor did the sequencing of Ki-ras reveal mutations. These results are in contrast to most findings in...... comparable rat studies. The deviations may be due to differences in exposure to the carcinogen or difference in strain and/or age. The lack of beta-catenin and Ki-ras alterations in this colon cancer model is unlike human sporadic colorectal cancers where these genetic changes are common findings....

  3. Environmental noise alters gastric myoelectrical activity: Effect of age

    Institute of Scientific and Technical Information of China (English)

    James S Castle; Jin-Hong Xing; Mark R Warner; Mark A Korsten

    2007-01-01

    AIM: To evaluate the effect of age and acoustic stress on gastric myoelectrical activity (GMA) and autonomic nervous system function,METHODS: Twenty-one male subjects (age range 22-71years, mean 44 years) were recruited and exposed, in random order, to three auditory stimuli (Hospital noise,conversation babble and traffic noise) after a 20-min baseline. All periods lasted 20 min and were interspersed with a 10 min of recovery. GMA was obtained using a Synectics Microdigitrapper. Autonomic nerve function was assessed by monitoring blood pressure and heart rate using an automatic recording device.RESULTS: Dominant power tended to decrease with increase of age (P<0.05). The overall percentage of three cycle per minute (CPM) activity decreased during exposure to hospital noise (12.0%, P < 0.05), traffic noise (13.9%, P < 0.05), and conversation babble(7.1%). The subjects in the younger group (< 50 years)showed a consistent reduction in the percentage of 3CPM activity during hospital noise (22.9%, P < 0.05),traffic noise (19.0%, P < 0.05), and conversation babble(15.5%). These observations were accompanied by a significant increase in bradygastria: hospital noise (P< 0.05) and traffic noise (P < 0.05). In contrast, the subjects over 50 years of age did not exhibit a significant decrease in 3 CPM activity. Regardless of age, noise did not alter blood pressure or heart rate.CONCLUSION: GMA changes with age. Loud noise can alter GMA, especially in younger individuals. Our data indicate that even short-term exposure to noise may alter the contractility of the stomach.

  4. The oncoprotein H-RasV12 increases mitochondrial metabolism

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    Arumugam Sengodagounder

    2007-12-01

    Full Text Available Abstract Background Neoplastic cells increase glycolysis in order to produce anabolic precursors and energy within the hypoxic environment of a tumor. Ras signaling is activated in several cancers and has been found to regulate metabolism by enhancing glycolytic flux to lactate. We examined the effects of sequential immortalization and H-RasV12-transformation of human bronchial epithelial cells on the anabolic fate of fully-labeled 13C-glucose-derived carbons using two-dimensional total correlated spectroscopic analysis-nuclear magnetic resonance spectroscopy (2D TOCSY-NMR. Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells unexpectedly increased tricarboxylic acid cycle activity as measured by the direct conversion of 13C-glucose carbons into the anabolic substrates glutamate/glutamine, aspartate and uridine. We then observed that immortalization and H-RasV12-transformation of bronchial epithelial cells caused a stepwise increase in oxygen consumption, a global measure of electron transport chain activity. Importantly, ectopic expression of H-RasV12 sensitized immortalized cells to the ATP-depleting and cytotoxic effects of electron transport perturbation using the complex I inhibitor rotenone. Conclusion Taken together, these data indicate that the oncoprotein H-RasV12 increases mitochondrial metabolism and provide new rationale for the targeting of the tricarboxylic acid cycle and electron transport chain as anti-neoplastic strategies.

  5. Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

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    Muñoz-Félix, José M; Fuentes-Calvo, Isabel; Cuesta, Cristina; Eleno, Nélida; Crespo, Piero; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-10-01

    The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-β1 (TGF-β1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-β1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-β1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc.

  6. Detection of a Point Mutation at Codon 12 of the Kirsten-Ras (K-ras Oncogen

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    Howida M. Sharaf, Nihal S. El-Kinawy, Ayman Omar* and Manal A. Ali

    2012-04-01

    Full Text Available Background: Mutations in ras genes have been observed in a variety of cancers and were found to play an important role in human leukemogenesis and in preleukemic disease as myelodysplastic syndrome (MDS. The purpose of this study was to determine the prevalence of mutated K-ras oncogene in myelodysplastic syndrome (MDS; with a special emphasis on their possible role in affecting clinical status, relation to karyotypic pattern; response to therapeutic measures; its impact on the fate of the disease and overall survival.Subjects & methods: Detection of point mutation of Kirsten-ras (K-ras gene in 30 patients suffering from myelodysplastic syndrome was carried out using quantitative enriched polymerase chain reaction (QEPCR and was confirmed by sequencing. QEPCR is a two- stage PCR procedure with modified primers that enriches mutant alleles, via restriction endonuclease digestion of normal alleles and enables identification of one mutant allele among 100,000 normal alleles. Results: Activating mutations of the codon 12 of K-ras gene were detected in 7/30 (23.3%cases of MDS, the most common mutation involved a substitution of aspartic acid for glycine (GGT→GAT. The incidence of K-ras mutations was found to be significantly associated with refractory anemia with excess blasts type II (RAEBII and unclassified (UC MDS than other subtypes (p=0.005, and was significantly associated with hypercellular bone marrow (p=0.04 showing marked dyserythropoitic changes. Furthermore, mutant K-ras gene was found to be significantly associated with abnormal karyotypes (p=0.04. Patients with mutated K-ras gene were significantly associated with either high or intermediate risk according to International Prognostic Scoring System (IPSS (p=0.001. 6/7(85.7% of those carrying the mutation showed poor response to treatment compared to non carriers with a statistical significant difference (p=0.009. Five out of eight (62.5% patients who were transformed to AML carried

  7. Altered brain activity for phonological manipulation in dyslexic Japanese children

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    Yamamoto, Hisako; Oba, Kentaro; Terasawa, Yuri; Moriguchi, Yoshiya; Uchiyama, Hitoshi; Seki, Ayumi; Koeda, Tatsuya; Inagaki, Masumi

    2013-01-01

    Because of unique linguistic characteristics, the prevalence rate of developmental dyslexia is relatively low in the Japanese language. Paradoxically, Japanese children have serious difficulty analysing phonological processes when they have dyslexia. Neurobiological deficits in Japanese dyslexia remain unclear and need to be identified, and may lead to better understanding of the commonality and diversity in the disorder among different linguistic systems. The present study investigated brain activity that underlies deficits in phonological awareness in Japanese dyslexic children using functional magnetic resonance imaging. We developed and conducted a phonological manipulation task to extract phonological processing skills and to minimize the influence of auditory working memory on healthy adults, typically developing children, and dyslexic children. Current experiments revealed that several brain regions participated in manipulating the phonological information including left inferior and middle frontal gyrus, left superior temporal gyrus, and bilateral basal ganglia. Moreover, dyslexic children showed altered activity in two brain regions. They showed hyperactivity in the basal ganglia compared with the two other groups, which reflects inefficient phonological processing. Hypoactivity in the left superior temporal gyrus was also found, suggesting difficulty in composing and processing phonological information. The altered brain activity shares similarity with those of dyslexic children in countries speaking alphabetical languages, but disparity also occurs between these two populations. These are initial findings concerning the neurobiological impairments in dyslexic Japanese children. PMID:24052613

  8. Altered brain activity for phonological manipulation in dyslexic Japanese children.

    Science.gov (United States)

    Kita, Yosuke; Yamamoto, Hisako; Oba, Kentaro; Terasawa, Yuri; Moriguchi, Yoshiya; Uchiyama, Hitoshi; Seki, Ayumi; Koeda, Tatsuya; Inagaki, Masumi

    2013-12-01

    Because of unique linguistic characteristics, the prevalence rate of developmental dyslexia is relatively low in the Japanese language. Paradoxically, Japanese children have serious difficulty analysing phonological processes when they have dyslexia. Neurobiological deficits in Japanese dyslexia remain unclear and need to be identified, and may lead to better understanding of the commonality and diversity in the disorder among different linguistic systems. The present study investigated brain activity that underlies deficits in phonological awareness in Japanese dyslexic children using functional magnetic resonance imaging. We developed and conducted a phonological manipulation task to extract phonological processing skills and to minimize the influence of auditory working memory on healthy adults, typically developing children, and dyslexic children. Current experiments revealed that several brain regions participated in manipulating the phonological information including left inferior and middle frontal gyrus, left superior temporal gyrus, and bilateral basal ganglia. Moreover, dyslexic children showed altered activity in two brain regions. They showed hyperactivity in the basal ganglia compared with the two other groups, which reflects inefficient phonological processing. Hypoactivity in the left superior temporal gyrus was also found, suggesting difficulty in composing and processing phonological information. The altered brain activity shares similarity with those of dyslexic children in countries speaking alphabetical languages, but disparity also occurs between these two populations. These are initial findings concerning the neurobiological impairments in dyslexic Japanese children.

  9. Society News: PhD theses could win prizes; Last chance for IYA2009 grants; New Fellows; RAS Fellows win prizes; Need a job? Need staff? RAS Library Saturdays

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    2009-08-01

    Fellows who are PhD student supervisors should be on the lookout for exceptionally good work from research students submitting their theses this year, for nomination for the RAS Michael Penston Astronomy Prize and the RAS Keith Runcorn Prize. The RAS is offering one last chance to apply for grants towards International Year of Astronomy activities, but you'll have to apply soon. The Society sends congratulations to Fellows of the RAS who have recently received prestigious awards for their work.

  10. Mice lacking Ras-GRF1 show contextual fear conditioning but not spatial memory impairments: convergent evidence from two independently generated mouse mutant lines

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    Raffaele ed'Isa

    2011-12-01

    Full Text Available Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning the Brambilla’s mice with a third mouse line (GENA53 in which a nonsense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in the Brambilla’s mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdalar functions but possibly to some distinct hippocampal connections specific to

  11. Thalidomide combined with irradiation alters the activity of two proteases.

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    Şimşek, Ece; Aydemir, Esra; Korcum, Aylin Fidan; Fişkın, Kayahan

    2015-02-01

    The aim of the present study was to investigate the effects of thalidomide, a drug known for its anti‑angiogenic and antitumor properties, at its cytotoxic dose previously determined as 40 µg/ml (according to four cytotoxic test results). The effect of the drug alone and in combination with radiotherapy using Cobalt 60 (60Co) at 45 Gy on the enzymatic activity of substance‑P degrading A disintegrin and metalloproteinase (ADAM)10 and neprilysin (NEP) was investigated in the mouse breast cancer cell lines 4T1 and 4T1 heart metastases post‑capsaicin (4THMpc). Thalidomide (40 µg/ml) exerted differing effects on the activities of ADAM10 and NEP enzymes. In 4T1 cells, 40 µg/ml thalidomide alone did not alter ADAM10 enzyme activity. 60Co irradiation at 45 Gy alone caused a 42% inhibition in ADAM10 activity, however, the inhibition increased to 89% when combined therapy was used. By contrast, in the 4THMpc cell line, 40 µg/ml thalidomide alone induced a 66.6% increase in ADAM10 enzyme activity. Radiotherapy alone and thalidomide with 60Co combined therapy caused a 33.3 and 40% inhibition of ADAM10 activity, respectively. In 4T1 cells, thalidomide alone caused a 40.9% increase in NEP activity. Radiation therapy alone or in combination with the drug caused a 40.7% increase in NEP activity. In more aggressive 4THMpc cells, thalidomide alone caused a 26.6% increase in NEP activity. Radiotherapy alone and combined therapy caused a 33.3 and 37% increase in enzyme activity, respectively. To the best of our knowledge, the present study is the first to demonstrate that thalidomide alone or in combination with radiotherapy exhibits significant cytotoxic effects on 4T1 and 4THMpc mouse breast cancer cell lines indicating that this drug affects the enzymatic activity of ADAM10 and NEP in vitro.

  12. A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc.

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    Li Sheng-Fowler

    Full Text Available As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM, DNA (100 µg was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA.

  13. Low HDL cholesterol, aggression and altered central serotonergic activity.

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    Buydens-Branchey, L; Branchey, M; Hudson, J; Fergeson, P

    2000-03-01

    Many studies support a significant relation between low cholesterol levels and poor impulse, aggression and mood control. Evidence exists also for a causal link between low brain serotonin (5-HT) activity and these behaviors. Mechanisms linking cholesterol and hostile or self-destructive behavior are unknown, but it has been suggested that low cholesterol influences 5-HT function. This study was designed to explore the relationship between plasma cholesterol, measures of impulsivity and aggression, and indices of 5-HT function in personality disordered cocaine addicts. Thirty-eight hospitalized male patients (age 36.8+/-7.1) were assessed with the DSM-III-R, the Buss-Durkee Hostility Inventory (BDHI), the Barratt Impulsiveness Scale (BIS) and the Brown-Goodwin Assessment for Life History of Aggression. Fasting basal cholesterol (total, LDL and HDL) was determined 2 weeks after cocaine discontinuation. On the same day 5-HT function was assessed by neuroendocrine (cortisol and prolactin) and psychological (NIMH and 'high' self-rating scales) responses following meta-chlorophenylpiperazine (m-CPP) challenges. Reduced neuroendocrine responses, 'high' feelings and increased 'activation-euphoria' following m-CPP have been interpreted as indicating 5-HT alterations in a variety of psychiatric conditions. Significantly lower levels of HDL cholesterol were found in patients who had a history of aggression (P=0.005). Lower levels of HDL cholesterol were also found to be significantly associated with more intense 'high' and 'activation-euphoria' responses as well as with blunted cortisol responses to m-CPP (P=0.033, P=0.025 and P=0.018, respectively). This study gives further support to existing evidence indicating that in some individuals, the probability of exhibiting impulsive and violent behaviors may be increased when cholesterol is low. It also suggests that low cholesterol and alterations in 5-HT activity may be causally related.

  14. The efficacy of Raf kinase recruitment to the GTPase H-ras depends on H-ras membrane conformer-specific nanoclustering.

    Science.gov (United States)

    Guzmán, Camilo; Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Andrade, Débora M; Reymond, Luc; Eggeling, Christian; Abankwa, Daniel

    2014-04-01

    Solution structures and biochemical data have provided a wealth of mechanistic insight into Ras GTPases. However, information on how much the membrane organization of these lipid-modified proteins impacts on their signaling is still scarce. Ras proteins are organized into membrane nanoclusters, which are necessary for Ras-MAPK signaling. Using quantitative conventional and super-resolution fluorescence methods, as well as mathematical modeling, we investigated nanoclustering of H-ras helix α4 and hypervariable region mutants that have different bona fide conformations on the membrane. By following the emergence of conformer-specific nanoclusters in the plasma membrane of mammalian cells, we found that conformers impart distinct nanoclustering responses depending on the cytoplasmic levels of the nanocluster scaffold galectin-1. Computational modeling revealed that complexes containing H-ras conformers and galectin-1 affect both the number and lifetime of nanoclusters and thus determine the specific Raf effector recruitment. Our results show that mutations in Ras can affect its nanoclustering response and thus allosterically effector recruitment and downstream signaling. We postulate that cancer- and developmental disease-linked mutations that are associated with the Ras membrane conformation may exhibit so far unrecognized Ras nanoclustering and therefore signaling alterations.

  15. Germline expression of H-Ras(G12V) causes neurological deficits associated to Costello syndrome.

    Science.gov (United States)

    Viosca, J; Schuhmacher, A J; Guerra, C; Barco, A

    2009-02-01

    Costello syndrome (CS) is a rare congenital disorder caused by germline activation of H-Ras oncogenes. A mouse model of CS generated by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in embryonic stem (ES) cells has been recently described. These mice phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. We investigated here their neurological and behavioral phenotype. The analysis of H-Ras(G12V) mice revealed phenotypes that resembled the hyperemotivity, hypersensibility and cognitive impairments observed in children with CS. Stronger neurological deficits were found in the analysis of mice homozygous for this mutation than in the analysis of heterozygous mice, suggesting the existence of a gene dose effect. These mice represent the first mouse model for CS, offering an experimental tool to study the molecular and physiological alterations underlying the neurological manifestations of CS and to test new therapies aimed at preventing or ameliorating the cognitive and emotional impairments associated to this condition. PMID:18823404

  16. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.

    Science.gov (United States)

    Yagoda, Nicholas; von Rechenberg, Moritz; Zaganjor, Elma; Bauer, Andras J; Yang, Wan Seok; Fridman, Daniel J; Wolpaw, Adam J; Smukste, Inese; Peltier, John M; Boniface, J Jay; Smith, Richard; Lessnick, Stephen L; Sahasrabudhe, Sudhir; Stockwell, Brent R

    2007-06-14

    Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.

  17. Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

    Science.gov (United States)

    Coyle, Scott M; Lim, Wendell A

    2016-01-01

    The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease. PMID:26765565

  18. KLF4 regulates adult lung tumor-initiating cells and represses K-Ras-mediated lung cancer.

    Science.gov (United States)

    Yu, T; Chen, X; Zhang, W; Liu, J; Avdiushko, R; Napier, D L; Liu, A X; Neltner, J M; Wang, C; Cohen, D; Liu, C

    2016-02-01

    Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. To identify novel factors that contribute to lung cancer pathogenesis, we analyzed a lung cancer database from The Cancer Genome Atlas and found that Krüppel-like Factor 4 (KLF4) expression is significantly lower in patients' lung cancer tissue than in normal lung tissue. In addition, we identified seven missense mutations in the KLF4 gene. KLF4 is a transcription factor that regulates cell proliferation and differentiation as well as the self-renewal of stem cells. To understand the role of KLF4 in the lung, we generated a tamoxifen-induced Klf4 knockout mouse model. We found that KLF4 inhibits lung cancer cell growth and that depletion of Klf4 altered the differentiation pattern in the developing lung. To understand how KLF4 functions during lung tumorigenesis, we generated the K-ras(LSL-G12D/+);Klf4(fl/fl) mouse model, and we used adenovirus-expressed Cre to induce K-ras activation and Klf4 depletion in the lung. Although Klf4 deletion alone or K-ras mutation alone can trigger lung tumor formation, Klf4 deletion combined with K-ras mutation significantly enhanced lung tumor formation. We also found that Klf4 deletion in conjunction with K-ras activation caused lung inflammation. To understand the mechanism whereby KLF4 is regulated during lung tumorigenesis, we analyzed KLF4 promoter methylation and the profiles of epigenetic factors. We found that Class I histone deacetylases (HDACs) are overexpressed in lung cancer and that HDAC inhibitors induced expression of KLF4 and inhibited proliferation of lung cancer cells, suggesting that KLF4 is probably repressed by histone acetylation and that HDACs are valuable drug targets for lung cancer treatment.

  19. A novel role for flotillin-1 in H-Ras-regulated breast cancer aggressiveness.

    Science.gov (United States)

    Koh, Minsoo; Yong, Hae-Young; Kim, Eun-Sook; Son, Hwajin; Jeon, You Rim; Hwang, Jin-Sun; Kim, Myeong-Ok; Cha, Yujin; Choi, Wahn Soo; Noh, Dong-Young; Lee, Kyung-Min; Kim, Ki-Bum; Lee, Jae-Seon; Kim, Hyung Joon; Kim, Haemin; Kim, Hong-Hee; Kim, Eun Joo; Park, So Yeon; Kim, Hoe Suk; Moon, Woo Kyung; Choi Kim, Hyeong-Reh; Moon, Aree

    2016-03-01

    Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p < 0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer.

  20. Effect of Boschniakia rossica on expression of GST-P, p53 and p21(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats.

    Science.gov (United States)

    Yin, Zong-Zhu; Jin, Hai-Ling; Yin, Xue-Zhe; Li, Tian-Zhu; Quan, Ji-Shu; Jin, Zeng-Nan

    2000-12-01

    AIM:To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21(ras) proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS:The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21(ras) proteins were investigated by immunohisto-chemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet induced mouse granuloma formation methods.RESULTS:The 500mg/kg of BR-H2O extract frac-tionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, P<0.05) and the expression of mutant p53 and p21(ras) protein was lower than that of hepatic preneoplastic lesions (33% and 22% positive respectively in DEN+AAF group vs negative in DEN+AAF+BR group). Both CH(2)Cl(2) and H(2)O extracts from BR had anti-inflamatory effect in xylene and crotonoil induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR H(2)O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.CONCLUSION:BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis.Both CH(2)Cl(2) and H(2)O extracts from BR exerted anti-inflammatory effect in rats and mice. PMID:11819701

  1. Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

    Directory of Open Access Journals (Sweden)

    Putty-Reddy Sudhir

    Full Text Available BACKGROUND: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale. METHODOLOGY/PRINCIPAL FINDINGS: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77 using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60% of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. CONCLUSIONS/SIGNIFICANCE: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

  2. Alteration of biophysical activity of pulmonary surfactant by aluminosilicate nanoparticles.

    Science.gov (United States)

    Kondej, Dorota; Sosnowski, Tomasz R

    2013-02-01

    The influence of five different types of aluminosilicate nanoparticles (NPs) on the dynamic surface activity of model pulmonary surfactant (PS) (Survanta) was studied experimentally using oscillating bubble tensiometry. Bentonite, halloysite and montmorillonite (MM) NPs, which are used as fillers of polymer composites, were characterized regarding the size distribution, morphology and surface area. Particle doses applied in the studies were estimated based on the inhalation rate and duration, taking into account the expected aerosol concentration and deposition efficiency after penetration of NPs into the alveolar region. The results indicate that aluminosilicate NPs at concentrations in the pulmonary liquid above 0.1 mg cm(-3) are capable of promoting alterations of the original dynamic biophysical activity of the PS. This effect is indicated by deviation of the minimum surface tension, stability index and the size of surface tension hysteresis. Such response is dependent on the type of NPs present in the system and is stronger when particle concentration increases. It is suggested that interactions between NPs and the PS must be related to the surfactant adsorption on the suspended particles, while in the case of surface-modified clay NPs the additional washout of surface-active components may be expected. It is speculated that observed changes in surface properties of the surfactant may be associated with undesired health effects following extensive inhalation of aluminosilicate NPs in the workplace. PMID:23363039

  3. Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

    Science.gov (United States)

    Dent, P; Reardon, D B; Morrison, D K; Sturgill, T W

    1995-08-01

    The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a

  4. Rational design of small molecule inhibitors targeting the Ras GEF, SOS1

    Science.gov (United States)

    Evelyn, Chris R.; Duan, Xin; Biesiada, Jacek; Seibel, William L.; Meller, Jaroslaw; Zheng, Yi

    2014-01-01

    Summary Ras GTPases regulate intracellular signaling involved in cell proliferation. Elevated Ras signaling activity has been associated with human cancers. Ras activation is catalyzed by guanine-nucleotide exchange factors (GEFs), of which SOS1 is a major member that transduces receptor tyrosine kinase signaling to Ras. We have developed a rational approach coupling virtual screening with experimental screening in identifying small-molecule inhibitors targeting the catalytic site of SOS1 and SOS1-regulated Ras activity. A lead inhibitor, NSC-658497, is found to bind to SOS1, competitively suppresses SOS1-Ras interaction, and dose-dependently inhibits SOS1 GEF activity. Mutagenesis and structure-activity relationship studies map the NSC-658497 site of action to the SOS1 catalytic site, and define the chemical moieties in the inhibitor essential for the activity. NSC-658497 showed dose-dependent efficacy in inhibiting Ras, downstream signaling activities, and associated cell proliferation. These studies establish a proof of principle for rational design of small-molecule inhibitors targeting Ras GEF enzymatic activity. PMID:25455859

  5. Rational design of small molecule inhibitors targeting the Ras GEF, SOS1.

    Science.gov (United States)

    Evelyn, Chris R; Duan, Xin; Biesiada, Jacek; Seibel, William L; Meller, Jaroslaw; Zheng, Yi

    2014-12-18

    Ras GTPases regulate intracellular signaling involved in cell proliferation. Elevated Ras signaling activity has been associated with human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major member that transduces receptor tyrosine kinase signaling to Ras. We have developed a rational approach coupling virtual screening with experimental screening in identifying small-molecule inhibitors targeting the catalytic site of SOS1 and SOS1-regulated Ras activity. A lead inhibitor, NSC-658497, was found to bind to SOS1, competitively suppress SOS1-Ras interaction, and dose-dependently inhibit SOS1 GEF activity. Mutagenesis and structure-activity relationship studies map the NSC-658497 site of action to the SOS1 catalytic site, and define the chemical moieties in the inhibitor essential for the activity. NSC-658497 showed dose-dependent efficacy in inhibiting Ras, downstream signaling activities, and associated cell proliferation. These studies establish a proof of principle for rational design of small-molecule inhibitors targeting Ras GEF enzymatic activity.

  6. Pseudorabies virus infection alters neuronal activity and connectivity in vitro.

    Directory of Open Access Journals (Sweden)

    Kelly M McCarthy

    2009-10-01

    Full Text Available Alpha-herpesviruses, including human herpes simplex virus 1 & 2, varicella zoster virus and the swine pseudorabies virus (PRV, infect the peripheral nervous system of their hosts. Symptoms of infection often include itching, numbness, or pain indicative of altered neurological function. To determine if there is an in vitro electrophysiological correlate to these characteristic in vivo symptoms, we infected cultured rat sympathetic neurons with well-characterized strains of PRV known to produce virulent or attenuated symptoms in animals. Whole-cell patch clamp recordings were made at various times after infection. By 8 hours of infection with virulent PRV, action potential (AP firing rates increased substantially and were accompanied by hyperpolarized resting membrane potentials and spikelet-like events. Coincident with the increase in AP firing rate, adjacent neurons exhibited coupled firing events, first with AP-spikelets and later with near identical resting membrane potentials and AP firing. Small fusion pores between adjacent cell bodies formed early after infection as demonstrated by transfer of the low molecular weight dye, Lucifer Yellow. Later, larger pores formed as demonstrated by transfer of high molecular weight Texas red-dextran conjugates between infected cells. Further evidence for viral-induced fusion pores was obtained by infecting neurons with a viral mutant defective for glycoprotein B, a component of the viral membrane fusion complex. These infected neurons were essentially identical to mock infected neurons: no increased AP firing, no spikelet-like events, and no electrical or dye transfer. Infection with PRV Bartha, an attenuated circuit-tracing strain delayed, but did not eliminate the increased neuronal activity and coupling events. We suggest that formation of fusion pores between infected neurons results in electrical coupling and elevated firing rates, and that these processes may contribute to the altered neural

  7. Physical activity alters antioxidant status in exercising elderly subjects.

    Science.gov (United States)

    Rousseau, Anne-Sophie; Margaritis, Irène; Arnaud, Josiane; Faure, Henri; Roussel, Anne-Marie

    2006-07-01

    Nutritional adequacy and physical activity are two aspects of a health-promoting lifestyle. Not much is known about antioxidant nutrient requirements for exercising elderly (EE) subjects. The question of whether exercise training alters the status of antioxidant vitamins as well as trace elements in elderly subjects and fails to balance the age-related increase in oxidative stress is addressed in this study. There were 18 EE (68.1+/-3.1 years), 7 sedentary elderly (SE; 70.4+/-5.0 years), 17 exercising young (EY; 31.2+/-7.1 years) and 8 sedentary young (SY; 27.1+/-5.8 years) subjects who completed 7-day food and activity records. Each subject's blood was sampled on Day 8. A similar selenium (Se) status but a higher erythrocyte glutathione peroxidase (GSH-Px) activity were found in EE subjects as compared with EY and SE subjects. Blood oxidized glutathione was higher and plasma total thiol was lower in EE subjects as compared with EY subjects. Mean vitamin C (167 vs. 106 mg/day), vitamin E (11.7 vs. 8.3 mg/day) and beta-carotene (4 vs. 2.4 mg/day) intakes were higher in EE subjects as compared with EY subjects. However, EE subjects exhibited the lowest plasma carotenoid concentrations, especially in beta-carotene, which was not related to intakes. Despite high intakes of antioxidant micronutrients, no adaptive mechanism able to counteract the increased oxidative stress in aging was found in EE subjects. Results on GSH-Px activity illustrate that the nature of the regulation of this biomarker of Se status is different in response to training and aging. These data also strongly suggest specific antioxidant requirements for athletes with advancing age, with a special attention to carotenoids.

  8. Radiosensitivity of small-cell lung cancer xenografts compared with activity of c-myc, N-myc, L-myc, c-raf-1 and K-ras proto-oncogenes

    DEFF Research Database (Denmark)

    Rygaard, K; Slebos, R J; Spang-Thomsen, M

    1991-01-01

    Oncogenes of the myc family c-raf-1 and K-ras have been reported to modulate radiosensitivity. We examined the possible relationship between in vivo radiosensitivity to single-dose irradiation with 3-10 Gy, and activity of these proto-oncogenes in 2 sets of small-cell lung cancer (SCLC) xenografts...... expressed identical amounts of c-raf-1 and high levels of c-myc mRNA, but neither expressed N-myc or L-myc. None of the tumours was mutated at codon 12 or K-ras. Our results show that SCLC xenografts with different radiosensitivity may express identical amounts of some of the proto-oncogenes reported...... to modulate radiosensitivity. Thus, factors other than activation of the examined proto-oncogenes must be involved in causing the differences in radiosensitivity found in the SCLC xenografts. Possible long-term effects of irradiation on proto-oncogene expression was examined in xenografts of GLC-16, following...

  9. A primary cardiac leiomyosarcoma with mutation at H-ras codon 12.

    Science.gov (United States)

    Parissis, J; Arvanitis, D; Sourvinos, G; Spandidos, D

    1997-01-01

    The presence of activating ras mutations in a cardiac leiomyosarcoma which occurred in the right atrium of the heart of a female patient was examined. The tumor had the appearance of leiomyosarcoma in rutine histopathological examination and the definite diagnosis was confirmed by a positive immunohistochemical reaction to smooth muscle actin. Molecular analysis by polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) technique showed a point mutation of H-ras gene at codon 12. To the best of our knowledge, this is the first report describing ras gene mutation in a cardiac leiomyosarcoma implying a role for the ras oncogenes in the development of this tumor.

  10. Evidence implicating the Ras pathway in multiple CD28 costimulatory functions in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Sujit V Janardhan

    Full Text Available CD28 costimulation is a critical event in the full activation of CD4(+ T cells that augments cytokine gene transcription, promotes cytokine mRNA stability, prevents induction of anergy, increases cellular metabolism, and increases cell survival. However, despite extensive biochemical analysis of the signaling events downstream of CD28, molecular pathways sufficient to functionally replace the diverse aspects of CD28-mediated costimulation in normal T cells have not been identified. Ras/MAPK signaling is a critical pathway downstream of T cell receptor stimulation, but its role in CD28-mediated costimulation has been controversial. We observed that physiologic CD28 costimulation caused a relocalization of the RasGEF RasGRP to the T cell-APC interface by confocal microscopy. In whole cell biochemical analysis, CD28 cross-linking with either anti-CD28 antibody or B7.1-Ig augmented TCR-induced Ras activation. To determine whether Ras signaling was sufficient to functionally mimic CD28 costimulation, we utilized an adenoviral vector encoding constitutively active H-Ras (61L to transduce normal, Coxsackie-Adenovirus Receptor (CAR transgenic CD4(+ T cells. Like costimulation via CD28, active Ras induced AKT, JNK and ERK phosphorylation. In addition, constitutive Ras signaling mimicked the ability of CD28 to costimulate IL-2 protein secretion, prevent anergy induction, increase glucose uptake, and promote cell survival. Importantly, we also found that active Ras mimicked the mechanism by which CD28 costimulates IL-2 production: by increasing IL-2 gene transcription, and promoting IL-2 mRNA stability. Finally, active Ras was able to induce IL-2 production when combined with ionomycin stimulation in a MEK-1-dependent fashion. Our results are consistent with a central role for Ras signaling in CD28-mediated costimulation.

  11. Regulation of prolactin in mice with altered hypothalamic melanocortin activity.

    Science.gov (United States)

    Dutia, Roxanne; Kim, Andrea J; Mosharov, Eugene; Savontaus, Eriika; Chua, Streamson C; Wardlaw, Sharon L

    2012-09-01

    This study used two mouse models with genetic manipulation of the melanocortin system to investigate prolactin regulation. Mice with overexpression of the melanocortin receptor (MC-R) agonist, α-melanocyte-stimulating hormone (Tg-MSH) or deletion of the MC-R antagonist agouti-related protein (AgRP KO) were studied. Male Tg-MSH mice had lower blood prolactin levels at baseline (2.9±0.3 vs. 4.7±0.7ng/ml) and after restraint stress (68±6.5 vs. 117±22ng/ml) vs. WT (pprolactin content was not different. Blood prolactin was also decreased in male AgRP KO mice at baseline (4.2±0.5 vs. 7.6±1.3ng/ml) and after stress (60±4.5 vs. 86.1±5.7ng/ml) vs. WT (pprolactin content was lower in male AgRP KO mice (4.3±0.3 vs. 6.7±0.5μg/pituitary, pprolactin levels were observed in female AgRP KO mice vs. WT. Hypothalamic dopamine activity was assessed as the potential mechanism responsible for changes in prolactin levels. Hypothalamic tyrosine hydroxylase mRNA was measured in both genetic models vs. WT mice and hypothalamic dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) content were measured in male AgRP KO and WT mice but neither were significantly different. However, these results do not preclude changes in dopamine activity as dopamine turnover was not directly investigated. This is the first study to show that baseline and stress-induced prolactin release and pituitary prolactin content are reduced in mice with genetic alterations of the melanocortin system and suggests that changes in hypothalamic melanocortin activity may be reflected in measurements of serum prolactin levels.

  12. Specificity in Ras and Rap signaling

    NARCIS (Netherlands)

    Raaijmakers, J.H.; Bos, Johannes L.

    2009-01-01

    Ras and Rap proteins are closely related small GTPases. Whereas Ras is known for its role in cell proliferation and survival, Rap1 is predominantly involved in cell adhesion and cell junction formation. Ras and Rap are regulated by different sets of guanine nucleotide exchange factors and GTPase-act

  13. Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study

    Directory of Open Access Journals (Sweden)

    de Bruïne Adriaan P

    2005-12-01

    Full Text Available Abstract Background The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1 and Ras (K-ras pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. Methods In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Results Mutations at the phosphorylation sites (codons 31, 33, 37, and 45 in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656 and 36% (235/656, respectively. Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656. Nine percent of all tumours (58/656 lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. Conclusion CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

  14. Disrupting the oncogenic synergism between nucleolin and Ras results in cell growth inhibition and cell death.

    Directory of Open Access Journals (Sweden)

    Sari Schokoroy

    Full Text Available BACKGROUND: The ErbB receptors, Ras proteins and nucleolin are major contributors to malignant transformation. The pleiotropic protein nucleolin can bind to both Ras protein and ErbB receptors. Previously, we have demonstrated a crosstalk between Ras, nucleolin and the ErbB1 receptor. Activated Ras facilitates nucleolin interaction with ErbB1 and stabilizes ErbB1 levels. The three oncogenes synergistically facilitate anchorage independent growth and tumor growth in nude mice. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we used several cancer cell lines. The effect of Ras and nucleolin inhibition was determined using cell growth, cell death and cell motility assays. Protein expression was determined by immunohistochemistry. We found that inhibition of Ras and nucleolin reduces tumor cell growth, enhances cell death and inhibits anchorage independent growth. Our results reveal that the combined treatment affects Ras and nucleolin levels and localization. Our study also indicates that Salirasib (FTS, Ras inhibitor reduces cell motility, which is not affected by the nucleolin inhibitor. CONCLUSIONS/SIGNIFICANCE: These results suggest that targeting both nucleolin and Ras may represent an additional avenue for inhibiting cancers driven by these oncogenes.

  15. Inhibition of RAF Isoforms and Active Dimers by LY3009120 Leads to Anti-tumor Activities in RAS or BRAF Mutant Cancers.

    Science.gov (United States)

    Peng, Sheng-Bin; Henry, James R; Kaufman, Michael D; Lu, Wei-Ping; Smith, Bryan D; Vogeti, Subha; Rutkoski, Thomas J; Wise, Scott; Chun, Lawrence; Zhang, Youyan; Van Horn, Robert D; Yin, Tinggui; Zhang, Xiaoyi; Yadav, Vipin; Chen, Shih-Hsun; Gong, Xueqian; Ma, Xiwen; Webster, Yue; Buchanan, Sean; Mochalkin, Igor; Huber, Lysiane; Kays, Lisa; Donoho, Gregory P; Walgren, Jennie; McCann, Denis; Patel, Phenil; Conti, Ilaria; Plowman, Gregory D; Starling, James J; Flynn, Daniel L

    2015-09-14

    LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

  16. Liver tumors induced in B6C3F{sub 1} mice by benz[a]anthracene and two of its halogenated derivatives contain K-RAS oncogene mutations

    Energy Technology Data Exchange (ETDEWEB)

    Xia, O.; Yi, P.; Zhan, D. [and others

    1997-10-01

    Polycyclic aromatic hydrocarbons (PAHs) and halogenated PAHs are genotoxic environmental contaminants. We previously examined the tumorigenicity of benz[a]anthracene (BA), 7-Cl-BA, and 7-Br-BA in the neonatal mouse tumorigenicity bioassay. Male B6C3F{sub 1} mice were administered i.p. injections at a total dose of 400 nmol per mouse on 1, 8, and 15 days after birth. BA, 7-Cl-BA, and 7-Br-BA induced hepatocellular adenoma in 67, 92, and 96% of the mice, respectively, and induced hepatocellular carcinoma in 15, 100 and 83% of the mice, respectively. In the present study, mRNA was isolated from each of the liver tumors induced by the three compounds, reversed-transcribed to cDNA, and portions of the K- and H-ras oncogene coding sequences were amplified and analyzed for DNA sequence alterations. 92% (11/12) of BA-induced, 79% (19/24) of 7-Cl-BA-induced and 86% (19/22) of 7-Br-BA-induced liver tumors had activated ras protooncogenes. In contrast to the general finding of H-ras mutations in B6C3F{sub 1} mouse liver tumors, all the mutations were at the first base of K-ras codon 13, resulting in a pattern of GGC{yields}CGC. No other ras oncogene mutations were detected. Our results clearly demonstrate that these chemicals induce a unique type of ras (K-ras) oncogene activation in the liver tumors of B6C3F{sub 1} mice.

  17. PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus

    Institute of Scientific and Technical Information of China (English)

    Ting Jin; Weizhong Liu; Yixuan Zhang; Yi Pan; Zhenzhen Wang; Walter G Thomas; Yan Chen; Qiurong Ding; Heng Huang; Daqian Xu; Yuhui Jiang; Ben Zhou; Zhenghu Li; Xiaomeng Jiang; Jing He

    2012-01-01

    Ras plays a pivotal role in many cellular activities,and its subcellular compartmentalization provides spatial and temporal selectivity.Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family.PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus.Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner.Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas,NRas and KRas4A,but not KRas4B.PAQR10 and PAQR11 can also interact with HRas,NRas and KRas4A,but not KRas4B.The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state.Consistently,knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus.Intriguingly,PAQR10 and PAQR11 are able to interact with RasGRP1,a guanine nucleotide exchange protein of Ras,and increase Golgi localization of RasGRP1.The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11.The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1.Furthermore,differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11.Collectively,this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.

  18. MUTASI K RAS PADA KARSINOGENESIS KANKER KOLOREKTAL

    Directory of Open Access Journals (Sweden)

    Ni Putu Sriwidyani

    2014-09-01

    Full Text Available Karsinogenesis kanker kolorektal merupakan proses multi-step, melibatkan berbagai abnormalitasgenetik. Mutasi gen K RAS sering ditemukan pada tumor ini. K RAS adalah gen yang menyandi proteinK ras, suatu produk proto-onkogen yang merupakan komponen penting pada jalur pensignalan darireseptor permukaan sel untuk mengontrol proliferasi, diferensiasi, dan kematian sel. Kebanyakanmutasi terjadi pada kodon 12 dan 13 dari ekson 1. Protein K ras mutan akan menyebabkan aktivasipersisten dari banyak signal downstream dari pertumbuhan dan survival sel. Pemeriksaan adanyamutasi pada gen K RAS memegang peranan penting pada prognosis dan terapi dari kanker kolorektal.[MEDICINA 2013;44:97-100].

  19. B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding.

    Science.gov (United States)

    Fischer, Andreas; Hekman, Mirko; Kuhlmann, Jürgen; Rubio, Ignacio; Wiese, Stefan; Rapp, Ulf R

    2007-09-01

    Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

  20. Coexistence of K-ras mutations and HPV infection in colon cancer

    Directory of Open Access Journals (Sweden)

    Tezol Ayda

    2006-05-01

    Full Text Available Abstract Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

  1. Novel Ras pathway inhibitor induces apoptosis and growth inhibition of K-ras-mutated cancer cells in vitro and in vivo.

    Science.gov (United States)

    Jasinski, Piotr; Zwolak, Pawel; Terai, Kaoru; Dudek, Arkadiusz Z

    2008-11-01

    MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind. PMID:19010291

  2. The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase.

    Science.gov (United States)

    Jaumot, Montserrat; Yan, Jun; Clyde-Smith, Jodi; Sluimer, Judith; Hancock, John F

    2002-01-01

    Ha-Ras and Ki-Ras have different distributions across plasma membrane microdomains. The Ras C-terminal anchors are primarily responsible for membrane micro-localization, but recent work has shown that the interaction of Ha-Ras with lipid rafts is modulated by GTP loading via a mechanism that requires the hypervariable region (HVR). We have now identified two regions in the HVR linker domain that regulate Ha-Ras raft association. Release of activated Ha-Ras from lipid rafts is blocked by deleting amino acids 173-179 or 166-172. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific N-terminal sequences of the linker domain operate in concert with a more C-terminal spacer domain to regulate Ha-Ras raft association. Mutations in the linker domain that confine activated Ha-RasG12V to lipid rafts abrogate Raf-1, phosphoinositide 3-kinase, and Akt activation and inhibit PC12 cell differentiation. N-Myristoylation also prevents the release of activated Ha-Ras from lipid rafts and inhibits Raf-1 activation. These results demonstrate that the correct modulation of Ha-Ras lateral segregation is critical for downstream signaling. Mutations in the linker domain also suppress the dominant negative phenotype of Ha-RasS17N, indicating that HVR sequences are essential for efficient interaction of Ha-Ras with exchange factors in intact cells.

  3. The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

    International Nuclear Information System (INIS)

    Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras tmΔ4A/tmΔ4A (exon 4A knock-out) ES cells which express K-ras4B only and K-ras -/- (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras tmΔ4A/tmΔ4A mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras tmΔ4A/tmΔ4A ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras tmΔ4A/tmΔ4A and K-ras -/- ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras -/- cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras tmΔ4A/tmΔ4A ES cells were more resistant to etoposide-induced apoptosis than K-ras -/- cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice

  4. Fragment N2, a caspase-3-generated RasGAP fragment, inhibits breast cancer metastatic progression.

    Science.gov (United States)

    Barras, David; Lorusso, Girieca; Lhermitte, Benoît; Viertl, David; Rüegg, Curzio; Widmann, Christian

    2014-07-01

    The p120 RasGAP protein negatively regulates Ras via its GAP domain. RasGAP carries several other domains that modulate several signaling molecules such as Rho. RasGAP is also a caspase-3 substrate. One of the caspase-3-generated RasGAP fragments, corresponding to amino acids 158-455 and called fragment N2, was previously reported to specifically sensitize cancer cells to death induced by various anticancer agents. Here, we show that fragment N2 inhibits migration in vitro and that it impairs metastatic progression of breast cancer to the lung. Hence, stress-activated caspase-3 might contribute to the suppression of metastasis through the generation of fragment N2. These results indicate that the activity borne by fragment N2 has a potential therapeutic relevance to counteract the metastatic process.

  5. Retraction: "Activated K-Ras and INK4a/Arf Deficiency Promote Aggressiveness of Pancreatic Cancer by Induction of EMT Consistent With Cancer Stem Cell Phenotype" by Wang et al.

    Science.gov (United States)

    2016-10-01

    The above article, published online on November 23, 2012 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 4B and C to be inappropriately manipulated and re-labeled. Literature Cited Wang Z, Ali S, Banerjee S, Bao B, Li Y, Azmi AS, Korc M, Sarkar FH. 2013. Activated K-Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype. J Cell Physiol 228:556-562; doi: 10.1002/jcp.24162. PMID:27315162

  6. Retraction: "Activated K-Ras and INK4a/Arf Deficiency Promote Aggressiveness of Pancreatic Cancer by Induction of EMT Consistent With Cancer Stem Cell Phenotype" by Wang et al.

    Science.gov (United States)

    2016-10-01

    The above article, published online on November 23, 2012 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 4B and C to be inappropriately manipulated and re-labeled. Literature Cited Wang Z, Ali S, Banerjee S, Bao B, Li Y, Azmi AS, Korc M, Sarkar FH. 2013. Activated K-Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype. J Cell Physiol 228:556-562; doi: 10.1002/jcp.24162.

  7. Altered expression of the IQGAP1 gene in human lung cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, C.E.; Palmisano, W.A.; Lechner, J.F. [and others

    1995-12-01

    IQGAP1 is a GTPase activation protein that accelerates GTP hydrolysis by normal p21 ras proteins. Therefore, IQGAP1 could act as an upstream affector of p21 ras activity by convert in excess amounts of active GTP-21 ras to inactive GDP-21 ras. IQGAP1 displays extensive sequence similarity to the catalytic domain of all previously reported ras GAPs, including the tumor suppressor gene protein neurofibromatosis type 1 (NF1). It has been shown that abnormal NF1 protein cannot negatively regulate the activity of ras proteins in neuroblast cells. This observation supports the hypothesis that NF1 is a tumor suppressor gene whose product acts upstream of ras. IQGAP1 is primarily expressed in lung, where it may play a role similar to NF1 in regulating the activity of H-ras or K-ras proteins. IQGAP1 functions as other GAPs by controlling the activity of ras.

  8. Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

    Science.gov (United States)

    Muñoz-Félix, José M; Fuentes-Calvo, Isabel; Cuesta, Cristina; Eleno, Nélida; Crespo, Piero; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-10-01

    The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-β1 (TGF-β1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-β1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-β1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc. PMID:26873620

  9. Alteration of swelling clay minerals by acid activation

    NARCIS (Netherlands)

    Steudel, A.; Batenburg, L.F.; Fischer, H.R.; Weidler, P.G.; Emmerich, K.

    2009-01-01

    The bulk material of six dioctahedral and two trioctahedral swellable clay minerals was leached in H2SO4 and HCl at concentrations of 1.0, 5.0 and 10.0 M at 80 °C for several hours. Alteration of the clay mineral structures was dependent on the individual character of each mineral (chemical composit

  10. Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

    Energy Technology Data Exchange (ETDEWEB)

    Buhrman, Greg; O; #8242; Connor, Casey; Zerbe, Brandon; Kearney, Bradley M.; Napoleon, Raeanne; Kovrigina, Elizaveta A.; Vajda, Sandor; Kozakov, Dima; Kovrigin, Evgenii L.; Mattos, Carla (NCSU); (MCW); (BU)

    2012-09-17

    We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.

  11. Effect of Boschniakia rossica on expression of GST-P, p53 and p21(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats.

    Science.gov (United States)

    Yin, Zong-Zhu; Jin, Hai-Ling; Yin, Xue-Zhe; Li, Tian-Zhu; Quan, Ji-Shu; Jin, Zeng-Nan

    2000-12-01

    AIM:To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21(ras) proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS:The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21(ras) proteins were investigated by immunohisto-chemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet induced mouse granuloma formation methods.RESULTS:The 500mg/kg of BR-H2O extract frac-tionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, Panti-inflamatory effect in xylene and crotonoil induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR H(2)O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.CONCLUSION:BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis.Both CH(2)Cl(2) and H(2)O extracts from BR exerted anti-inflammatory effect in rats and mice.

  12. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Copp& #233; , Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua; Nelson, Peter; Desprez, Pierre-Yves; Campisi, Judith

    2008-10-24

    Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  13. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Coppé

    2008-12-01

    Full Text Available Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial-mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.

  14. Dexras1 a unique ras-GTPase interacts with NMDA receptor activity and provides a novel dissociation between anxiety, working memory and sensory gating.

    Science.gov (United States)

    Carlson, G C; Lin, R E; Chen, Y; Brookshire, B R; White, R S; Lucki, I; Siegel, S J; Kim, S F

    2016-05-13

    Dexras1 is a novel GTPase that acts at a confluence of signaling mechanisms associated with psychiatric and neurological disease including NMDA receptors, NOS1AP and nNOS. Recent work has shown that Dexras1 mediates iron trafficking and NMDA-dependent neurodegeneration but a role for Dexras1 in normal brain function or psychiatric disease has not been studied. To test for such a role, mice with germline knockout (KO) of Dexras1 were assayed for behavioral abnormalities as well as changes in NMDA receptor subunit protein expression. Because Dexras1 is up-regulated during stress or by dexamethasone treatment, we included measures associated with emotion including anxiety and depression. Baseline anxiety-like measures (open field and zero maze) were not altered, nor were depression-like behavior (tail suspension). Measures of memory function yielded mixed results, with no changes in episodic memory (novel object recognition) but a significant decrement on working memory (T-maze). Alternatively, there was an increase in pre-pulse inhibition (PPI), without concomitant changes in either startle amplitude or locomotor activity. PPI data are consistent with the direction of change seen following exposure to dopamine D2 antagonists. An examination of NMDA subunit expression levels revealed an increased expression of the NR2A subunit, contrary to previous studies demonstrating down-regulation of the receptor following antipsychotic exposure (Schmitt et al., 2003) and up-regulation after exposure to isolation rearing (Turnock-Jones et al., 2009). These findings suggest a potential role for Dexras1 in modulating a selective subset of psychiatric symptoms, possibly via its interaction with NMDARs and/or other disease-related binding-partners. Furthermore, data suggest that modulating Dexras1 activity has contrasting effects on emotional, sensory and cognitive domains. PMID:26946266

  15. Mutational analysis of PI3K/AKT and RAS/RAF pathway activation in malignant salivary gland tumours with a new mutation of PIK3CA.

    Science.gov (United States)

    Shalmon, B; Drendel, M; Wolf, M; Hirshberg, A; Cohen, Y

    2016-06-01

    The phosphoinositide 3-kinase (PIK3)/v-akt murine thymoma (AKT) oncogene pathway and the RAS/RAF pathway are involved in regulating the signalling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth. Mutations in the genes within these pathways are frequently found in several tumours. The aim of this study was to investigate the frequency of mutations in the PIK3CA, BRAF, and KRAS genes in cases of malignant salivary gland tumours. Mutational analysis of the PIK3CA, KRAS, and BRAF genes was performed by direct sequencing of material from 21 patients with malignant salivary gland tumours who underwent surgery between 1992 and 2001. No mutations were found in the KRAS exon 2, BRAF exon 15, or PIK3CA exon 9 genes. However, an unpublished mutation of the PIK3CA gene in exon 20 (W1051 stop mutation) was found in one case of adenocarcinoma NOS. The impact of this mutation on the biological behaviour of the tumour has yet to be explored, however the patient with adenocarcinoma NOS harbouring this mutation has survived for over 20 years following surgery despite a high stage at presentation. Further studies with more homogeneous patient cohorts are needed to address whether this mutation reflects a different clinical presentation and may benefit from targeted treatment strategies. PMID:26811072

  16. Hyperglycemia promotes K-Ras-induced lung tumorigenesis through BASCs amplification.

    Directory of Open Access Journals (Sweden)

    Carla Micucci

    Full Text Available Oncogenic K-Ras represents the most common molecular change in human lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC. The presence of K-Ras mutation is associated with a poor prognosis, but no effective treatment strategies are available for K-Ras -mutant NSCLC. Epidemiological studies report higher lung cancer mortality rates in patients with type 2 diabetes. Here, we use a mouse model of K-Ras-mediated lung cancer on a background of chronic hyperglycemia to determine whether elevated circulating glycemic levels could influence oncogenic K-Ras-mediated tumor development. Inducible oncogenic K-Ras mouse model was treated with subtoxic doses of streptozotocin (STZ to induce chronic hyperglycemia. We observed increased tumor mass and higher grade of malignancy in STZ treated diabetic mice analyzed at 4, 12 and 24 weeks, suggesting that oncogenic K-Ras increased lung tumorigenesis in hyperglycemic condition. This promoting effect is achieved by expansion of tumor-initiating lung bronchio-alveolar stem cells (BASCs in bronchio-alveolar duct junction, indicating a role of hyperglycemia in the activity of K-Ras-transformed putative lung stem cells. Notably, after oncogene K-Ras activation, BASCs show upregulation of the glucose transporter (Glut1/Slc2a1, considered as an important player of the active control of tumor cell metabolism by oncogenic K-Ras. Our novel findings suggest that anti-hyperglycemic drugs, such as metformin, may act as therapeutic agent to restrict lung neoplasia promotion and progression.

  17. High physical activity in young children suggests positive effects by altering autoantigen-induced immune activity.

    Science.gov (United States)

    Carlsson, E; Ludvigsson, J; Huus, K; Faresjö, M

    2016-04-01

    Physical activity in children is associated with several positive health outcomes such as decreased cardiovascular risk factors, improved lung function, enhanced motor skill development, healthier body composition, and also improved defense against inflammatory diseases. We examined how high physical activity vs a sedentary lifestyle in young children influences the immune response with focus on autoimmunity. Peripheral blood mononuclear cells, collected from 55 5-year-old children with either high physical activity (n = 14), average physical activity (n = 27), or low physical activity (n = 14), from the All Babies In Southeast Sweden (ABIS) cohort, were stimulated with antigens (tetanus toxoid and beta-lactoglobulin) and autoantigens (GAD65 , insulin, HSP60, and IA-2). Immune markers (cytokines and chemokines), C-peptide and proinsulin were analyzed. Children with high physical activity showed decreased immune activity toward the autoantigens GAD65 (IL-5, P < 0.05), HSP60 and IA-2 (IL-10, P < 0.05) and also low spontaneous pro-inflammatory immune activity (IL-6, IL-13, IFN-γ, TNF-α, and CCL2 (P < 0.05)) compared with children with an average or low physical activity. High physical activity in young children seems to have positive effects on the immune system by altering autoantigen-induced immune activity. PMID:25892449

  18. ANALYSIS OF C-HA-RAS GENE AMPLIFICATION AND MUTATION IN LARYNGEAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    刘世喜; 林代诚; 洪邦泰; 黄光琦

    1995-01-01

    In order to study the ahered molecular events during laryngeal carcinogenesis and elucidate the role of Ha-ras oncogene amplification and mutation, we have examined their profile by polymerase chain reaction (PCR) and selective oligonucleoride hybridization. We analyzed the mutational status of codon 12 of Ha-ras in 22 laryngeal carcinomas and 10 normal tissues, and found that 7 of 22 laryngeal carcinomas con-tained a Ha-ras mutation at codon 12. The frequency of mutation was 32%. None of the normal tissues re-vealed mutation. Moreover, no amplification was found in cancers when compared to the normal. Our findings indicated that the aefivmed Ha-ras gene existed in laryngeal carcinoma, and activation of the Ha-ras gene by mutation at codon 12 might play a key role in laryngeal carcinogenesis.

  19. A RAS renaissance: emerging targeted therapies for KRAS-mutated non-small cell lung cancer.

    Science.gov (United States)

    Vasan, Neil; Boyer, Julie L; Herbst, Roy S

    2014-08-01

    Of the numerous oncogenes implicated in human cancer, the most common and perhaps the most elusive to target pharmacologically is RAS. Since the discovery of RAS in the 1960s, numerous studies have elucidated the mechanism of activity, regulation, and intracellular trafficking of the RAS gene products, and of its regulatory pathways. These pathways yielded druggable targets, such as farnesyltransferase, during the 1980s to 1990s. Unfortunately, early clinical trials investigating farnesyltransferase inhibitors yielded disappointing results, and subsequent interest by pharmaceutical companies in targeting RAS waned. However, recent advances including the identification of novel regulatory enzymes (e.g., Rce1, Icmt, Pdeδ), siRNA-based synthetic lethality screens, and fragment-based small-molecule screens, have resulted in a "Ras renaissance," signified by new Ras and Ras pathway-targeted therapies that have led to new clinical trials of patients with Ras-driven cancers. This review gives an overview of KRas signaling pathways with an emphasis on novel targets and targeted therapies, using non-small cell lung cancer as a case example.

  20. 1,25-Dihydroxyvitamin D inhibits glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cell.

    Science.gov (United States)

    Zhou, Xuanzhu; Zheng, Wei; Nagana Gowda, G A; Raftery, Daniel; Donkin, Shawn S; Bequette, Brian; Teegarden, Dorothy

    2016-10-01

    Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes and to prevent breast cancer. The current studies investigated the effect of 1,25(OH)2D on glutamine metabolism during cancer progression employing Harvey-ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Treatment with 1,25(OH)2D significantly reduced intracellular glutamine and glutamate levels measured by nuclear magnetic resonance (NMR) by 23±2% each. Further, 1,25(OH)2D treatment reduced glutamine and glutamate flux, determined by [U-(13)C5] glutamine tracer kinetics, into the TCA cycle by 31±0.2% and 17±0.4%, respectively. The relative levels of mRNA and protein abundance of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D treatment in both MCF10A-ras cells and MCF10A which overexpress ErbB2 (HER-2/neu). Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D treatment and the impact was eliminated with the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA). A consensus sequence to the vitamin D responsive element (VDRE) was identified in silico in the SLC1A5 gene promoter, and site-directed mutagenesis analyses with reporter gene studies demonstrate a functional negative VDRE in the promoter of the SLC1A5 gene. siRNA-SLC1A5 transfection in MCF10A-ras cells significantly reduced SLC1A5 mRNA expression as well as decreased viable cell number similar to 1,25(OH)2D treatment. SLC1A5 knockdown also induced an increase in apoptotic cells in MCF10A-ras cells. These results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. Thus, 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may facilitate vitamin D prevention of breast

  1. Reduced Expression of the Extracellular Calcium-Sensing Receptor (CaSR) Is Associated with Activation of the Renin-Angiotensin System (RAS) to Promote Vascular Remodeling in the Pathogenesis of Essential Hypertension

    Science.gov (United States)

    Wang, La-mei; Tang, Na; Zhong, Hua; Liu, Yong-min; Li, Zhen; Feng, Qian; He, Fang

    2016-01-01

    The proliferation of vascular smooth muscle cells (VSMCs), remodeling of the vasculature, and the renin-angiotensin system (RAS) play important roles in the development of essential hypertension (EH), which is defined as high blood pressure (BP) in which secondary causes, such as renovascular disease, are absent. The calcium-sensing receptor (CaSR) is involved in the regulation of BP. However, the underlying mechanisms by which the CaSR regulates BP are poorly understood. In the present study, the role of the CaSR in EH was investigated using male spontaneously hypertensive rats (SHRs) and rat and human plasma samples. The percentages of medial wall thickness to external diameter (WT%), total vessel wall cross-sectional area to the total area (WA%) of thoracic arteries, as well as the percentage of wall area occupied by collagen to total vessel wall area (CA%) were determined. Tissue protein expression and plasma concentrations of the CaSR, cyclic adenosine monophosphate (cAMP), renin, and angiotensin II (Ang II) were additionally assessed. WT%, WA%, and CA% were found to increase with increasing BP, whereas the plasma concentration of CaSR was found to decrease. With increasing BP, the levels of smooth muscle actin and calponin decreased, whereas those of osteopontin and proliferating cell nuclear antigen increased. The CaSR level negatively correlated with the levels of cAMP and Ang II, but positively correlated with those of renin. Our data suggest that reduced expression of the CaSR is correlated with activation of the RAS, which induces increased vascular remodeling and VSMC proliferation, and thereby associated with EH in the SHR model and in the Han Chinese population. Our findings provide new insights into the pathogenesis of EH. PMID:27391973

  2. Loss of RASSF1A Expression in Colorectal Cancer and Its Association with K-ras Status

    Directory of Open Access Journals (Sweden)

    Dan Cao

    2013-01-01

    Full Text Available Background. The RAS-association domain family 1 A (RASSF1A is a classical member of RAS effectors regulating cell proliferation and apoptosis. Loss of RASSF1A expression may shift the balance towards a growth-promoting effect without the necessity of activating K-ras mutations. Its potential association with K-ras mutations in colorectal cancer (CRC is unclear. Methods. RASSF1A expression was examined in normal mucosa, adenoma, and tumor tissues of colon and rectum, respectively. We examined the association of RASSF1A expression, mutations of K-ras, and EGFR status in 76 primary CRCs. The relationship between clinicopathological characteristics and RASSF1A expression was also analyzed. Results. RASSF1A expression level decreased progressively in normal mucosa, adenoma and, tumor tissues, and the loss of RASSF1A expression occurred more frequently in tumor tissues. Of 76 primary CRCs, loss of RASSF1A expression and/or K-ras mutations were detected in 77% cases. Loss of RASSF1A expression was more frequent in K-ras wild-type than in mutation cases (63% versus 32%, . Conclusions. Our study indicates that loss of RASSF1A may be involved in pathogenesis of CRC, its expression was found predominantly in K-ras wild-type CRCs, suggesting that it may be another way of affecting RAS signaling, in addition to K-ras mutations.

  3. The research progress on function of Ras guanyl nucleotide releasing protein family%Ras 鸟苷酸释放蛋白家族的功能研究新进展

    Institute of Scientific and Technical Information of China (English)

    杨柳; 陶娟; 涂亚庭

    2015-01-01

    Ras鸟苷酸释放蛋白家族(Ras guanyl nucleotide releasing proteins ,RasGRPs)是鸟嘌呤核苷酸交换因子(guanine nucleotide exchange factors ,GEFs)中的一员,由4种类型的蛋白质组成。 RasGRPs能通过交换鸟嘌呤核苷酸将Ras蛋白从无活性的GDP形式变成有活性的GTP形式,从而活化Ras。4种不同类型的RasGRP蛋白拥有共同的分子结构。其结构包括一个由Ras 交换中心和CDC25区域组成的催化中心,同时还拥有一对非典型的EF臂及C1端。异常表达的不同RasGRP蛋白在不同的疾病发病过程中发挥作用。%Ras guanyl nucleotide releasing proteins ( RasGRPs) were one of guanine nucleotide exchange factors .They consist of four types of proteins .RasGRPs can activate Ras from GDP form to GTP form through the exchange of guanine nucleotide .Four differ-ent types of RasGRP proteins share a common molecular structure .The structure consists of a catalytic center including Ras exchange centers and CDC25 region.They also have a pair of atypical EF arms and C 1 side.The abnormal expressions of different RasGRP pro-teins play a role in different diseases .

  4. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

    Science.gov (United States)

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

    2016-01-22

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

  5. Implication of K-ras and p53 in colorectal cancer carcinogenesis in Tunisian population cohort.

    Science.gov (United States)

    Ines, Chaar; Donia, Ounissi; Rahma, Boughriba; Ben Ammar, Azza; Sameh, Amara; Khalfallah, Taher; Abdelmajid, Ben Hmida; Sabeh, Mzabi; Saadia, Bouraoui

    2014-07-01

    According to the multistep route of genetic alterations in the colorectal adenoma-carcinoma sequence, the complex K-ras/p53 mutation is one of the first alterations to occur and represent an important genetic event in colorectal cancer (CRC). An evaluation of the mutation spectra in K-ras and p53 gene was effected in 167 Tunisian patients with sporadic CRC to determine whether our populations have similar pattern of genetic alteration as in Maghrebin's population. Mutation patterns of codon 12-13 of K-ras and exon 5-8 of p53 were analyzed by immunohistochemistry and PCR-SSCP and confirmed by sequencing. Mutations in the K-ras gene were detected in 31.13 % and affect the women more than the men (p = 0.008). Immunostaining showed that expression of p21 ras was correlated with the advanced age (p = 0.004), whereas loss of signal was associated with mucinous histotype (p = 0.003). Kaplan-Meier survival curve found that patients with the K-ras mutation had a shorter survival compared with patients without mutation (p = 0.005). Alteration in p53 was seen in 17.4 % of patients and affects three hot spot codons such as 175, 245, and 248. Overexpression of p53 was seen in 34.1 % and correlated with tumor node metastasis (TNM) advanced stage (p = 0.037) and mucinous histotype (p = 0.001). A high concordance between p53 expression and alteration (p<0.005) was shown. Concomitant mutations in K-ras and p53 gene were detected in only 4 % of tumors. K-ras and p53 undergo separate pathways in colorectal tumorogenesis. Interestingly, mutations in the K-ras gene might be considered a valuable prognostic factor correlated to poor outcome. p53 gene alterations were rather low in our set, and methylation pattern of p53 is required to elucidate the molecular basis of this protein in CRC. PMID:24763823

  6. RasGRPs are targets of the anti-cancer agent ingenol-3-angelate.

    Directory of Open Access Journals (Sweden)

    Xiaohua Song

    Full Text Available Ingenol-3-angelate (I3A is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A's effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP's C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin's lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.

  7. The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

    OpenAIRE

    Jones, S; Vignais, M L; Broach, J R

    1991-01-01

    The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also o...

  8. Altered Erythrocyte Glycolytic Enzyme Activities in Type-II Diabetes.

    Science.gov (United States)

    Mali, Aniket V; Bhise, Sunita S; Hegde, Mahabaleshwar V; Katyare, Surendra S

    2016-07-01

    The activity of enzymes of glycolysis has been studied in erythrocytes from type-II diabetic patients in comparison with control. RBC lysate was the source of enzymes. In the diabetics the hexokinase (HK) activity increased 50 % while activities of phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and aldolase (ALD) decreased by 37, 75 and 64 % respectively but were still several folds higher than that of HK. Hence, it is possible that in the diabetic erythrocytes the process of glycolysis could proceed in an unimpaired or in fact may be augmented due to increased levels of G6P. The lactate dehydrogenase (LDH) activity was comparatively high in both the groups; the diabetic group showed 85 % increase. In control group the HK, PFK and ALD activities showed strong positive correlation with blood sugar level while PGI activity did not show any correlation. In the diabetic group only PFK activity showed positive correlation. The LDH activity only in the control group showed positive correlation with marginal increase with increasing concentrations of glucose. PMID:27382204

  9. Heparin alters viral serpin, serp-1, anti-thrombolytic activity to anti-thrombotic activity.

    Science.gov (United States)

    Li, Xing; Schneider, Heather; Peters, Andrew; Macaulay, Colin; King, Elaine; Sun, Yunming; Liu, Liying; Dai, Erbin; Davids, Jennifer A; McFadden, Grant; Lucas, Alexandra

    2008-01-01

    Serine protease inhibitors (serpins) regulate coagulation and inflammation. Heparin, a glycosaminoglycan, is an important cofactor for modulation of the inhibitory function of mammalian serpins. The secreted myxoma viral serpin, Serp-1 exerts profound anti-inflammatory activity in a wide range of animal models. Serp-1 anti-inflammatory and anti-atherogenic activity is dependent upon inhibition of the uPA / uPA receptor thrombolytic complex. We demonstrate here that heparin binds to Serp-1 and enhances Serp-1 inhibition of thrombin, a human pro-thrombotic serine protease, in vitro, altering inhibitory activity to a more predominant anti-thrombotic activity. Heparin also facilitates the simultaneous thrombin-mediated cleavage of Serp-1 and prevents formation of a serpin-typical SDS-resistant complex, implying mutual neutralization of Serp-1 and thrombin. In a cell-based assay, heparin facilitates Serp-1 reversal of cellular activation by stabilizing cellular membrane fluidity in thrombin-activated monocytes. In conclusion, heparin and other GAGs serve as cofactors enhancing Serp-1 regulation of local thrombotic and inflammatory pathways. PMID:18949070

  10. Optimizing depuration of salmon in RAS

    Science.gov (United States)

    Fish cultured within water recirculating aquaculture systems (RAS) can acquire "earthy" or "musty" off-flavors due to bioaccumulation of the compounds geosmin and 2-methylisoborneol (MIB), respectively, which are produced by certain bacterial species present in RAS biosolids and microbial biofilms. ...

  11. Sensitization of human pancreatic cancer cells harboring mutated K-ras to apoptosis.

    Directory of Open Access Journals (Sweden)

    Ling Shen

    Full Text Available Pancreatic cancer is a devastating human malignancy and gain of functional mutations in K-ras oncogene is observed in 75%-90% of the patients. Studies have shown that oncogenic ras is not only able to promote cell growth or survival, but also apoptosis, depending upon circumstances. Using pancreatic cancer cell lines with or without expressing mutated K-ras, we demonstrated that the inhibition of endogenous PKC activity sensitized human pancreatic cancer cells (MIA and PANC-1 expressing mutated K-ras to apoptosis, which had no apoptotic effect on BxPC-3 pancreatic cancer cells that contain a normal Ras as well as human lung epithelial BAES-2B cells. In this apoptotic process, the level of ROS was increased and PUMA was upregulated in a p73-dependent fashion in MIA and PANC-1 cells. Subsequently, caspase-3 was cleaved. A full induction of apoptosis required the activation of both ROS- and p73-mediated pathways. The data suggest that PKC is a crucial factor that copes with aberrant K-ras to maintain the homeostasis of the pancreatic cancer cells harboring mutated K-ras. However, the suppression or loss of PKC disrupts the balance and initiates an apoptotic crisis, in which ROS and p73 appear the potential, key targets.

  12. Amygdala kindling alters protein kinase C activity in dentate gyrus.

    Science.gov (United States)

    Chen, S J; Desai, M A; Klann, E; Winder, D G; Sweatt, J D; Conn, P J

    1992-11-01

    Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.

  13. Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

    International Nuclear Information System (INIS)

    Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms

  14. Analysis of RAS oncogene mutations in human lymphoid malignancies.

    OpenAIRE

    Neri, A.; Knowles, D M; Greco, A.; McCormick, F; Dalla-Favera, R

    1988-01-01

    We investigated the frequency of mutations activating RAS oncogenes in human lymphoid malignancies, including B- and T-cell-derived acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. By the polymerase chain reaction/oligonucleotide hybridization method, DNA from 178 cases was analyzed for activating mutations involving codons 12 and 61 of the HRAS, KRAS and NRAS genes and codon 13 of the NRAS gene. Mutations involving codons 12 or 13 of the NRAS gene were de...

  15. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

    Science.gov (United States)

    Czikora, Agnes; Lundberg, Daniel J; Abramovitz, Adelle; Lewin, Nancy E; Kedei, Noemi; Peach, Megan L; Zhou, Xiaoling; Merritt, Raymond C; Craft, Elizabeth A; Braun, Derek C; Blumberg, Peter M

    2016-05-20

    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

  16. Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

    Science.gov (United States)

    Stanhill, A; Levin, V; Hendel, A; Shachar, I; Kazanov, D; Arber, N; Kaminski, N; Engelberg, D

    2006-03-01

    Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

  17. Ras1CA overexpression in the posterior silk gland improves silk yield

    Institute of Scientific and Technical Information of China (English)

    Li Ma; Hanfu Xu; Jinqi Zhu; Sanyuan Ma; Yan Liu; Rong-Jing Jiang; Qingyou Xia; Sheng Li

    2011-01-01

    Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm,Bombyx mori,but has reached a plateau during the last decades. For the first time,we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1CA oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%,while increasing food consumption by only 20%. Ras activation by Ras1CA overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins,up-regulated fibroin mRNA levels,increased total DNA content,and stimulated endoreplication. Moreover,Rasl activation increased cell and nuclei sizes,enriched subcellular organelles related to protein synthesis,and stimulated ribosome biogenesis for mRNA translation. We conclude that Rasl activation increases cell size and protein synthesis in the posterior silk gland,leading to silk yield improvement.

  18. Oncogenic Ras modulates p38 MAPK-mediated inflammatory cytokine production in glioblastoma cells.

    Science.gov (United States)

    Munoz, Lenka; Yeung, Yiu To; Grewal, Thomas

    2016-04-01

    Inflammation is an important factor promoting the progression of glioblastoma. In the present study we examined the contribution of Ras signaling and TNFα/IL-1β cytokines to the development of the glioblastoma inflammatory microenvironment. Enhanced activation of Ras through de-regulated activation of receptor tyrosine kinases, such as EGFR, PDGFR and cMet, is a hallmark of the majority of glioblastomas. Glioblastoma microenvironment contains high levels of TNFα and IL-1β, which mediate inflammation through induction of a local network of cytokines and chemokines. While many studies have focused on Ras- and TNFα/IL-1β-driven inflammation in isolation, little is known about the co-operation between these oncogenic and microenvironment-derived stimuli. Using constitutively active HRasG12V that mimics enhanced Ras activation, we demonstrate that elevated Ras activity in glioblastoma cells leads to up-regulation of IL-6 and IL-8. Furthermore, Ras synergizes with the microenvironment-derived TNFα and IL-1β resulting in amplified IL-6/IL-8 secretion. IL-8 secretion induced by Ras and TNFα/IL-1β is attenuated by inhibitors targeting Erk, JNK and p38 MAPK pathways. IL-6 secretion significantly decreased upon inhibition of JNK and p38 MAPK pathways. Interestingly, although constitutively active HRasG12V does not increase basal or TNFα/IL-1β stimulated p38 MAPK activity, HRasG12V increased the efficacy of the p38 MAPK inhibitor SB203580 to inhibit IL-1β-induced IL-6 secretion. In summary, oncogenic Ras co-operates with the microenvironment-derived TNFα/IL-1β to sustain inflammatory microenvironment, which was effectively attenuated via inhibition of p38 MAPK signaling. PMID:26794430

  19. Repression and activation by multiprotein complexes that alter chromatin structure.

    Science.gov (United States)

    Kingston, R E; Bunker, C A; Imbalzano, A N

    1996-04-15

    Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby. It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation. These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable. As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated. In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes. This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.

  20. Silver and Gold Nanoparticles Alter Cathepsin Activity In vitro

    Science.gov (United States)

    Speshock, Janice L.; Braydich-Stolle, Laura K.; Szymanski, Eric R.; Hussain, Saber M.

    2011-12-01

    Nanomaterials are being incorporated into many biological applications for use as therapeutics, sensors, or labels. Silver nanomaterials are being utilized for biological implants and wound dressings as an antiviral material, whereas gold nanomaterials are being used as biological labels or sensors due to their surface properties and biocompatibility. Cytotoxicity data of these materials are becoming more prevalent; however, little research has been performed to understand how the introduction of these materials into cells affects cellular processes. Here, we demonstrate the impact that silver and gold nanoparticles have on cathepsin activity in vitro. Cathepsins are important cellular proteases that are imperative for proper immune system function. We have selected to examine gold and silver nanoparticles due to the increased use of these materials in biological applications. This manuscript depicts how both of these types of nanomaterials affect cathepsin activity, which could impact the host's immune system and its ability to respond to pathogens. Cathepsin B activity decreases in a dose-dependent manner with all nanoparticles tested. Alternatively, the impact of nanoparticles on cathepsin L activity depends greatly on the type and size of the material.

  1. Altered Error-Related Activity in Patients with Schizophrenia

    Science.gov (United States)

    Koch, Kathrin; Wagner, Gerd; Schultz, Christoph; Schachtzabel, Claudia; Nenadic, Igor; Axer, Martina; Reichenbach, Jurgen R.; Sauer, Heinrich; Schlosser, Ralf G. M.

    2009-01-01

    Deficits in working memory (WM) and executive cognitive control are core features of schizophrenia. However, findings regarding functional activation strengths are heterogeneous, partly due to differences in task demands and behavioral performance. Previous investigators proposed integrating these heterogeneous findings into a comprehensive model…

  2. Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF.

    Science.gov (United States)

    Baljuls, Angela; Dobrzyński, Maciej; Rauch, Jens; Rauch, Nora; Kolch, Walter

    2016-10-01

    RAF family kinases are central components of the Ras-RAF-MEK-ERK cascade. Dimerization is a key mechanism of RAF activation in response to physiological, pathological and pharmacological signals. It is mediated by a dimer interface region in the RAF kinase domain that is also conserved in KSR, a scaffolding protein that binds RAF, MEK and ERK. The regulation of RAF dimerization is incompletely understood. Especially little is known about the molecular mechanism involved in the selection of the dimerization partner. Previously, we reported that Ras-dependent binding of the tumour suppressor DiRas3 to C-RAF inhibits the C-RAF:B-RAF heterodimerization. Here we show that DiRas3 binds to KSR1 independently of its interaction with activated Ras and RAF. Our data also suggest that depending on the local stoichiometry between DiRas3 and oncogenic Ras, DiRas3 can either enhance homodimerization of KSR1 or recruit KSR1 to the Ras:C-RAF complex and thereby reduce the availability of C-RAF for binding to B-RAF. This mechanism, which is shared between A-RAF and C-RAF, may be involved in the regulation of Ras12V-induced cell transformation by DiRas3. PMID:27368419

  3. The Ras-Erk-ETS-Signaling Pathway Is a Drug Target for Longevity.

    Science.gov (United States)

    Slack, Cathy; Alic, Nazif; Foley, Andrea; Cabecinha, Melissa; Hoddinott, Matthew P; Partridge, Linda

    2015-07-01

    Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. Here, we identify a critical role for Ras-Erk-ETS signaling in aging in Drosophila. We show that inhibition of Ras is sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. We identify the E-twenty six (ETS) transcriptional repressor, Anterior open (Aop), as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, we demonstrate that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals.

  4. ALTERED ENZYMATIC ACTIVITY OF LYSOZYMES BOUND TO VARIOUSLY SULFATED CHITOSANS

    Institute of Scientific and Technical Information of China (English)

    Hong-wei Wang; Lin Yuan; Tie-liang Zhao; He Huang; Hong Chen; Di Wu

    2012-01-01

    The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure.It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan (6S-chitosan) and 3,6-O-sulfated chitosan (3,6S-chitosan),but decreased greatly after being bound to 2-N-6-O-sulfated chitosan (2,6S-chitosan).Meanwhile,among these sulfated chitosans,2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra.These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme,lysozyme undergoes conformational change of different magnitudes,which results in corresponding levels of lysozyme activity.Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance (SPR) suggested that their affinities might be determined by their molecular structures.

  5. Leukotriene B(4) BLT receptor signaling regulates the level and stability of cyclooxygenase-2 (COX-2) mRNA through restricted activation of Ras/Raf/ERK/p42 AUF1 pathway.

    Science.gov (United States)

    Zhai, Beibei; Yang, Huiqing; Mancini, Arturo; He, QingWen; Antoniou, John; Di Battista, John A

    2010-07-30

    Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid

  6. Postnatal foraging demands alter adrenocortical activity and psychosocial development.

    Science.gov (United States)

    Lyons, D M; Kim, S; Schatzberg, A F; Levine, S

    1998-05-01

    Mother squirrel monkeys stop carrying infants at earlier ages in high-demand (HD) conditions where food is difficult to find relative to low-demand (LD) conditions. To characterize these transitions in psychosocial development, from 10- to 21-weeks postpartum we collected measures of behavior, adrenocortical activity, and social transactions coded for initiator (mother or infant), goal (make-contact or break-contact), and outcome (success or failure). Make-contact attempts were most often initiated by HD infants, but mothers often opposed these attempts and less than 50% were successful. Break-contact attempts were most often initiated by LD infants, but mothers often opposed these attempts and fewer LD than HD infant break-contact attempts were successful. Plasma levels of cortisol were significantly higher in HD than LD mothers, but differences in adrenocortical activity were less consistent in their infants. HD and LD infants also spent similar amounts of time nursing on their mothers and feeding on solid foods. By rescheduling some transitions in development (carry-->self-transport), and not others (nursing-->self-feeding), mothers may have partially protected infants from the immediate impact of an otherwise stressful foraging task. PMID:9589217

  7. Altered Activities of Antioxidant Enzymes in Patients with Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Lucie Vávrová

    2013-02-01

    Full Text Available Objective: In the pathogenesis of the metabolic syndrome (MetS, an increase of oxidative stress could play an important role which is closely linked with insulin resistance, endothelial dysfunction, and chronic inflammation. The aim of our study was to assess several parameters of the antioxidant status in MetS. Methods: 40 subjects with MetS and 40 age- and sex-matched volunteers without MetS were examined for activities of superoxide dismutase (CuZnSOD, catalase (CAT, glutathione peroxidase 1 (GPX1, glutathione reductase (GR, paraoxonase1 (PON1, concentrations of reduced glutathione (GSH, and conjugated dienes in low-density lipoprotein (CD-LDL. Results: Subjects with MetS had higher activities of CuZnSOD (p Conclusions: Our results implicated an increased oxidative stress in MetS and a decreased antioxidative defense that correlated with some laboratory (triglycerides, high-density lipoprotein cholesterol (HDL-C and clinical (waist circumference, blood pressure components of MetS.

  8. Alterations in electrodermal activity and cardiac parasympathetic tone during hypnosis.

    Science.gov (United States)

    Kekecs, Zoltán; Szekely, Anna; Varga, Katalin

    2016-02-01

    Exploring autonomic nervous system (ANS) changes during hypnosis is critical for understanding the nature and extent of the hypnotic phenomenon and for identifying the mechanisms underlying the effects of hypnosis in different medical conditions. To assess ANS changes during hypnosis, electrodermal activity and pulse rate variability (PRV) were measured in 121 young adults. Participants either received hypnotic induction (hypnosis condition) or listened to music (control condition), and both groups were exposed to test suggestions. Blocks of silence and experimental sound stimuli were presented at baseline, after induction, and after de-induction. Skin conductance level (SCL) and high frequency (HF) power of PRV measured at each phase were compared between groups. Hypnosis decreased SCL compared to the control condition; however, there were no group differences in HF power. Furthermore, hypnotic suggestibility did not moderate ANS changes in the hypnosis group. These findings indicate that hypnosis reduces tonic sympathetic nervous system activity, which might explain why hypnosis is effective in the treatment of disorders with strong sympathetic nervous system involvement, such as rheumatoid arthritis, hot flashes, hypertension, and chronic pain. Further studies with different control conditions are required to examine the specificity of the sympathetic effects of hypnosis.

  9. Alterations in electrodermal activity and cardiac parasympathetic tone during hypnosis.

    Science.gov (United States)

    Kekecs, Zoltán; Szekely, Anna; Varga, Katalin

    2016-02-01

    Exploring autonomic nervous system (ANS) changes during hypnosis is critical for understanding the nature and extent of the hypnotic phenomenon and for identifying the mechanisms underlying the effects of hypnosis in different medical conditions. To assess ANS changes during hypnosis, electrodermal activity and pulse rate variability (PRV) were measured in 121 young adults. Participants either received hypnotic induction (hypnosis condition) or listened to music (control condition), and both groups were exposed to test suggestions. Blocks of silence and experimental sound stimuli were presented at baseline, after induction, and after de-induction. Skin conductance level (SCL) and high frequency (HF) power of PRV measured at each phase were compared between groups. Hypnosis decreased SCL compared to the control condition; however, there were no group differences in HF power. Furthermore, hypnotic suggestibility did not moderate ANS changes in the hypnosis group. These findings indicate that hypnosis reduces tonic sympathetic nervous system activity, which might explain why hypnosis is effective in the treatment of disorders with strong sympathetic nervous system involvement, such as rheumatoid arthritis, hot flashes, hypertension, and chronic pain. Further studies with different control conditions are required to examine the specificity of the sympathetic effects of hypnosis. PMID:26488759

  10. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Naumov, Inna [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Kazanov, Dina [Integrated Cancer Prevention Center, Tel Aviv (Israel); Lisiansky, Victoria [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Starr, Alex [Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Aroch, Ilan; Shapira, Shiran; Kraus, Sarah [Integrated Cancer Prevention Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel); Arber, Nadir, E-mail: narber@post.tau.ac.il [Integrated Cancer Prevention Center, Tel Aviv (Israel); Department of Gastroenterology, Tel Aviv Sourasky Medical Center, Tel Aviv (Israel); Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv (Israel)

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  11. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    International Nuclear Information System (INIS)

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35–40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our “gene therapy” approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ∼ 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ∼ 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  12. Colon cancer and gene alterations: their immunological implications and suggestions for prognostic indices and improvements in biotherapy.

    Science.gov (United States)

    Contasta, Ida; Pellegrini, Patrizia; Berghella, Anna Maria; Del Beato, Tiziana; Adorno, Domenico

    2006-10-01

    Studies have shown that changes occur in c-Ki-ras, p53, and Bcl2 gene structure and function during the various stages of human colon carcinogenesis. Alterations of these genes are responsible for the establishment of a state of continuous stimulus for cell division and apoptotic inhibition at physiological and pharmacological levels. This paper focuses on the results of our research aimed at investigating how these gene alterations influence tumoral mechanisms on an immunological level and how immunological parameters can be used as prognostic markers for the passage of normal tissue to adenoma and adenoma to carcinoma. Overall, our data suggest that an alteration in the c-Ki-ras gene results in a switch to a suppressive type of immune response, determining an impairment of immune cell activation at both antigen- presenting-cell and T-cell levels. c-Ki-ras gene mutations, p53 deletions, and Bc12 expression, on the other hand, can be used as prognostic markers for the passage of normal tissue to adenoma and adenoma to carcinoma. The p53 oncogene does not appear to impair patients' immunological response further. In conclusion, an evaluation of c-Ki-ras, rather than p53 gene alterations, would seem to be more relevant in colon cancer prevention programs and biotherapy improvement.

  13. Altered behavior in spotted hyenas associated with increased human activity

    Science.gov (United States)

    Boydston, Erin E.; Kapheim, Karen M.; Watts, Heather E.; Szykman, Micaela; Holekamp, Kay E.

    2003-01-01

    To investigate how anthropogenic activity might affect large carnivores, we studied the behaviour of spotted hyenas (Crocuta crocuta) during two time periods. From 1996 to 1998, we documented the ecological correlates of space utilization patterns exhibited by adult female hyenas defending a territory at the edge of a wildlife reserve in Kenya. Hyenas preferred areas near dense vegetation but appeared to avoid areas containing the greatest abundance of prey, perhaps because these were also the areas of most intensive livestock grazing. We then compared hyena behaviour observed in 1996–98 with that observed several years earlier and found many differences. Female hyenas in 1996–98 were found farther from dens, but closer to dense vegetation and to the edges of their territory, than in 1988–90. Recent females also had larger home ranges, travelled farther between consecutive sightings, and were more nocturnal than in 1988–90. Finally, hyenas occurred in smaller groups in 1996–98 than in 1988–90. We also found several changes in hyena demography between periods. We next attempted to explain differences observed between time periods by testing predictions of hypotheses invoking prey abundance, climate, interactions with lions, tourism and livestock grazing. Our data were consistent with the hypothesis that increased reliance on the reserve for livestock grazing was responsible for observed changes. That behavioural changes were not associated with decreased hyena population density suggests the behavioural plasticity typical of this species may protect it from extinction.

  14. Altered behaviour in spotted hyenas associated with increased human activity

    Science.gov (United States)

    Boydston, E.E.; Kapheim, K.M.; Watts, H.E.; Szykman, M.; Holekamp, K.E.

    2003-01-01

    To investigate how anthropogenic activity might affect large carnivores, we studied the behaviour of spotted hyenas (Crocuta crocuta) during two time periods. From 1996 to 1998, we documented the ecological correlates of space utilization patterns exhibited by adult female hyenas defending a territory at the edge of a wildlife reserve in Kenya. Hyenas preferred areas near dense vegetation but appeared to avoid areas containing the greatest abundance of prey, perhaps because these were also the areas of most intensive livestock grazing. We then compared hyena behaviour observed in 1996-98 with that observed several years earlier and found many differences. Female hyenas in 1996-98 were found farther from dens, but closer to dense vegetation and to the edges of their territory, than in 1988-90. Recent females also had larger home ranges, travelled farther between consecutive sightings, and were more nocturnal than in 1988-90. Finally, hyenas occurred in smaller groups in 1996-98 than in 1988-90. We also found several changes in hyena demography between periods. We next attempted to explain differences observed between time periods by testing predictions of hypotheses invoking prey abundance, climate, interactions with lions, tourism and livestock grazing. Our data were consistent with the hypothesis that increased reliance on the reserve for livestock grazing was responsible for observed changes. That behavioural changes were not associated with decreased hyena population density suggests the behavioural plasticity typical of this species may protect it from extinction. ?? 2003 The Zoological Society of London.

  15. Transcriptional Profile of Ki-Ras-Induced Transformation of Thyroid Cells

    DEFF Research Database (Denmark)

    Visconti, Roberta; Federico, Antonella; Coppola, Valeria;

    2007-01-01

    Abstract In the last years, an increasing number of experiments has provided compelling evidence for a casual role of Ras protein mutations, resulting in their constitutive activation, in thyroid carcinogenesis. However, despite the clear involvement of Ras proteins in thyroid carcinogenesis, the...... in human thyroid carcinoma cell lines and tumor samples, our results, therefore, providing a new molecular profile of the genes involved in thyroid neoplastic transformation....

  16. The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH

    DEFF Research Database (Denmark)

    Willumsen, B M; Vass, W C; Velu, T J;

    1991-01-01

    We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass......, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming...... activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated...

  17. A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene

    DEFF Research Database (Denmark)

    Papageorge, A G; Willumsen, B M; Johnsen, M;

    1986-01-01

    several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast...... to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When...

  18. Low concentrations of hydrogen peroxide or nitrite induced of Paracoccidioides brasiliensis cell proliferation in a Ras-dependent manner.

    Directory of Open Access Journals (Sweden)

    Ana Eliza Coronel Janu Haniu

    Full Text Available Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM, should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS and nitrogen (RNS species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. In lower eukaryotes, Byr2 kinase represents a Ras target. The present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM or NO2 (0.1-0.25 µM stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD fused with GST (RBD-Byr2-GST to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. In conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.

  19. Expression of ras oncogene and major histocompatibility complex (MHC) antigen in carcinomas of the uterine cervix

    International Nuclear Information System (INIS)

    Consecutive 50 cases of squamous cell carcinomas of the uterine cervix diagnosed in 1992 were subjected to immunohistochemical study for ras oncogene product (p21) and MHC class II (DR) antigen using a microprobe immunostainer. Activated ras and aberrant DR expression were noted in 26 cases (52%) and 11 cases (22%) of cervical squamous cell carcinomas, respectively, without difference among histologic types. The reaction was mainly intracytoplasmic, with granular staining pattern and diffuse distribution. No direct histologic correlation between ras and DR expression was found. Four cases with HPV 16/18 DNA in superficial koilocytotic cells, revealed by in situ hybridization, showed various expression of ras and DR, and these 3 factors histologically did not seem to be affected one another. (Author)

  20. NAM: The 2004 RAS National Astronomy Meeting

    Science.gov (United States)

    Jones, Barrie; Norton, Andrew

    2004-06-01

    This year's RAS National Astronomy Meeting was held at the Open University's Milton Keynes campus from 29 March to 2 April. The event was organized by members of the OU Physics & Astronomy Department and Planetary & Space Science Research Institute. Around 450 people attended the meeting, at which more than 220 talks were presented, along with around 90 posters. Co-chairs of RAS NAM04, Barrie Jones and Andrew Norton, summarize.

  1. Gene expression profiling of tumours derived from rasV12/E1A-transformed mouse embryonic fibroblasts to identify genes required for tumour development

    Directory of Open Access Journals (Sweden)

    Dagorn Jean

    2005-01-01

    Full Text Available Abstract Background In cancer, cellular transformation is followed by tumour development. Knowledge on the mechanisms of transformation, involving activation of proto-oncogenes and inactivation of tumour-suppressor genes has considerably improved whereas tumour development remains poorly understood. An interesting way of gaining information on tumour progression mechanisms would be to identify genes whose expression is altered during tumour formation. We used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumours derived from rasV12/E1A-transformed mouse embryo fibroblasts in order to identify the genes that could be involved in tumour development. Results Among the 12,000 genes analyzed in this study, only 489 showed altered expression during tumour development, 213 being up-regulated and 276 down-regulated. The genes differentially expressed are involved in a variety of cellular functions, including control of transcription, regulation of mRNA maturation and processing, regulation of protein translation, activation of interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell-to-cell interaction, extracellular matrix formation, metabolism and production of secretory factors. Conclusions Some of the genes identified in this work, whose expression is altered upon rasV12/E1A transformation of MEFs, could be new cancer therapeutic targets.

  2. A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.

    Science.gov (United States)

    Cherkasova, Vera A; McCully, Ryan; Wang, Yunmei; Hinnebusch, Alan; Elion, Elaine A

    2003-07-15

    In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks. PMID:12867033

  3. Activated R-Ras, Rac1, Pi 3-Kinase and Pkcε Can Each Restore Cell Spreading Inhibited by Isolated Integrin β1 Cytoplasmic Domains

    OpenAIRE

    Berrier, Allison L.; Mastrangelo, Anthony M.; Downward, Julian; Ginsberg, Mark; LaFlamme, Susan E.

    2000-01-01

    Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact β cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spread...

  4. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

    Science.gov (United States)

    Londino, James D; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F; Noah, James W; Matalon, Sadis

    2013-05-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

  5. Post-transcriptional regulation of connexin43 in H-Ras-transformed cells.

    Directory of Open Access Journals (Sweden)

    Mustapha Kandouz

    Full Text Available Connexin43 (Cx43 expression is lost in cancer cells and many studies have reported that Cx43 is a tumor suppressor gene. Paradoxically, in a cellular NIH3T3 model, we have previously shown that Ha-Ras-mediated oncogenic transformation results in increased Cx43 expression. Although the examination of transcriptional regulation revealed essential regulatory elements, it could not solve this paradox. Here we studied post-transcriptional regulation of Cx43 expression in cancer using the same model in search of novel gene regulatory elements. Upon Ras transformation, both Cx43 mRNA stability and translation efficiency were increased. We investigated the role of Cx43 mRNA 3' and 5'Untranslated regions (UTRs and found an opposing effect; a 5'UTR-driven positive regulation is observed in Ras-transformed cells (NIH-3T3(Ras, while the 3'UTR is active only in normal NIH-3T3(Neo cells and completely silenced in NIH-3T3(Ras cells. Most importantly, we identified a previously unknown regulatory element within the 3'UTR, named S1516, which accounts for this 3'UTR-mediated regulation. We also examined the effect of other oncogenes and found that Ras- and Src-transformed cells show a different Cx43 UTRs post-transcriptional regulation than ErbB2-transformed cells, suggesting distinct regulatory pathways. Next, we detected different patterns of S1516 RNA-protein complexes in NIH-3T3(Neo compared to NIH-3T3(Ras cells. A proteomic approach identified most of the S1516-binding proteins as factors involved in post-transcriptional regulation. Building on our new findings, we propose a model to explain the discrepancy between the Cx43 expression in Ras-transformed NIH3T3 cells and the data in clinical specimens.

  6. Epidermal Growth Factor Receptor (EGFR gene copy number (GCN correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH and chromogenic in situ hybridization (CISH analysis

    Directory of Open Access Journals (Sweden)

    Scartozzi Mario

    2009-08-01

    Full Text Available Abstract Background K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Methods Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Results Forty-four patients were available for analysis. We observed a partial remission in 9 (60% and 2 (9% cases with a FISH EGFR GCN ≥ 2.6 and Conclusion FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

  7. Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

    Energy Technology Data Exchange (ETDEWEB)

    Bitencourt, C.S. [Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Duarte, C.G.; Azzolini, A.E.C.S.; Assis-Pandochi, A.I. [Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-03-02

    Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

  8. Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

    Directory of Open Access Journals (Sweden)

    Pathan AAK

    2016-05-01

    Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the

  9. Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory.

    Science.gov (United States)

    Gyurkó, M Dávid; Csermely, Péter; Sőti, Csaba; Steták, Attila

    2015-01-01

    The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans. PMID:26469632

  10. Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli.

    OpenAIRE

    Uno, I.; Mitsuzawa, H.; Matsumoto, K.; Tanaka, K; Oshima, T.; Ishikawa, T

    1985-01-01

    Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 pr...

  11. Basal but not luminal mammary epithelial cells require PI3K/mTOR signaling for Ras-driven overgrowth.

    Science.gov (United States)

    Plichta, Kristin A; Mathers, Jessica L; Gestl, Shelley A; Glick, Adam B; Gunther, Edward J

    2012-11-15

    The mammary ducts of humans and mice are comprised of two main mammary epithelial cell (MEC) subtypes: a surrounding layer of basal MECs and an inner layer of luminal MECs. Breast cancer subtypes show divergent clinical behavior that may reflect properties inherent in their MEC compartment of origin. How the response to a cancer-initiating genetic event is shaped by MEC subtype remains largely unexplored. Using the mouse mammary gland, we designed organotypic three-dimensional culture models that permit challenge of discrete MEC compartments with the same oncogenic insult. Mammary organoids were prepared from mice engineered for compartment-restricted coexpression of oncogenic H-RAS(G12V) together with a nuclear fluorescent reporter. Monitoring of H-RAS(G12V)-expressing MECs during extended live cell imaging permitted visualization of Ras-driven phenotypes via video microscopy. Challenging either basal or luminal MECs with H-RAS(G12V) drove MEC proliferation and survival, culminating in aberrant organoid overgrowth. In each compartment, Ras activation triggered modes of collective MEC migration and invasion that contrasted with physiologic modes used during growth factor-initiated branching morphogenesis. Although basal and luminal Ras activation produced similar overgrowth phenotypes, inhibitor studies revealed divergent use of Ras effector pathways. Blocking either the phosphoinositide 3-kinase or the mammalian target of rapamycin pathway completely suppressed Ras-driven invasion and overgrowth of basal MECs, but only modestly attenuated Ras-driven phenotypes in luminal MECs. We show that MEC subtype defines signaling pathway dependencies downstream of Ras. Thus, cells-of-origin may critically determine the drug sensitivity profiles of mammary neoplasia. PMID:23010075

  12. SY 09-3 ROLE OF INTRARENAL RAS IN THE PATHOGENESIS OF SALT-DEPENDENT HYPERTENSION.

    Science.gov (United States)

    Nishiyama, Akira

    2016-09-01

    The renin-angiotensin system (RAS) plays a critical role in the regulation of blood pressure and body fluid homeostasis. In addition to its physiological roles, angiotensin II (AngII) induces inflammation, cell growth, mitogenesis, apoptosis, migration, and differentiation, regulates the gene expression of bioactive substances, and activates multiple intracellular signaling pathways, all of which contribute to renal tissue injuries. In the kidney, all of the RAS components are present and intrarenal AngII is formed by independent multiple mechanisms. In particular, a series of our studies have revealed that intrarenal angiotensinogen plays a predominant role in the regulation of AngII production in the kidney. Consequently, AngII is compartmentalized in the renal interstitial fluid and the proximal tubular compartments with much higher concentrations than those existing in the circulation. Recent evidence has also revealed that inappropriate activation of the intrarenal RAS is an important contributor to the pathogenesis of hypertension and renal injury. For example, circulating plasma AngII levels are reduced in CKD patients with salt-dependent hypertension; however, intrarenal RAS is activated by inappropriately augmentation of angiotensinogen in the kidney. Thus, it is necessary to understand the mechanisms responsible for independent regulation of the intrarenal RAS. In this symposium, I will briefly summarize our current understanding of independent regulation of the intrarenal RAS and discuss how inappropriate activation of this system contributes to the development of salt-dependent hypertension in patients with CKD. We will also discuss the possible biomarker of the intrarenal RAS. PMID:27642896

  13. Altered lower leg muscle activation patterns in patients with cerebral palsy during cycling on an ergometer

    Science.gov (United States)

    Alves-Pinto, Ana; Blumenstein, Tobias; Turova, Varvara; Lampe, Renée

    2016-01-01

    Objective Cycling on a recumbent ergometer constitutes one of the most popular rehabilitation exercises in cerebral palsy (CP). However, no control is performed on how muscles are being used during training. Given that patients with CP present altered muscular activity patterns during cycling or walking, it is possible that an incorrect pattern of muscle activation is being promoted during rehabilitation cycling. This study investigated patterns of muscular activation during cycling on a recumbent ergometer in patients with CP and whether those patterns are determined by the degree of spasticity and of mobility. Methods Electromyographic (EMG) recordings of lower leg muscle activation during cycling on a recumbent ergometer were performed in 14 adult patients diagnosed with CP and five adult healthy participants. EMG recordings were done with an eight-channel EMG system built in the laboratory. The activity of the following muscles was recorded: Musculus rectus femoris, Musculus biceps femoris, Musculus tibialis anterior, and Musculus gastrocnemius. The degree of muscle spasticity and mobility was assessed using the Modified Ashworth Scale and the Gross Motor Function Classification System, respectively. Muscle activation patterns were described in terms of onset and duration of activation as well as duration of cocontractions. Results Muscle activation in CP was characterized by earlier onsets, longer periods of activation, a higher occurrence of agonist–antagonist cocontractions, and a more variable cycling tempo in comparison to healthy participants. The degree of altered muscle activation pattern correlated significantly with the degree of spasticity. Conclusion This study confirmed the occurrence of altered lower leg muscle activation patterns in patients with CP during cycling on a recumbent ergometer. There is a need to develop feedback systems that can inform patients and therapists of an incorrect muscle activation during cycling and support the training

  14. Platelet Activation in Human Immunodeficiency Virus Type-1 Patients Is Not Altered with Cocaine Abuse.

    Directory of Open Access Journals (Sweden)

    Michelle Kiebala

    Full Text Available Recent work has indicated that platelets, which are anucleate blood cells, significantly contribute to inflammatory disorders. Importantly, platelets also likely contribute to various inflammatory secondary disorders that are increasingly associated with Human Immunodeficiency Virus Type-1 (HIV infection including neurological impairments and cardiovascular complications. Indeed, HIV infection is often associated with increased levels of platelet activators. Additionally, cocaine, a drug commonly abused by HIV-infected individuals, leads to increased platelet activation in humans. Considering that orchestrated signaling mechanisms are essential for platelet activation, and that nuclear factor-kappa B (NF-κB inhibitors can alter platelet function, the role of NF-κB signaling in platelet activation during HIV infection warrants further investigation. Here we tested the hypothesis that inhibitory kappa B kinase complex (IKK activation would be central for platelet activation induced by HIV and cocaine. Whole blood from HIV-positive and HIV-negative individuals, with or without cocaine abuse was used to assess platelet activation via flow cytometry whereas IKK activation was analyzed by performing immunoblotting and in vitro kinase assays. We demonstrate that increased platelet activation in HIV patients, as measured by CD62P expression, is not altered with reported cocaine use. Furthermore, cocaine and HIV do not activate platelets in whole blood when treated ex vivo. Finally, HIV-induced platelet activation does not involve the NF-κB signaling intermediate, IKKβ. Platelet activation in HIV patients is not altered with cocaine abuse. These results support the notion that non-IKK targeting approaches will be better suited for the treatment of HIV-associated inflammatory disorders.

  15. CD8(+) T cells mediate RAS-induced psoriasis-like skin inflammation through IFN-γ.

    Science.gov (United States)

    Gunderson, Andrew J; Mohammed, Javed; Horvath, Frank J; Podolsky, Michael A; Anderson, Cherie R; Glick, Adam B

    2013-04-01

    The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RAS(V12G) in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses, and increased T helper type 1 (Th1)/Th17 and T cell type 1 (Tc1)/Tc17 skin infiltration. The majority of skin-infiltrating CD8(+) T cells coexpressed IFN-γ and IL-17A. When RAS was expressed on a Rag1-/- background, microabscess formation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation were suppressed. Depletion of CD8(+), but not CD4(+), T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17(+) γδ T cells, and epidermal hyperproliferation to levels similar to a Rag1-/- background. Reconstitution of Rag1-/- inducible RAS mice with purified CD8(+) T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ, but not of IL-17A, in CD8(+) T-cell-reconstituted Rag1-/- mice expressing RAS blocked CD8-mediated skin inflammation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation. These results show that CD8(+) T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ. PMID:23151849

  16. Oncogenic Ras-Induced Morphologic Change Is through MEK/ERK Signaling Pathway to Downregulate Stat3 at a Posttranslational Level in NIH3T3 Cells

    Directory of Open Access Journals (Sweden)

    Hsuan-Heng Yeh

    2008-01-01

    Full Text Available Ras is a key regulator of the MAP kinase-signaling cascade and may cause morphologic change of Ras-transformed cells. Signal transducer and activator of transcription 3 (Stat3 can be activated by cytokine stimulation. In this study, we unravel that Ha-rasV12 overexpression can downregulate the expression of Stat3 protein at a posttranslational level in NIH3T3 cells. Furthermore, we demonstrate that Stat3 expression downregulated by Ha-rasV12 overexpression is through proteosome degradation and not through a mTOR/p70S6K-related signaling pathway. The suppression of Stat3 accompanied by the morphologic change induced by Ha-rasV12 was through mitogen extracellular kinase (MEK/extracellular-regulated kinase (ERK signaling pathway. Microtubule disruption is involved in Ha-rasV12-induced morphologic change, which could be reversed by overexpression of Stat3. Taken together, we are the first to demonstrate that Stat3 protein plays a critical role in Ha-rasV12-induced morphologic change. Oncogenic Ras-triggered morphologic change is through the activation of MEK/ERK to posttranslationally downregulate Stat3 expression. Our finding may shed light on developing novel therapeutic strategies against Ras-related tumorigenesis.

  17. Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling

    International Nuclear Information System (INIS)

    Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients

  18. K-Ras and mitochondria: Dangerous liaisons

    Institute of Scientific and Technical Information of China (English)

    Jiri Neuzil; Jakub Rohlena; Lan-Feng Dong

    2012-01-01

    It is well documented that the KRAS oncogene efficiently transforms non-malignant cells,and there is some evidence for the role of mitochondria in this process.Now Peng Huang and colleagues show that K-Ras induction results early on in mitochondria assuming the phenotype consistent with the so-called Warburg effect,i.e.,increased glycolysis and attenuated oxidative phosphorylation.Thus the K-Ras protein capable of swift induction of phenotypic changes typical of cancer cells,yet these changes are reversible,and for cells to irreversibly reach their full malignant potential a much longer K-Ras expression is required,implicating mitochondria in the longer-term effects of the oncogene.

  19. Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

    Science.gov (United States)

    Baldwin, Kenneth M.; Haddad, Fadia

    2002-01-01

    The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

  20. Non-enzymatic Glycation of Almond Cystatin Leads to Conformational Changes and Altered Activity.

    Science.gov (United States)

    Siddiqui, Azad A; Sohail, Aamir; Bhat, Sheraz A; Rehman, Md T; Bano, Bilqees

    2015-01-01

    The non-enzymatic reaction between proteins and reducing sugars, known as glycation, leads to the formation of inter and intramolecular cross-links of proteins. Stable end products called as advanced Maillard products or advanced glycation end products (AGEs) have received tremendous attention since last decades. It was suggested that the formation of AGEs not only modify the conformation of proteins but also induces altered biological activity. In this study, cystatin purified from almond was incubated with three different sugars namely D-ribose, fructose and lactose to monitor the glycation process. Structural changes induced in cystatin on glycation were studied using UV-visible spectroscopy, fluorescence spectroscopy, CD and FTIR techniques. Glycated cystatin was found to migrate slower on electrophoresis as compared to control cystatin. Biological activity data of glycated cystatin showed that D-ribose was most effective in inducing conformational changes with maximum altered activity.

  1. Cortisol's effects on hippocampal activation in depressed patients are related to alterations in memory formation.

    Science.gov (United States)

    Abercrombie, Heather C; Jahn, Allison L; Davidson, Richard J; Kern, Simone; Kirschbaum, Clemens; Halverson, Jerry

    2011-01-01

    Many investigators have hypothesized that brain response to cortisol is altered in depression. However, neural activation in response to exogenously manipulated cortisol elevations has not yet been directly examined in depressed humans. Animal research shows that glucocorticoids have robust effects on hippocampal function, and can either enhance or suppress neuroplastic events in the hippocampus depending on a number of factors. We hypothesized that depressed individuals would show 1) altered hippocampal response to exogenous administration of cortisol, and 2) altered effects of cortisol on learning. In a repeated-measures design, 19 unmedicated depressed and 41 healthy individuals completed two fMRI scans. Fifteen mg oral hydrocortisone (i.e., cortisol) or placebo (order randomized and double-blind) was administered 1 h prior to encoding of emotional and neutral words during fMRI scans. Data analysis examined the effects of cortisol administration on 1) brain activation during encoding, and 2) subsequent free recall for words. Cortisol affected subsequent recall performance in depressed but not healthy individuals. We found alterations in hippocampal response to cortisol in depressed women, but not in depressed men (who showed altered response to cortisol in other regions, including subgenual prefrontal cortex). In both depressed men and women, cortisol's effects on hippocampal function were positively correlated with its effects on recall performance assessed days later. Our data provide evidence that in depressed compared to healthy women, cortisol's effects on hippocampal function are altered. Our data also show that in both depressed men and women, cortisol's effects on emotional memory formation and hippocampal function are related.

  2. Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

    Institute of Scientific and Technical Information of China (English)

    Soon-Jeong JEONG; Su-Gwan KIM; Jiyun YOO; Mi-Young HAN; Joo-Cheol PARK; Heung-Joong KIM; Seong Soo KANG; Baik-Dong CHOI; Moon-Jin JEONG

    2006-01-01

    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells.Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins.The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ interacted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway,was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  3. Evidence for altered amygdala activation in schizophrenia in an adaptive emotion recognition task.

    Science.gov (United States)

    Mier, Daniela; Lis, Stefanie; Zygrodnik, Karina; Sauer, Carina; Ulferts, Jens; Gallhofer, Bernd; Kirsch, Peter

    2014-03-30

    Deficits in social cognition seem to present an intermediate phenotype for schizophrenia, and are known to be associated with an altered amygdala response to faces. However, current results are heterogeneous with respect to whether this altered amygdala response in schizophrenia is hypoactive or hyperactive in nature. The present study used functional magnetic resonance imaging to investigate emotion-specific amygdala activation in schizophrenia using a novel adaptive emotion recognition paradigm. Participants comprised 11 schizophrenia outpatients and 16 healthy controls who viewed face stimuli expressing emotions of anger, fear, happiness, and disgust, as well as neutral expressions. The adaptive emotion recognition approach allows the assessment of group differences in both emotion recognition performance and associated neuronal activity while also ensuring a comparable number of correctly recognized emotions between groups. Schizophrenia participants were slower and had a negative bias in emotion recognition. In addition, they showed reduced differential activation during recognition of emotional compared with neutral expressions. Correlation analyses revealed an association of a negative bias with amygdala activation for neutral facial expressions that was specific to the patient group. We replicated previous findings of affected emotion recognition in schizophrenia. Furthermore, we demonstrated that altered amygdala activation in the patient group was associated with the occurrence of a negative bias. These results provide further evidence for impaired social cognition in schizophrenia and point to a central role of the amygdala in negative misperceptions of facial stimuli in schizophrenia.

  4. Prenatal immune activation alters hippocampal place cell firing characteristics in adult animals.

    Science.gov (United States)

    Wolff, Amy R; Bilkey, David K

    2015-08-01

    Prenatal maternal immune activation (MIA) is a risk factor for several developmental neuropsychiatric disorders, including autism, bipolar disorder and schizophrenia. Adults with these disorders display alterations in memory function that may result from changes in the structure and function of the hippocampus. In the present study we use an animal model to investigate the effect that a transient prenatal maternal immune activation episode has on the spatially-modulated firing activity of hippocampal neurons in adult animals. MIA was induced in pregnant rat dams with a single injection of the synthetic cytokine inducer polyinosinic:polycytidylic acid (poly I:C) on gestational day 15. Control dams were given a saline equivalent. Firing activity and local field potentials (LFPs) were recorded from the CA1 region of the adult male offspring of these dams as they moved freely in an open arena. Most neurons displayed characteristic spatially-modulated 'place cell' firing activity and while there was no between-group difference in mean firing rate between groups, place cells had smaller place fields in MIA-exposed animals when compared to control-group cells. Cells recorded in MIA-group animals also displayed an altered firing-phase synchrony relationship to simultaneously recorded LFPs. When the floor of the arena was rotated, the place fields of MIA-group cells were more likely to shift in the same direction as the floor rotation, suggesting that local cues may have been more salient for these animals. In contrast, place fields in control group cells were more likely to shift firing position to novel spatial locations suggesting an altered response to contextual cues. These findings show that a single MIA intervention is sufficient to change several important characteristics of hippocampal place cell activity in adult offspring. These changes could contribute to the memory dysfunction that is associated with MIA, by altering the encoding of spatial context and by

  5. RAS in Pregnancy and Preeclampsia and Eclampsia

    Directory of Open Access Journals (Sweden)

    M. Rodriguez

    2012-01-01

    Full Text Available Preeclampsia is a common disease of pregnancy characterized by the presence of hypertension and commitment of many organs, including the brain, secondary to generalized endothelial dysfunction. Its etiology is not known precisely, but it involved several factors, highlighting the renin angiotensin system (RAS, which would have an important role in the origin of multisystem involvement. This paper reviews the evidence supporting the involvement of RAS in triggering the disease, in addition to the components of this system that would be involved and how it eventually produces brain engagement.

  6. Byzantine seals from the Ras fortress

    Directory of Open Access Journals (Sweden)

    Ivanišević Vujadin

    2013-01-01

    Full Text Available In this paper, seals found at the location of the Ras fortress (Tvrđava Ras have been published. Inscriptions on these seals show that they used to belong to persons which could be identified with certain military commanders who served under Alexios I Komnenos. The seals in question are: the seals of protonobelissimos Eustathios Kamytzes, Constantine Dalassenos Doukas, protoproedros and doux Constantine Kekaumenos and a certain person called Alexios. [Projekat Ministarstva nauke Republike Srbije, br. 177021 i br. 177032

  7. Experimenting with Request Assignment Simulator (RAS

    Directory of Open Access Journals (Sweden)

    R. Arokia Paul Rajan

    2014-11-01

    Full Text Available There is no existence of dedicated simulators on the Internet that studies the impact of load balancing principles of the cloud architectures. Request Assignment Simulator (RAS is a customizable, visual tool that helps to understand the request assignment to the resources based on the load balancing principles. We have designed this simulator to fit into Infrastructure as a Service (IaaS cloud model. In this paper, we present a working manual useful for the conduct of experiment with RAS. The objective of this paper is to instill the user to understand the pertinent parameters in the cloud, their metrics, load balancing principles, and their impact on the performance.

  8. Society News: Monica Grady awarded CBE; Grubb Parsons Lecture 2012; Join the RAS; Astronomy on radio for kids; New Fellows; Peter D Hingley

    Science.gov (United States)

    2012-08-01

    RAS Fellow Prof. Monica Grady has been made a Commander of the Most Excellent Order of the British Empire (CBE), in recognition of her services to space science. The RAS sponsors the annual Grubb Parsons Lecture, which this year took place on 6 June at the University of Durham. If you are a professional astronomer, geophysicist, or similar, a student studying these disciplines, or simply someone with a serious interest in them, we urge you to apply for membership of the RAS. Outreach is an important activity for the RAS. We recently supported an astronomy series called Deep Space High on the digital radio channel Fun Kids.

  9. The RAS Problem: Turning Off a Broken Switch

    Science.gov (United States)

    The RAS gene is commonly mutated in cancer and researchers are working to better understand how to develop drugs that can target the RAS protein, which for many years has been considered to be “undruggable.”

  10. RAS Projects at the NCI Frederick National Lab

    Science.gov (United States)

    The RAS Initiative involves a number of projects focusing on ways to inhibit the proliferation of cancer cells by mutant RAS proteins. Projects are conducted at the FNLCR hub, with collaboration from the research community nationwide.

  11. Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways.

    Science.gov (United States)

    Zhang, Jing; Liu, Yangang; Beard, Caroline; Tuveson, David A; Jaenisch, Rudolf; Jacks, Tyler E; Lodish, Harvey F

    2007-06-15

    When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.

  12. CD8+ T cells Mediate RAS-induced Psoriasis-like Skin Inflammation Through IFN-γ

    OpenAIRE

    Gunderson, Andrew J.; Mohammed, Javed; Horvath, Frank; Podolsky, Michael, J.; Anderson, Cherie; Glick, Adam B.

    2012-01-01

    The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RASV12G in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses and increased Th1/Th17 and Tc1/Tc17 skin infiltration. The majority of skin infiltrating CD8+ T cells co-expressed IFN-γ and IL-17A. When RAS was exp...

  13. The ras1 protein of S. pombe mediates pheromone-induced transcription. Abstract

    DEFF Research Database (Denmark)

    Nielsen, Olaf; Davey, John; Egel, Richard

    1991-01-01

    Differentiering, signaltransduktion, parringstype feromon, Schizosaccharomyces pombe, ras homolog, Transkription......Differentiering, signaltransduktion, parringstype feromon, Schizosaccharomyces pombe, ras homolog, Transkription...

  14. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents.

    Directory of Open Access Journals (Sweden)

    Melville B Vaughan

    Full Text Available BACKGROUND: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent. METHODOLOGY/PRINCIPAL FINDINGS: Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras, were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63, basement-membrane formation (collagen IV, laminin-5, and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes. CONCLUSIONS/SIGNIFICANCE: The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.

  15. Alteration and modulation of protein activity by varying post-translational modification

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N; Reed, David W; Thompson, Vicki S; Lacey, Jeffrey A; Apel, William A

    2015-03-03

    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

  16. Alteration and modulation of protein activity by varying post-translational modification

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David N.; Reed, David W.; Thompson, Vicki S.; Lacey, Jeffrey A.; Apel, William A.

    2016-07-12

    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

  17. Fecal Protease Activity Is Associated with Compositional Alterations in the Intestinal Microbiota

    OpenAIRE

    Carroll, Ian M.; Ringel-Kulka, Tamar; Ferrier, Laurent; Wu, Michael C.; Siddle, Jennica P.; Bueno, Lionel; Ringel, Yehuda

    2013-01-01

    Objective: Intestinal proteases carry out a variety of functions in the gastrointestinal (GI) tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial) of elevated proteases in patients with GI disease is unclear. Aim: The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples. Design: In order to capture a wide range of fec...

  18. Altering the activation mechanism in Thermomyces lanuginosus lipase

    DEFF Research Database (Denmark)

    Skjold-Jørgensen, Jakob; Vind, Jesper; Svendsen, Allan;

    2014-01-01

    , and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region...... solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities....

  19. Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Katherine K Slemmons

    Full Text Available Rhabdomyosarcoma (RMS, a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS and pleomorphic (pRMS histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways.

  20. Status of neutron complex of INR RAS

    Energy Technology Data Exchange (ETDEWEB)

    Grachev, M.I.; Koptelov, E.A.; Kravchuk, L.V.; Matveev, V.A.; Perekrestenko, A.D.; Sidorkin, S.F. [Institute for Nuclear Research of Russian Academy of Sciences, Prospekt, Moscow (Russian Federation); Stavissky, Y.Y.

    2001-03-01

    The neutron complex of INR RAS consists of two sources of neutrons, beam stop, lead slowing down spectrometer and solid state spectrometers. The description of objects and their condition, the program of planned researches, co-operation with other institutes of the Moscow Region, progress reached for last two years are introduced in the article. (author)

  1. Status of neutron complex of INR RAS

    International Nuclear Information System (INIS)

    The neutron complex of INR RAS consists of two sources of neutrons, beam stop, lead slowing down spectrometer and solid state spectrometers. The description of objects and their condition, the program of planned researches, co-operation with other institutes of the Moscow Region, progress reached for last two years are introduced in the article. (author)

  2. Ras-Related Tumorigenesis Is Suppressed by BNIP3-Mediated Autophagy through Inhibition of Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Shan-Ying Wu

    2011-12-01

    Full Text Available Autophagy plays diverse roles in Ras-related tumorigenesis. H-rasval12 induces autophagy through multiple signaling pathways including Raf-1/ERK pathway, and various ERK downstream molecules of autophagy have been reported. In this study, Bcl-2/adenovirus E1B 19-kDa–interacting protein 3 (BNIP3 is identified as a downstream transducer of the Ras/Raf/ERK signaling pathway to induce autophagy. BNIP3 was upregulated by H-rasval12 at the transcriptional level to compete with Beclin 1 for binding with Bcl-2. H-rasval12–induced autophagy suppresses cell proliferation demonstrated both in vitro and in vivo by expression of ectopic BNIP3, Atg5, or interference RNA of BNIP3 (siBNIP3 and Atg5 (shAtg5 using mouse NIH3T3 and embryo fibroblast cells. H-rasval12 induces different autophagic responses depending on the duration of Ras overexpression. After a short time (48 hours of Ras overexpression, autophagy inhibits cell proliferation. In contrast, a longer time (2 weeks of Ras overexpression, cell proliferation was enhanced by autophagy. Furthermore, overexpression of mutant Ras, BNIP3, and LC3-II was detected in bladder cancer T24 cells and the tumor parts of 75% of bladder cancer specimens indicating a positive correlation between autophagy and tumorigenesis. Taken together, our mouse model demonstrates a balance between BNIP3-mediated autophagy and H-rasval12–induced tumor formation and reveals that H-rasval12 induces autophagy in a BNIP3-dependent manner, and the threshold of autophagy plays a decisive role in H-rasval12–induced tumorigenesis. Our findings combined with others’ reports suggest a new therapeutic strategy against Ras-related tumorigenesis by negative or positive regulation of autophagic activity, which is determined by the level of autophagy and tumor progression stages.

  3. Ras Al Khaimah (RAK): World Oil Report 1991

    Energy Technology Data Exchange (ETDEWEB)

    1991-08-01

    This paper reports on Ras Al Khalmah (RAK) that had no drilling activity in 1990. In 1991, International Petroleum Ltd., (IPL), which holds a 1,100 sq mi concession onshore, may spud their West Jiri prospect. Amoco holds an offshore concession and was to have completed seismic last year. the RAK Gas Commission was reported to be operating the offshore Saleh gas field in 1990, which a former foreign concessionaire relinquished in mid-1989 since it had reached its economic limit. Production from Saleh last year is estimated at 1,000 bcpd and about 9 MMcfgd.

  4. Alterations in Neuronal Activity in Basal Ganglia-Thalamocortical Circuits in the Parkinsonian State

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    Adriana eGalvan

    2015-02-01

    Full Text Available In patients with Parkinson’s disease and in animal models of this disorder, neurons in the basal ganglia and related regions in thalamus and cortex show changes that can be recorded by using electrophysiologic single-cell recording techniques, including altered firing rates and patterns, pathologic oscillatory activity and increased inter-neuronal synchronization. In addition, changes in synaptic potentials or in the joint spiking activities of populations of neurons can be monitored as alterations in local field potentials, electroencephalograms or electrocorticograms. Most of the mentioned electrophysiologic changes are probably related to the degeneration of diencephalic dopaminergic neurons, leading to dopamine loss in the striatum and other basal ganglia nuclei, although degeneration of non-dopaminergic cell groups may also have a role. The altered electrical activity of the basal ganglia and associated nuclei may contribute to some of the motor signs of the disease. We here review the current knowledge of the electrophysiologic changes at the single cell level, the level of local populations of neural elements, and the level of the entire basal ganglia-thalamocortical network in parkinsonism, and discuss the possible use of this information to optimize treatment approaches to Parkinson’s disease, such as deep brain stimulation therapy.

  5. Utilizing ras signaling pathway to direct selective replication of herpes simplex virus-1.

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    Weihong Pan

    Full Text Available Re-engineering the tropism of viruses is an attractive translational strategy for targeting cancer cells. The Ras signal transduction pathway is a central hub for a variety of pro-oncogenic events with a fundamental role in normal and neoplastic physiology. In this work we were interested in linking Ras activation to HSV-1 replication in a direct manner in order to generate a novel oncolytic herpes virus which can target cancer cells. To establish such link, we developed a mutant HSV-1 in which the expression of ICP4 (infected cell protein-4, a viral protein necessary for replication is controlled by activation of ELK, a transcription factor down-stream of the Ras pathway and mainly activated by ERK (extracellular signal-regulated kinase, an important Ras effector pathway. This mutant HSV-1 was named as Signal-Smart 1 (SS1. A series of prostate cells were infected with the SS1 virus. Cells with elevated levels of ELK activation were preferentially infected by the SS1 virus, as demonstrated by increased levels of viral progeny, herpetic glycoprotein C and overall SS1 viral protein production. Upon exposure to SS1, the proliferation, invasiveness and colony formation capabilities of prostate cancer cells with increased ELK activation were significantly decreased (p<0.05, while the rate of apoptosis/necrosis in these cells was increased. Additionally, high Ras signaling cells infected with SS1 showed a prominent arrest in the G1 phase of the cell cycle as compared to cells exposed to parental HSV-1. The results of this study reveal the potential for re-modeling the host-herpes interaction to specifically interfere with the life of cancer cells with increased Ras signaling. SS1 also serves as a "prototype" for development of a family of signal-smart viruses which can target cancer cells on the basis of their signaling portfolio.

  6. Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Hoon; Chennakrishnaiah, Shilpa [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Audemard, Eric [McGill University and Genome Quebec Innovation Centre, Montreal, Quebec (Canada); Montermini, Laura; Meehan, Brian [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Rak, Janusz, E-mail: janusz.rak@mcgill.ca [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

    2014-08-22

    Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellular vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.

  7. Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate.

    Science.gov (United States)

    Sayyed, Katia; Vee, Marc Le; Abdel-Razzak, Ziad; Jouan, Elodie; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-07-01

    Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRG cells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking. PMID:27450509

  8. Design, Evaluation and GCM-Performance of a New Parameterization for Microphysics of Clouds with Relaxed Arakawa-Schubert Scheme (McRas)

    Science.gov (United States)

    Sud, Y. C.; Walker, G. K.

    1998-01-01

    A prognostic cloud scheme named McRAS (Microphysics of clouds with Relaxed Arakawa-Schubert Scheme) was developed with the aim of improving cloud-microphysics, and cloud-radiation interactions in GCMs. McRAS distinguishes convective, stratiform, and boundary-layer clouds. The convective clouds merge into stratiform clouds on an hourly time-scale, while the boundary-layer clouds do so instantly. The cloud condensate transforms into precipitation following the auto-conversion relations of Sundqvist that contain a parametric adaptation for the Bergeron-Findeisen process of ice crystal growth and collection of cloud condensate by precipitation. All clouds convect, advect, and diffuse both horizontally and vertically with a fully active cloud-microphysics throughout its life-cycle, while the optical properties of clouds are derived from the statistical distribution of hydrometeors and idealized cloud geometry. An evaluation of McRAS in a single column model (SCM) with the GATE Phase III data has shown that McRAS can simulate the observed temperature, humidity, and precipitation without discernible systematic errors. An evaluation with the ARM-CART SCM data in a cloud model intercomparison exercise shows reasonable but not an outstanding accurate simulation. Such a discrepancy is common to almost all models and is related, in part, to the input data quality. McRAS was implemented in the GEOS II GCM. A 50 month integration that was initialized with the ECMWF analysis of observations for January 1, 1987 and forced with the observed sea-surface temperatures and sea-ice distribution and vegetation properties (biomes, and soils), with prognostic soil moisture, snow-cover, and hydrology showed a very realistic simulation of cloud process, incloud water and ice, and cloud-radiative forcing (CRF). The simulated ITCZ showed a realistic time-mean structure and seasonal cycle, while the simulated CRF showed sensitivity to vertical distribution of cloud water which can be easily

  9. Involvement of MINK, a Ste20 Family Kinase, in Ras Oncogene-Induced Growth Arrest in Human Ovarian Surface Epithelial Cells

    NARCIS (Netherlands)

    Nicke, B.; Bastien, J.; Khanna, S.J.; Warne, P.H.; Cowling, V.; Cook, S.J.; Peters, G.; Delpuech, O.; Schulze, A.; Berns, K.; Mullenders, J.; Beijersbergen, R.L.; Bernards, R.A.; Ganesan, T.S.; Downward, J.; Hancock, D.C.

    2005-01-01

    The ability of activated Ras to induce growth arrest of human ovarian surface epithelial (HOSE) cells via induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 has been used to screen for Ras pathway signaling components using a library of RNA interference (RNAi) vectors targeting the kino

  10. RasGrf1 deficiency delays aging in mice

    OpenAIRE

    Borrás, Consuelo; Monleón, Daniel; López-Grueso, Raul; Gambini, Juan; Orlando, Leonardo; Federico V Pallardó; Santos, Eugenio; Viña, José; Font de Mora, Jaime

    2011-01-01

    RasGRF1 is a Ras-guanine nucleotide exchange factor implicated in a variety of physiological processes including learning and memory and glucose homeostasis. To determine the role of RASGRF1 in aging, lifespan and metabolic parameters were analyzed in aged RasGrf1−/− mice. We observed that mice deficient for RasGrf1−/− display an increase in average and most importantly, in maximal lifespan (20% higher than controls). This was not due to the role of Ras in cancer because tumor-free survival w...

  11. The impacts of altered tropical cyclone activity on climate mitigation strategies

    Science.gov (United States)

    Fisk, J. P.; Hurtt, G. C.; LePage, Y.; Patel, P.; Chini, L. P.; Thomson, A. M.; Clarke, L.; Calvin, K. V.; Wise, M.; Chambers, J. Q.; Negron Juarez, R. I.

    2012-12-01

    There is growing evidence that anthropogenic climate change may alter patterns of tropical cyclone frequency, intensity and spatial distribution, which in turn will alter the carbon balance of terrestrial systems in the large regions impacted by these storms. Recent studies project up to a doubling of major storms (Saffir-Simpson Scale 3-5) over the next century. Single large storms have been shown to be capable of causing committed carbon emissions equivalent to the annual U.S. carbon sink. These changes have the potential to affect climate mitigation strategies, most of which rely on maintaining or enhancing the terrestrial carbon sink to restrain the accumulation of atmospheric greenhouse gases. Altered patterns of disturbances and the resulting changes to the carbon balance of terrestrial systems could impact the magnitude of emissions to mitigate, the economic value of ecosystem carbon storage, and thus future land-use patterns, food prices and energy technology. Here we investigate the potential consequences of altered tropical cyclone activity on climate mitigation strategies using a fully integrated model (iED) that links advanced ecological and socio-economic models. The model combines the regional integrated assessment algorithms of the Global Change Assessment Model (GCAM), with the climate- sensitive ecosystem and carbon modeling in the Ecosystem Demography (ED) model, and the land-use mapping algorithms of the Global Land-use Model (GLM). We explore a range of scenarios of altered future tropical cyclone frequency, intensity and spatial pattern, the resulting effects on the terrestrial carbon balance, and the coupled effects on the food and energy sector under a range of future climate mitigation goals.

  12. Repeated Episodes of Heroin Cause Enduring Alterations of Circadian Activity in Protracted Abstinence

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    Luis Stinus

    2012-09-01

    Full Text Available Opiate withdrawal is followed by a protracted abstinence syndrome consisting of craving and physiological changes. However, few studies have been dedicated to both the characterization and understanding of these long-term alterations in post-dependent subjects. The aim of the present study was to develop an opiate dependence model, which induces long-lasting behavioral changes in abstinent rats. Here, we first compared the effects of several protocols for the induction of opiate dependence (morphine pellets, repeated morphine or heroin injections on the subsequent response to heroin challenges (0.25 mg/kg at different time points during abstinence (3, 6, 9 and 18 weeks. In a second set of experiments, rats were exposed to increasing doses of heroin and subsequently monitored for general circadian activity up to 20 weeks of abstinence. Results show that heroin injections rather than the other methods of opiate administration have long-term consequences on rats’ sensitivity to heroin with its psychostimulant effects persisting up to 18 weeks of abstinence. Moreover, intermittent episodes of heroin dependence rather than a single exposure produce enduring alteration of the basal circadian activity both upon heroin cessation and protracted abstinence. Altogether, these findings suggest that the induction of heroin dependence through intermittent increasing heroin injections is the optimal method to model long-term behavioral alterations during protracted abstinence in rats. This animal model would be useful in further characterizing long-lasting changes in post-dependent subjects to help understand the prolonged vulnerability to relapse.

  13. Repeated episodes of heroin cause enduring alterations of circadian activity in protracted abstinence.

    Science.gov (United States)

    Stinus, Luis; Cador, Martine; Caille, Stephanie

    2012-01-01

    Opiate withdrawal is followed by a protracted abstinence syndrome consisting of craving and physiological changes. However, few studies have been dedicated to both the characterization and understanding of these long-term alterations in post-dependent subjects. The aim of the present study was to develop an opiate dependence model, which induces long-lasting behavioral changes in abstinent rats. Here, we first compared the effects of several protocols for the induction of opiate dependence (morphine pellets, repeated morphine or heroin injections) on the subsequent response to heroin challenges (0.25 mg/kg) at different time points during abstinence (3, 6, 9 and 18 weeks). In a second set of experiments, rats were exposed to increasing doses of heroin and subsequently monitored for general circadian activity up to 20 weeks of abstinence. Results show that heroin injections rather than the other methods of opiate administration have long-term consequences on rats' sensitivity to heroin with its psychostimulant effects persisting up to 18 weeks of abstinence. Moreover, intermittent episodes of heroin dependence rather than a single exposure produce enduring alteration of the basal circadian activity both upon heroin cessation and protracted abstinence. Altogether, these findings suggest that the induction of heroin dependence through intermittent increasing heroin injections is the optimal method to model long-term behavioral alterations during protracted abstinence in rats. This animal model would be useful in further characterizing long-lasting changes in post-dependent subjects to help understand the prolonged vulnerability to relapse. PMID:24961201

  14. Complete denture base assessments using holograms: dimensional alterations after different activation methods

    Science.gov (United States)

    Dughir, Ciprian; Popovschi, Ana Maria; Cojocariu, Andreea Codruta; Topala, Florin Ionel; Negrutiu, Meda Lavinia; Sinescu, Cosmin; de Sabata, Aldo; Duma, Virgil-Florin

    2016-03-01

    Holography is a well-developed method with a large range of applications, including dentistry. This study uses holographic methods for the study of total dental prosthesis. The issue is that the transformation of wax denture base in polymethylacrylate causes dimensional alterations and retractions in the final dental constructs. These could cause the failure of the stability of the complete denture in the oral cavity. Thus, the aim of this study is to determine and to compare using holography, total prosthesis obtained using three different manufacturing methods: pressing, injection, and polymerization. Each of the three types of dentures thus produced were recorded over the previously wax complete base holographic plates. The dimensional alterations that appear after using the different activation methods were thus determined. The most significant modification was remarked in the custom press technology, while the smallest variations were detected in the injection alternative.

  15. Alteration of membrane phospholipid methylation by adenosine analogs does not affect T lymphocyte activation

    International Nuclear Information System (INIS)

    Membrane phospholipid methylation has been described during activation of various immune cells. Moreover recent data indicated modulation of immune cells functions by adenosine. As S-adenosyl-methionine and S-adenosyl-homocysteine are adenosine analogs and modulators of transmethylation reactions, the effects of SAH and SAM were investigated on membrane phospholipid methylation and lymphocyte activation. SAM was shown to induce the membrane phospholipid methylation as assessed by the 3Hmethyl-incorporation in membrane extract. This effect was inhibited by SAH. In contrast SAM and SAH did not affect the phytohemagglutinin-induced proliferative response of peripheral blood mononuclear cells. SAH neither modified the early internalization of membrane CD3 antigens nor did it prevent the late expression of HLA-DR antigens on lymphocytes activated by phytohemagglutinin. These results indicate that in vitro alteration of phospholipid methylation does not affect subsequent steps of human T lymphocyte activation and proliferation

  16. EGFR-Ras-Raf Signaling in Epidermal Stem Cells: Roles in Hair Follicle Development, Regeneration, Tissue Remodeling and Epidermal Cancers

    OpenAIRE

    Manuela Baccarini; Christian Rupp; Eszter Doma

    2013-01-01

    The mammalian skin is the largest organ of the body and its outermost layer, the epidermis, undergoes dynamic lifetime renewal through the activity of somatic stem cell populations. The EGFR-Ras-Raf pathway has a well-described role in skin development and tumor formation. While research mainly focuses on its role in cutaneous tumor initiation and maintenance, much less is known about Ras signaling in the epidermal stem cells, which are the main targets of skin carcinogenesis. In this review,...

  17. Systems-wide Analysis of K-Ras, Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics*

    OpenAIRE

    Gnad, Florian; Young, Amy; Zhou, Wei; Lyle, Karen; Ong, Christy C.; Matthew P. Stokes; Silva, Jeffrey C.; Belvin, Marcia; Friedman, Lori S.; Koeppen, Hartmut; Minden, Audrey; Hoeflich, Klaus P.

    2013-01-01

    Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphop...

  18. A Drosophila immune response against Ras-induced overgrowth

    Directory of Open Access Journals (Sweden)

    Thomas Hauling

    2014-03-01

    Full Text Available Our goal is to characterize the innate immune response against the early stage of tumor development. For this, animal models where genetic changes in specific cells and tissues can be performed in a controlled way have become increasingly important, including the fruitfly Drosophila melanogaster. Many tumor mutants in Drosophila affect the germline and, as a consequence, also the immune system itself, making it difficult to ascribe their phenotype to a specific tissue. Only during the past decade, mutations have been induced systematically in somatic cells to study the control of tumorous growth by neighboring cells and by immune cells. Here we show that upon ectopic expression of a dominant-active form of the Ras oncogene (RasV12, both imaginal discs and salivary glands are affected. Particularly, the glands increase in size, express metalloproteinases and display apoptotic markers. This leads to a strong cellular response, which has many hallmarks of the granuloma-like encapsulation reaction, usually mounted by the insect against larger foreign objects. RNA sequencing of the fat body reveals a characteristic humoral immune response. In addition we also identify genes that are specifically induced upon expression of RasV12. As a proof-of-principle, we show that one of the induced genes (santa-maria, which encodes a scavenger receptor, modulates damage to the salivary glands. The list of genes we have identified provides a rich source for further functional characterization. Our hope is that this will lead to a better understanding of the earliest stage of innate immune responses against tumors with implications for mammalian immunity.

  19. Altered cognition-related brain activity and interactions with acute pain in migraine

    Directory of Open Access Journals (Sweden)

    Vani A. Mathur

    2015-01-01

    Full Text Available Little is known about the effect of migraine on neural cognitive networks. However, cognitive dysfunction is increasingly being recognized as a comorbidity of chronic pain. Pain appears to affect cognitive ability and the function of cognitive networks over time, and decrements in cognitive function can exacerbate affective and sensory components of pain. We investigated differences in cognitive processing and pain–cognition interactions between 14 migraine patients and 14 matched healthy controls using an fMRI block-design with two levels of task difficulty and concurrent heat (painful and not painful stimuli. Across groups, cognitive networks were recruited in response to a difficult cognitive task, and a pain–task interaction was found in the right (contralateral to pain stimulus posterior insula (pINS, such that activity was modulated by decreasing the thermal pain stimulus or by engaging the difficult cognitive task. Migraine patients had less task-related deactivation within the left dorsolateral prefrontal cortex (DLPFC and left dorsal anterior midcingulate cortex (aMCC compared to controls. These regions have been reported to have decreased cortical thickness and cognitive-related deactivation within other pain populations, and are also associated with pain regulation, suggesting that the current findings may reflect altered cognitive function and top-down regulation of pain. During pain conditions, patients had decreased task-related activity, but more widespread task-related reductions in pain-related activity, compared to controls, suggesting cognitive resources may be diverted from task-related to pain-reduction-related processes in migraine. Overall, these findings suggest that migraine is associated with altered cognitive-related neural activity, which may reflect altered pain regulatory processes as well as broader functional restructuring.

  20. Altered muscular activation during prone hip extension in women with and without low back pain

    Directory of Open Access Journals (Sweden)

    Arab Amir M

    2011-08-01

    Full Text Available Abstract Background Altered movement pattern has been associated with the development of low back pain (LBP. The purpose of this study was to investigate the activity pattern of the ipsilateral erector spinae (IES and contralateral erectorspinae (CES, gluteus maximus (GM and hamstring (HAM muscles during prone hip extension (PHE test in women with and without LBP. A cross-sectional non-experimental design was used. Methods Convenience sample of 20 female participated in the study. Subjects were categorized into two groups: with LBP (n = 10 and without LBP (n = 10. The electromyography (EMG signal amplitude of the tested muscles during PHE (normalized to maximum voluntary electrical activity (MVE was measured in the dominant lower extremity in all subjects. Results Statistical analysis revealed greater normalized EMG signal amplitude in women with LBP compared to non-LBP women. There was significant difference in EMG activity of the IES (P = 0.03 and CES (P = 0.03 between two groups. However, no significant difference was found in EMG signals of the GM (P = 0.11 and HAM (P = 0.14 among two groups. Conclusion The findings of this study demonstrated altered activation pattern of the lumbo-pelvic muscles during PHE in the women with chronic LBP. This information is important for investigators using PHE as either an evaluation tool or a rehabilitation exercise.

  1. Impact of antibiotic treatments on the expression of the R plasmid tra genes and on the host innate immune activity during pRAS1 bearing Aeromonas hydrophila infection in zebrafish (Danio rerio

    Directory of Open Access Journals (Sweden)

    Cantas Leon

    2012-03-01

    Full Text Available Abstract Background The transfer of R plasmids between bacteria has been well studied under laboratory conditions and the transfer frequency has been found to vary between plasmids and under various physical conditions. For the first time, we here study the expression of the selected plasmid mobility genes traD, virB11 and virD4 in the 45 kb IncU plasmid, pRAS1, conferring resistance to tetracycline, trimethoprim and sulphonamide, using an in vivo zebrafish infection- treatment model. Results Three days after oral infection of adult zebrafish with Aeromonas hydrophila harboring pRAS1, elevated expression of pro-inflammatory cytokine (TNF α, IL-1β and IL-8 and complement C3 genes in the intestine coincided with disease symptoms. Tetracycline, trimethoprim and an ineffective concentration of flumequine given 48 h prior to sampling, strongly increased expression of plasmid mobility genes, whereas an effective dosage of flumequine resulted in lower levels of mRNA copies of these genes relative to placebo treatment. Following effective treatment with flumequine, and ineffective treatments with a low concentration of flumequine, with trimethoprim or with sulphonamide, the intestinal expression of immune genes was strongly induced compared to placebo treated control fish. Conclusions Treatment of zebrafish infected with an antibiotic resistant (TcR, TmR, SuR A. hydrophila with ineffective concentrations of flumequine or the ineffective antimicrobials tetracycline and trimethoprim strongly induced expression of genes mediating conjugative transfer of the R-plasmid pRAS1. Simultaneously, there was a strong induction of selected inflammatory and immune response genes, which was again evident in fish subjected to ineffective treatment protocols. Our findings point to the essential role of therapeutic practices in escalation or control of antibiotic resistance transfer, and suggest that antibiotic substances, even in sub-inhibitory concentrations, may

  2. Ets Factors Regulate Neural Stem Cell Depletion and Gliogenesis in Ras Pathway Glioma.

    Science.gov (United States)

    Breunig, Joshua J; Levy, Rachelle; Antonuk, C Danielle; Molina, Jessica; Dutra-Clarke, Marina; Park, Hannah; Akhtar, Aslam Abbasi; Kim, Gi Bum; Hu, Xin; Bannykh, Serguei I; Verhaak, Roel G W; Danielpour, Moise

    2015-07-14

    As the list of putative driver mutations in glioma grows, we are just beginning to elucidate the effects of dysregulated developmental signaling pathways on the transformation of neural cells. We have employed a postnatal, mosaic, autochthonous glioma model that captures the first hours and days of gliomagenesis in more resolution than conventional genetically engineered mouse models of cancer. We provide evidence that disruption of the Nf1-Ras pathway in the ventricular zone at multiple signaling nodes uniformly results in rapid neural stem cell depletion, progenitor hyperproliferation, and gliogenic lineage restriction. Abolishing Ets subfamily activity, which is upregulated downstream of Ras, rescues these phenotypes and blocks glioma initiation. Thus, the Nf1-Ras-Ets axis might be one of the select molecular pathways that are perturbed for initiation and maintenance in glioma.

  3. Bioinformatics of non small cell lung cancer and the ras proto-oncogene

    CERN Document Server

    Kashyap, Amita; Babu M, Naresh

    2015-01-01

    Cancer is initiated by activation of oncogenes or inactivation of tumor suppressor genes. Mutations in the K-ras proto-oncogene are responsible for 10–30% of adenocarcinomas. Clinical Findings point to a wide variety of other cancers contributing to lung cancer incidence. Such a scenario makes identification of lung cancer difficult and thus identifying its mechanisms can contribute to the society. Identifying unique conserved patterns common to contributing proto-oncogenes may further be a boon to Pharmacogenomics and pharmacoinformatics. This calls for ab initio/de novo drug discovery that in turn will require a comprehensive in silico approach of Sequence, Domain, Phylogenetic and Structural analysis of the receptors, ligand screening and optimization and detailed Docking studies. This brief involves extensive role of the RAS subfamily that includes a set of proteins, which cause an over expression of cancer-causing genes like M-ras and initiate tumour formation in lungs. SNP Studies and Structure based ...

  4. Intrarenal alterations of the angiotensin-converting enzyme type 2/angiotensin 1-7 complex of the renin-angiotensin system do not alter the course of malignant hypertension in Cyp1a1-Ren-2 transgenic rats.

    Science.gov (United States)

    Husková, Zuzana; Kopkan, Libor; Červenková, Lenka; Doleželová, Šárka; Vaňourková, Zdeňka; Škaroupková, Petra; Nishiyama, Akira; Kompanowska-Jezierska, Elzbieta; Sadowski, Janusz; Kramer, Herbert J; Červenka, Luděk

    2016-04-01

    The role of the intrarenal renin-angiotensin system (RAS) in the pathophysiology of malignant hypertension is not fully understood. Accumulating evidence indicates that the recently discovered vasodilator axis of the RAS, angiotensin-converting enzyme (ACE) type 2 (ACE2)/angiotensin 1-7 (ANG 1-7), constitutes an endogenous system counterbalancing the hypertensiogenic axis, ACE/angiotensin II (ANG II)/AT1 receptor. This study aimed to evaluate the role of the intrarenal vasodilator RAS axis in the pathophysiology of ANG II-dependent malignant hypertension in Cyp1a1-Ren-2 transgenic rats. ANG II-dependent malignant hypertension was induced by 13 days' dietary administration of indole-3-carbinol (I3C), a natural xenobiotic that activates the mouse renin gene in Cyp1a1-Ren-2 transgenic rats. It was hypothesized that pharmacologically-induced inhibition of the ACE2/ANG 1-7 complex should aggravate, and activation of this axis should attenuate, the course of ANG II-dependent malignant hypertension. Blood pressure (BP) was monitored by radiotelemetry. ACE2 inhibitor (DX 600, 0.2 μg/day) and ACE2 activator (DIZE, 1 mg/day) were administrated via osmotic minipumps. Even though ACE2 inhibitor significantly decreased and ACE2 activator increased intrarenal ANG 1-7 concentrations, the course of BP, as well as of albuminuria, cardiac hypertrophy and renal glomerular damage, were not altered. It was shown that intrarenal alterations in the ACE2/ANG 1-7 complex did not significantly modify the course of malignant hypertension in I3C-induced Cyp1a1-Ren-2 transgenic rats. Thus, in our experimental setting alterations of this intrarenal vasodilator complex of the RAS do not significantly modify the form of malignant hypertension that clearly depends on the inappropriately increased activity of the ACE/ANG II/AT1 receptor axis.

  5. Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64.

    Directory of Open Access Journals (Sweden)

    Taylor C Strong

    Full Text Available The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.

  6. Changes in stomatal function and water use efficiency in potato plants with altered sucrolytic activity.

    Science.gov (United States)

    Antunes, Werner C; Provart, Nicholas J; Williams, Thomas C R; Loureiro, Marcelo E

    2012-04-01

    As water availability for agriculture decreases, breeding or engineering of crops with improved water use efficiency (WUE) will be necessary. As stomata are responsible for controlling gas exchange across the plant epidermis, metabolic processes influencing solute accumulation in guard cells are potential targets for engineering. In addition to its role as an osmoticum, sucrose breakdown may be required for synthesis of other osmotica or generation of the ATP needed for solute uptake. Thus, alterations in partitioning of sucrose between storage and breakdown may affect stomatal function. In agreement with this hypothesis, potato (Solanum tuberosum) plants expressing an antisense construct targeted against sucrose synthase 3 (SuSy3) exhibited decreased stomatal conductance, a slight reduction in CO(2) fixation and increased WUE. Conversely, plants with increased guard cell acid invertase activity caused by the introduction of the SUC2 gene from yeast had increased stomatal conductance, increased CO(2) fixation and decreased WUE. (14)CO(2) feeding experiments indicated that these effects cannot be attributed to alterations in photosynthetic capacity, and most likely reflect alterations in stomatal function. These results highlight the important role that sucrose breakdown may play in guard cell function and indicate the feasibility of manipulating plant WUE through engineering of guard cell sucrose metabolism.

  7. Chemotaxis: new role for Ras revealed

    Institute of Scientific and Technical Information of China (English)

    Jianshe Yan; Dale Hereld; Tian Jin

    2010-01-01

    @@ A recent study of chemotaxis revealed a new role for the proto-oncogene Ras in the social ameba Dictyostelium discoideum.Chemotaxis,the directional movement of cells toward chemokines and other chemoattractants,plays critical roles in diverse physiological processes,such as mobilization of immune cells to fight invading microorganisms,targeting of metastatic cancer cells to specific tissues,and guidance of sperm cells to ova during fertilization.This work,published in the July 26 issue of The Journal of Cell Biology,was conducted in Dr.Devreotes' lab at John Hopkins University and Dr.Parent's lab at National Cancer Institute.This research team demonstrated that RasC functions as an upstream regulator of TORC2 and thereby governs the effects of TORC2-PKB signaling on the cytoskeleton and cell migration.

  8. Plant adaptation to extreme environments: the example of Cistus salviifolius of an active geothermal alteration field.

    Science.gov (United States)

    Bartoli, Giacomo; Bottega, Stefania; Forino, Laura M C; Ciccarelli, Daniela; Spanò, Carmelina

    2014-02-01

    Cistus salviifolius is able to colonise one of the most extreme active geothermal alteration fields in terms of both soil acidity and hot temperatures. The analyses of morpho-functional and physiological characters, investigated in leaves of plants growing around fumaroles (G leaves) and in leaves developed by the same plants after transfer into growth chamber under controlled conditions (C leaves) evidenced the main adaptive traits developed by this pioneer plant in a stressful environment. These traits involved leaf shape and thickness, mesophyll compactness, stomatal and trichome densities, chloroplast size. Changes of functional and physiological traits concerned dry matter content, peroxide and lipid peroxidation, leaf area, relative water and pigment contents. A higher reducing power and antioxidant enzymatic activity were typical of G leaves. Though the high levels of stress parameters, G leaves showed stress-induced specific morphogenic and physiological responses putatively involved in their surviving in active geothermal habitats.

  9. Altered gene expression in highly purified enterocytes from patients with active coeliac disease

    Directory of Open Access Journals (Sweden)

    Jackson John

    2008-08-01

    Full Text Available Abstract Background Coeliac disease is a multifactorial inflammatory disorder of the intestine caused by ingestion of gluten in genetically susceptible individuals. Genes within the HLA-DQ locus are considered to contribute some 40% of the genetic influence on this disease. However, information on other disease causing genes is sparse. Since enterocytes are considered to play a central role in coeliac pathology, the aim of this study was to examine gene expression in a highly purified isolate of these cells taken from patients with active disease. Epithelial cells were isolated from duodenal biopsies taken from five coeliac patients with active disease and five non-coeliac control subjects. Contaminating T cells were removed by magnetic sorting. The gene expression profile of the cells was examined using microarray analysis. Validation of significantly altered genes was performed by real-time RT-PCR and immunohistochemistry. Results Enterocyte suspensions of high purity (98–99% were isolated from intestinal biopsies. Of the 3,800 genes investigated, 102 genes were found to have significantly altered expression between coeliac disease patients and controls (p Conclusion This study provides a profile of the molecular changes that occur in the intestinal epithelium of coeliac patients with active disease. Novel candidate genes were revealed which highlight the contribution of the epithelial cell to the pathogenesis of coeliac disease.

  10. The crowded sea: incorporating multiple marine activities in conservation plans can significantly alter spatial priorities.

    Directory of Open Access Journals (Sweden)

    Tessa Mazor

    Full Text Available Successful implementation of marine conservation plans is largely inhibited by inadequate consideration of the broader social and economic context within which conservation operates. Marine waters and their biodiversity are shared by a host of stakeholders, such as commercial fishers, recreational users and offshore developers. Hence, to improve implementation success of conservation plans, we must incorporate other marine activities while explicitly examining trade-offs that may be required. In this study, we test how the inclusion of multiple marine activities can shape conservation plans. We used the entire Mediterranean territorial waters of Israel as a case study to compare four planning scenarios with increasing levels of complexity, where additional zones, threats and activities were added (e.g., commercial fisheries, hydrocarbon exploration interests, aquaculture, and shipping lanes. We applied the marine zoning decision support tool Marxan to each planning scenario and tested a the ability of each scenario to reach biodiversity targets, b the change in opportunity cost and c the alteration of spatial conservation priorities. We found that by including increasing numbers of marine activities and zones in the planning process, greater compromises are required to reach conservation objectives. Complex plans with more activities incurred greater opportunity cost and did not reach biodiversity targets as easily as simplified plans with less marine activities. We discovered that including hydrocarbon data in the planning process significantly alters spatial priorities. For the territorial waters of Israel we found that in order to protect at least 10% of the range of 166 marine biodiversity features there would be a loss of ∼15% of annual commercial fishery revenue and ∼5% of prospective hydrocarbon revenue. This case study follows an illustrated framework for adopting a transparent systematic process to balance biodiversity goals and

  11. Heparin Alters Viral Serpin, Serp-1, Anti-Thrombolytic Activity to Anti-Thrombotic Activity

    OpenAIRE

    Li, Xing; Schneider, Heather; Peters, Andrew; Macaulay, Colin; King, Elaine; Sun, Yunming; Liu, Liying; Dai, Erbin; Davids, Jennifer A; McFadden, Grant; Lucas, Alexandra

    2008-01-01

    Serine protease inhibitors (serpins) regulate coagulation and inflammation. Heparin, a glycosaminoglycan, is an important cofactor for modulation of the inhibitory function of mammalian serpins. The secreted myxoma viral serpin, Serp-1 exerts profound anti-inflammatory activity in a wide range of animal models. Serp-1 anti-inflammatory and anti-atherogenic activity is dependent upon inhibition of the uPA / uPA receptor thrombolytic complex. We demonstrate here that heparin binds to Serp-1 and...

  12. Anabolic steroids alter the physiological activity of aggression circuits in the lateral anterior hypothalamus.

    Science.gov (United States)

    Morrison, T R; Sikes, R W; Melloni, R H

    2016-02-19

    Syrian hamsters exposed to anabolic/androgenic steroids (AAS) during adolescence consistently show increased aggressive behavior across studies. Although the behavioral and anatomical profiles of AAS-induced alterations have been well characterized, there is a lack of data describing physiological changes that accompany these alterations. For instance, behavioral pharmacology and neuroanatomical studies show that AAS-induced changes in the vasopressin (AVP) neural system within the latero-anterior hypothalamus (LAH) interact with the serotonin (5HT) and dopamine (DA) systems to modulate aggression. To characterize the electrophysiological profile of the AAS aggression circuit, we recorded LAH neurons in adolescent male hamsters in vivo and microiontophoretically applied agonists and antagonists of aggressive behavior. The interspike interval (ISI) of neurons from AAS-treated animals correlated positively with aggressive behaviors, and adolescent AAS exposure altered parameters of activity in regular firing neurons while also changing the proportion of neuron types (i.e., bursting, regular, irregular). AAS-treated animals had more responsive neurons that were excited by AVP application, while cells from control animals showed the opposite effect and were predominantly inhibited by AVP. Both DA D2 antagonists and 5HT increased the firing frequency of AVP-responsive cells from AAS animals and dual application of AVP and D2 antagonists doubled the excitatory effect of AVP or D2 antagonist administration alone. These data suggest that multiple DA circuits in the LAH modulate AAS-induced aggressive responding. More broadly, these data show that multiple neurochemical interactions at the neurophysiological level are altered by adolescent AAS exposure. PMID:26691962

  13. K-ras oncogene mutations in sporadic colorectal cancer in The Netherlands Cohort Study

    NARCIS (Netherlands)

    Brink, M.; Goeij, A.F.P.M. de; Weijenberg, M.P.; Roemen, G.M.J.M.; Lentjes, M.H.F.M.; Pachen, M.M.M.; Smits, K.M.; Bruïne, A.P. de; Goldbohm, R.A.; Brandt, P.A. van den

    2003-01-01

    Activation of K-ras oncogene has been implicated in colorectal carcinogenesis, being mutated in 30-60% of the adenocarcinomas. In this study, 737 incident colorectal cancer (CRC) patients, originating from 120 852 men and women (55-69 years at baseline) participating in the Netherlands Cohort Study

  14. Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

    DEFF Research Database (Denmark)

    Andersson, Ulrika; Treebak, Jonas Thue; Nielsen, Jakob Nis;

    2005-01-01

    Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested...

  15. Simulated microgravity alters multipotential differentiation of rat mesenchymal stem cells in association with reduced telomerase activity

    Science.gov (United States)

    Sun, Lianwen; Gan, Bo; Fan, Yubo; Xie, Tian; Hu, Qinghua; Zhuang, Fengyuan

    Microgravity is one of the most important characteristics in space flight. Exposure to microgravity results in extensive physiological changes in humans. Bone loss is one of the changes with serious consequences; however, the mechanism retains unclear. As the origin of osteoprogenitors, mesenchymal stem cells (MSCs) may play an important role in it. After cultured under simulated microgravity (in a rotary cell culture system, RCCS), MSCs were stained using oil red O to identify adipocytes. The mRNA level of bone morphogenetic protein (BMP)-2 and peroxisome proliferators-activated receptor (PPAR) γ2 was determined by RT-PCR. Otherwise, MSCs were induced to osteogenic differentiation after microgravity culture, and then the activity of alkaline phosphatase (ALP) was determined by PNPP and the content of osteocalcin (OC) by ELISA. Furthermore, the telomerase activity in MSCs was measured by TRAP. The results showed that simulated microgravity inhibited osteoblastic differentiation and induced adipogenic differentiation accompanied by the change of gene expression of BMP-2 and PPARγ2 in MSCs. Meanwhile, the telomerase activity decreased significantly in MSCs under simulated microgravity. The reduced bone formation in space flight may partly be due to the altered potential differentiation of MSCs associated with telomerase activity which plays a key role in regulating the lifespan of cell proliferation and differentiation. Therefore, telomerase activation/replacement may act as a potential countermeasure for microgravity-induced bone loss.

  16. Altered Brain Activities Associated with Neural Repetition Effects in Mild Cognitive Impairment Patients.

    Science.gov (United States)

    Yu, Jing; Li, Rui; Jiang, Yang; Broster, Lucas S; Li, Juan

    2016-05-11

    Older adults with mild cognitive impairment (MCI) manifest impaired explicit memory. However, studies on implicit memory such as repetition effects in persons with MCI have been limited. In the present study, 17 MCI patients and 16 healthy normal controls (NC) completed a modified delayed-match-to-sample task while undergoing functional magnetic resonance imaging. We aim to examine the neural basis of repetition; specifically, to elucidate whether and how repetition-related brain responses are altered in participants with MCI. When repeatedly rejecting distracters, both NC and MCI showed similar behavioral repetition effects; however, in both whole-brain and region-of-interest analyses of functional data, persons with MCI showed reduced repetition-driven suppression in the middle occipital and middle frontal gyrus. Further, individual difference analysis found that activation in the left middle occipital gyrus was positively correlated with rejecting reaction time and negatively correlated with accuracy rate, suggesting a predictor of repetition behavioral performance. These findings provide new evidence to support the view that neural mechanisms of repetition effect are altered in MCI who manifests compensatory repetition-related brain activities along with their neuropathology. PMID:27176074

  17. Ras-Mek-Erk signaling regulates Nf1 heterozygous neointima formation.

    Science.gov (United States)

    Stansfield, Brian K; Bessler, Waylan K; Mali, Raghuveer; Mund, Julie A; Downing, Brandon D; Kapur, Reuben; Ingram, David A

    2014-01-01

    Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1(+/-)) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1(+/-) macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1(+/-) neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1(+/-) mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1(+/-) neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation. PMID:24211110

  18. Vitamin D3 alters microglia immune activation by an IL-10 dependent SOCS3 mechanism.

    Science.gov (United States)

    Boontanrart, Mandy; Hall, Samuel D; Spanier, Justin A; Hayes, Colleen E; Olson, Julie K

    2016-03-15

    Microglia become activated immune cells during infection or disease in the central nervous system (CNS). However, the mechanisms that downregulate activated microglia to prevent immune-mediated damage are not completely understood. Vitamin D3 has been suggested to have immunomodulatory affects, and high levels of vitamin D3 have been correlated with a decreased risk for developing some neurological diseases. Recent studies have demonstrated the synthesis of active vitamin D3, 1,25-dihydroxyvitamin D3, within the CNS, but its cellular source and neuroprotective actions remain unknown. Therefore, we wanted to determine whether microglia can respond to vitamin D3 and whether vitamin D3 alters immune activation of microglia. We have previously shown that microglia become activated by IFNγ or LPS or by infection with virus to express pro-inflammatory cytokines, chemokines, and effector molecules. In this study, activated microglia increased the expression of the vitamin D receptor and Cyp27b1, which encodes the enzyme for converting vitamin D3 into its active form, thereby enhancing their responsiveness to vitamin D3. Most importantly, the activated microglia exposed to vitamin D3 had reduced expression of pro-inflammatory cytokines, IL-6, IL-12, and TNFα, and increased expression of IL-10. The reduction in pro-inflammatory cytokines was dependent on IL-10 induction of suppressor of cytokine signaling-3 (SOCS3). Therefore, vitamin D3 increases the expression of IL-10 creating a feedback loop via SOCS3 that downregulates the pro-inflammatory immune response by activated microglia which would likewise prevent immune mediated damage in the CNS.

  19. Fecal protease activity is associated with compositional alterations in the intestinal microbiota.

    Directory of Open Access Journals (Sweden)

    Ian M Carroll

    Full Text Available OBJECTIVE: Intestinal proteases carry out a variety of functions in the gastrointestinal (GI tract. Studies have reported that elevated enteric proteases in patients with GI disease can alter intestinal physiology, however the origin (human vs. microbial of elevated proteases in patients with GI disease is unclear. AIM: The aim of this study was to investigate the association between protease activity and the microbiota in human fecal samples. DESIGN: In order to capture a wide range of fecal protease (FP activity stool samples were collected from 30 IBS patients and 24 healthy controls. The intestinal microbiota was characterized using 454 high throughput pyro-sequencing of the 16S rRNA gene. The composition and diversity of microbial communities were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME pipeline. FP activity levels were determined using an ELISA-based method. FP activity was ranked and top and bottom quartiles (n=13 per quartile were identified as having high and low FP activity, respectively. RESULTS: The overall diversity of the intestinal microbiota displayed significant clustering separation (p = 0.001 between samples with high vs. low FP activity. The Lactobacillales, Lachnospiraceae, and Streptococcaceae groups were positively associated with FP activity across the entire study population, whilst the Ruminococcaceae family and an unclassified Coriobacteriales family were negatively associated with FP activity. CONCLUSIONS: These data demonstrate significant associations between specific intestinal bacterial groups and fecal protease activity and provide a basis for further causative studies investigating the role of enteric microbes and GI diseases.

  20. Upregulated Ras/Raf/ERK1/2 signaling pathway:a new hope in the repair of spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Tao Liu; Fu-jiang Cao; Dong-dong Xu; Yun-qiang Xu; Shi-qing Feng

    2015-01-01

    An increasing number of studies report that the Ras/Raf/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway has a death-promoting apoptotic function in neural cells. We hypothesized that the Ras/Raf/ERK1/2 signaling pathway may be abnormally regulated in rat injured spinal cord models. The weight drop method was used to establish rat spinal cord injury at T9. Western blot analysis and immunohistochemical staining revealed Ras expression was dramatically elevated, and the phosphorylations of A-Raf, B-Raf and C-Raf were all upregulated in the injured spinal cord. Both mitogen-activated protein kinase kinase 1/2 and ERK1/2, which belong to the Ras/Raf signaling kinases, were upregulated. These results indicate that Ras/Raf/ERK1/2 signaling may be upregulated in injured spinal cord and are involved in recovery after spinal cord injury.

  1. A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation

    DEFF Research Database (Denmark)

    Grassilli, E; Pisano, F; Cialdella, A;

    2016-01-01

    -transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer...... tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising...

  2. Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Andres, A.C.; Schoenenberger, C.A.; Groner, B.; Henninghausen, L.; LeMeur, M.; Gelinger, P.

    1987-03-01

    The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras - i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors.

  3. Ha-ras oncogene expression directed by a milk protein gene promoter: tissue specificity, hormonal regulation, and tumor induction in transgenic mice

    International Nuclear Information System (INIS)

    The activated human Ha-ras oncogene was subjected to the control of the promoter region of the murine whey acidic protein (Wap) gene, which is expressed in mammary epithelial cells in response to lactogenic hormones. The Wap-ras gene was stably introduced into the mouse germ line of five transgenic mice (one male and four females). Wap-ras expression was observed in the mammary glands of lactating females in two lines derived from female founders. The tissue-directed and hormone-dependent Wap expression was conferred on the Ha-ras oncogene. The signals governing Wap expression are located within 2.5 kilobases of 5' flanking sequence. The other two lines derived from female founders did not express the chimeric gene. In the line derived from the male founder the Wap-ras gene is integrated into the Y chromosome. Expression was found in the salivary gland of male animals only. After a long latency, Wap-ras-expressing mice developed tumors. The tumors arose in tissues expressing Wap-ras - i.e., mammary or salivary glands. Compared to the corresponding nonmalignant tissues, Wap-ras expression was enhanced in the tumors

  4. The Ras antagonist, farnesylthiosalicylic acid (FTS, decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Yoram Nevo

    Full Text Available The Ras superfamily of guanosine-triphosphate (GTP-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J/dy(2J mouse model of merosin deficient congenital muscular dystrophy. The dy(2J/dy(2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J/dy(2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J/dy(2J mouse model of congenital muscular dystrophy.

  5. Regulation and activity of secretory leukoprotease inhibitor (SLPI) is altered in smokers.

    Science.gov (United States)

    Meyer, Megan; Bauer, Rebecca N; Letang, Blanche D; Brighton, Luisa; Thompson, Elizabeth; Simmen, Rosalia C M; Bonner, James; Jaspers, Ilona

    2014-02-01

    A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection.

  6. Hyperactive Ras/MAPK signaling is critical for tibial nonunion fracture in neurofibromin-deficient mice.

    Science.gov (United States)

    Sharma, Richa; Wu, Xiaohua; Rhodes, Steven D; Chen, Shi; He, Yongzheng; Yuan, Jin; Li, Jiliang; Yang, Xianlin; Li, Xiaohong; Jiang, Li; Kim, Edward T; Stevenson, David A; Viskochil, David; Xu, Mingjiang; Yang, Feng-Chun

    2013-12-01

    Neurofibromatosis type 1 (NF1) is a common genetic disorder affecting 1 in 3500 individuals. Patients with NF1 are predisposed to debilitating skeletal manifestations, including osteopenia/osteoporosis and long bone pseudarthrosis (nonunion fracture). Hyperactivation of the Ras/mitogen-activated protein kinase (MAPK) pathway in NF1 is known to underlie aberrant proliferation and differentiation in cell lineages, including osteoclast progenitors and mesenchymal stem cells (MSCs) also known as osteoblast progenitors (pro-OBLs). Our current study demonstrates the hyper Ras/MAPK as a critical pathway underlying the pathogenesis of NF1-associated fracture repair deficits. Nf1-deficient pro-OBLs exhibit Ras/MAPK hyperactivation. Introduction of the NF1 GTPase activating-related domain (NF1 GAP-related domain) in vitro is sufficient to rescue hyper Ras activity and enhance osteoblast (OBL) differentiation in Nf1(-/-) pro-OBLs and NF1 human (h) MSCs cultured from NF1 patients with skeletal abnormalities, including pseudarthrosis or scoliosis. Pharmacologic inhibition of mitogen-activated protein kinase kinase (MEK) signaling with PD98059 partially rescues aberrant Erk activation while enhancing OBL differentiation and expression of OBL markers, osterix and osteocalcin, in Nf1-deficient murine pro-OBLs. Similarly, MEK inhibition enhances OBL differentiation of hMSCs. In addition, PD98059 rescues aberrant osteoclast maturation in Nf1 haploinsufficient bone marrow mononuclear cells (BMMNCs). Importantly, MEK inhibitor significantly improves fracture healing in an NF1 murine model, Col2.3Cre;Nf1(flox/-). Collectively, these data indicate the Ras/MAPK cascade as a critical pathway in the pathogenesis of bone loss and pseudarthrosis related to NF1 mutations. These studies provide evidence for targeting the MAPK pathway to improve bone mass and treat pseudarthrosis in NF1. PMID:23863460

  7. The alterations in biochemical signaling of hippocampal network activity in the autism brain The alterations in biochemical signaling of hippocampal network activity in the autism brain The alterations in biochemical signaling of hippocampal network activity in the autism brain

    Institute of Scientific and Technical Information of China (English)

    田允; 黄继云; 王锐; 陶蓉蓉; 卢应梅; 廖美华; 陆楠楠; 李静; 芦博; 韩峰

    2012-01-01

    Autism is a highly heritable neurodevelopmental condition characterized by impaired social interaction and communication. However, the role of synaptic dysfunction during development of autism remains unclear. In the present study, we address the alterations of biochemical signaling in hippocampal network following induction of the autism in experimental animals. Here, the an- imal disease model and DNA array being used to investigate the differences in transcriptome or- ganization between autistic and normal brain by gene co--expression network analysis.

  8. Altered activity of the medial prefrontal cortex and amygdala during acquisition and extinction of an active avoidance task

    Directory of Open Access Journals (Sweden)

    Xilu eJiao

    2015-09-01

    Full Text Available Altered medial prefrontal cortex (mPFC and amygdala function is associated with anxiety-related disorders. While the mPFC-amygdala pathway has a clear role in fear conditioning, these structures are also involved in active avoidance. Given that avoidance perseveration represents a core symptom of anxiety disorders, the neural substrate of avoidance, especially its extinction, requires better understanding. The present study was designed to investigate the activity of mPFC and amygdala neurons during acquisition and extinction of lever-press avoidance in rats. In particular, neural activity was examined in the mPFC, intercalated cell clusters (ITCs, lateral (LA, basal (BA and central (CeA amygdala, at various time points during acquisition and extinction, using induction of the immediate early gene product, c-Fos. Neural activity was greater in the mPFC, LA, BA, and ITC during the extinction phase as compared to the acquisition phase. In contrast, the CeA was the only region that was more activated during acquisition than during extinction. Our results indicate that elevated activity in the mPFC, BA, LA and ITCs, and reduced CeA activity is associated with extinction of active avoidance. Moreover, inhibitory neurons are activated differently in the mPFC and BA during early and late phase of acquisition and extinction, suggesting their dynamic involvement in the development of avoidance response. Together, these data start to identify the key brain regions important in active avoidance behavior, areas that could be associated with avoidance perseveration in anxiety disorders.

  9. Psychoneuroendocrine immunology: perception of stress can alter body temperature and natural killer cell activity.

    Science.gov (United States)

    Hiramoto, R N; Solvason, H B; Hsueh, C M; Rogers, C F; Demissie, S; Hiramoto, N S; Gauthier, D K; Lorden, J F; Ghanta, V K

    1999-01-01

    Psychoimmunology has been credited with using the mind as a way to alter immunity. The problem with this concept is that many of the current psychoimmunology techniques in use are aimed at alleviating stress effects on the immune system rather than at direct augmentation of immunity by the brain. Studies in animals provide a model that permits us to approach the difficulties associated with gaining an understanding of the CNS-immune system connection. A particular advantage of using animals over humans is that psychological and social contributions play a less prominent role for animals than for human subjects, since the animals are all inbred and reared under identical controlled conditions. If the insightful information provided by animal studies is correct, then psychotherapy for the treatment of diseases might be made more effective if some aspect of this knowledge is included in the design of the treatment. We emphasize conditioning as a regimen and an acceptable way to train the brain to remember an output pathway to raise immunity. We propose that a specific drug or perception (mild stress, represented by rotation, total body heating or handling) could substitute and kindle the same output pathway without the need for conditioning. If this view is correct, then instead of using conditioning, it may be possible to use an antigen to activate desired immune cells, and substitute a drug or an external environmental sensory stimulus (perception) to energize the output pathway to these cells. Alternatively, monitoring alterations of body temperature in response to a drug or perception might allow us to follow how effectively the brain is performing in altering immunity. Studies with animals suggest that there are alternative ways to use the mind to raise natural or acquired immunity in man.

  10. Leptin in nucleus of the solitary tract alters the cardiovascular responses to aortic baroreceptor activation.

    Science.gov (United States)

    Ciriello, John

    2013-06-01

    Recent data suggests that neurons expressing the long form of the leptin receptor form at least two distinct groups within the caudal nucleus of the solitary tract (NTS): a group within the lateral NTS (Slt) and one within the medial (Sm) and gelantinosa (Sg) NTS. Discrete injections of leptin into Sm and Sg, a region that receives chemoreceptor input, elicit increases in arterial pressure (AP) and renal sympathetic nerve activity (RSNA). However, the effect of microinjections of leptin into Slt, a region that receives baroreceptor input is unknown. Experiments were done in the urethane-chloralose anesthetized, paralyzed and artificially ventilated Wistar or Zucker obese rat to determine leptin's effect in Slt on heart rate (HR), AP and RSNA during electrical stimulation of the aortic depressor nerve (ADN). Depressor sites within Slt were first identified by the microinjection of l-glutamate (Glu; 0.25M; 10nl) followed by leptin microinjections. In the Wistar rat leptin microinjection (50ng; 20nl) into depressor sites within the lateral Slt elicited increases in HR and RSNA, but no changes in AP. Additionally, leptin injections into Slt prior to Glu injections at the same site or to stimulation of the ADN were found to attenuate the decreases in HR, AP and RSNA to both the Glu injection and ADN stimulation. In Zucker obese rats, leptin injections into NTS depressor sites did not elicit cardiovascular responses, nor altered the cardiovascular responses elicited by stimulation of ADN. Those data suggest that leptin acts at the level of NTS to alter the activity of neurons that mediate the cardiovascular responses to activation of the aortic baroreceptor reflex. PMID:23535030

  11. Effects of mutant human Ki-rasG12C gene dosage on murine lung tumorigenesis and signaling to its downstream effectors

    International Nuclear Information System (INIS)

    Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-rasG12C allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 μg/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-rasG12C allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 μg/ml of DOX resulted in dose-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 μg/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 μg/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-rasG12C expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models

  12. Altered default mode network activity in patient with anxiety disorders: An fMRI study

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Xiaohu [Imaging Department of Tong Ji Hospital of Tong Ji University, Shanghai 200065 (China) and Bio-X lab, Department of Physics, Zhe Jiang University, Hangzhou 310027 (China)], E-mail: xhzhao999@263.net; Wang Peijun [Imaging Department of Tong Ji Hospital of Tong Ji University, Shanghai 200065 (China)], E-mail: tongjipjwang@vip.sina.com; Li Chunbo [Department of Psychiatry, Tong Ji Hospital of Tong Ji University, Shanghai 200065 (China)], E-mail: licb@mail.tongji.edu.cn; Hu Zhenghui [Department of Electrical and Engineering, Hong Kong University of Science and Technology, Hong Kong (China)], E-mail: eezhhu@ust.hk; Xi Qian [Imaging Department of Tong Ji Hospital of Tong Ji University, Shanghai 200065 (China)], E-mail: 96125007@sina.com.cn; Wu Wenyuan [Department of Psychiatry, Tong Ji Hospital of Tong Ji University, Shanghai 200065 (China)], E-mail: wuwy@mail.tongji.edu.cn; Tang Xiaowei [Bio-X lab, Department of Physics, Zhe Jiang University, Hangzhou 310027 (China)], E-mail: tangxw@zju.edu.cn

    2007-09-15

    Anxiety disorder, a common mental disorder in our clinical practice, is characterized by unprovoked anxiety. Medial prefrontal cortex (MPFC) and posterior cingulate cortex (PCC), which closely involved in emotional processing, are critical regions in the default mode network. We used functional magnetic resonance imaging (fMRI) to investigate whether default mode network activity is altered in patients with anxiety disorder. Ten anxiety patients and 10 healthy controls underwent fMRI while listening to emotionally neutral words alternating with rest (Experiment 1) and threat-related words alternating with emotionally neutral words (Experiment 2). In Experiment 1, regions of deactivation were observed in patients and controls. In Experiment 2, regions of deactivation were observed only in patients. The observed deactivation patterns in the two experiments, which included MPFC, PCC, and inferior parietal cortex, were similar and consistent with the default model network. Less deactivation in MPFC and greater deactivation in PCC were observed for patients group comparing to controls in Experiment 1. Our observations suggest that the default model network is altered in anxiety patients and dysfunction in MPFC and PCC may play an important role in anxiety psychopathology.

  13. Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.

    Science.gov (United States)

    Jaumot, M; Hancock, J F

    2001-07-01

    Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium fluoride, or ss-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I(1) or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

  14. A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dariel Ashton-Beaucage

    2014-03-01

    Full Text Available The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing

  15. Fluvoxamine alters the activity of energy metabolism enzymes in the brain

    Directory of Open Access Journals (Sweden)

    Gabriela K. Ferreira

    2014-09-01

    Full Text Available Objective: Several studies support the hypothesis that metabolism impairment is involved in the pathophysiology of depression and that some antidepressants act by modulating brain energy metabolism. Thus, we evaluated the activity of Krebs cycle enzymes, the mitochondrial respiratory chain, and creatine kinase in the brain of rats subjected to prolonged administration of fluvoxamine. Methods: Wistar rats received daily administration of fluvoxamine in saline (10, 30, and 60 mg/kg for 14 days. Twelve hours after the last administration, rats were killed by decapitation and the prefrontal cortex, cerebral cortex, hippocampus, striatum, and cerebellum were rapidly isolated. Results: The activities of citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV were decreased after prolonged administration of fluvoxamine in rats. However, the activities of complex II, succinate dehydrogenase, and creatine kinase were increased. Conclusions: Alterations in activity of energy metabolism enzymes were observed in most brain areas analyzed. Thus, we suggest that the decrease in citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV can be related to adverse effects of pharmacotherapy, but long-term molecular adaptations cannot be ruled out. In addition, we demonstrated that these changes varied according to brain structure or biochemical analysis and were not dose-dependent.

  16. Guanidination of notexin alters its membrane-damaging activity in response to sphingomyelin and cholesterol

    Indian Academy of Sciences (India)

    Pei-Hsiu Kao; Yi-Ling Chiou; Shinne-Ren Lin; Long-Sen Chang

    2010-12-01

    To elucidate the contribution of phospholipase A2 (PLA2) activity of notexin to its ability to perturb membranes, comparative studies on the interaction of notexin and guanidinated notexin (Gu-notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of notexin against EYPC vesicles, but had an insignificant influence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA2 activity by EDTA led to a reduction in the ability of Gu-notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of notexin and Gu-notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of notexin and Gu-notexin differed in either Ca2+-bound or metal-free states. These results indicate that notexin and Gu-notexin could induce membrane permeability without the involvement of PLA2 activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.

  17. Acrylamide alters glycogen content and enzyme activities in the liver of juvenile rat.

    Science.gov (United States)

    Kovac, Renata; Rajkovic, Vesna; Koledin, Ivana; Matavulj, Milica

    2015-10-01

    Acrylamide (AA) is spontaneously formed in carbohydrate-rich food during high-temperature processing. It is neurotoxic and potentially cancer causing chemical. Its harmful effects on the liver, especially in a young organism, are still to be elucidated. The study aimed to examine main liver histology, its glycogen content and enzyme activities in juvenile rats treated with 25 or 50mg/kg bw of AA for 3 weeks. Liver samples were fixed in formalin, routinely processed for paraffin embedding, sectioning and histochemical staining. Examination of haematoxylin and eosin (H&E)-stained sections showed an increase in the volume of hepatocytes, their nuclei and cytoplasm in both AA-treated groups compared to the control. In Periodic acid-Schiff (PAS)-stained sections in low-dose group was noticed glycogen reduction, while in high-dose group was present its accumulation compared to the control, respectively. Serum analysis showed increased activity of aspartate aminotransferase (AST), and decreased activity of alanine aminotransferase (ALT) in both AA-treated groups, while the activity of alkaline phosphatase (ALP) was increased in low-dose, but decreased in high-dose group compared to the control, respectively. Present results suggest a prominent hepatotoxic potential of AA which might alter the microstructural features and functional status in hepatocytes of immature liver.

  18. BRAF, K-ras and BAT26 mutations in colorectal polyps and stool

    Institute of Scientific and Technical Information of China (English)

    Ying-Min Jin; Bao-Jie Li; Bo Qu; Ya-Ju Du

    2006-01-01

    AIM: To assess the feasibility of using BRAF, K-ras and BAT26 genes as stool-based molecular markers for detection of colorectal adenomas and hyperplastic polyps (HPs).METHODS: We applied PCR-SSCP and direct sequencing to detect BRAF mutations of polyps and paired stool samples. Primer-mediated restriction fragment lengthpolymorphism (RFLP) analysis and mutant-enriched PCR were used in detection of K-ras mutations of polyp tissues and paired stool samples respectively. BAT26, a microsatellite instability marker was examined by detection of small unstable alleles in a poly (A) repeat. RESULTS: No genetic alterations were detected in the 36 colonoscopically normal patients in either tissues or stools. BRAF, K-ras and BAT26 mutations were found in 4 (16%), 10 (40%) and 3 (12%) of 25 adenoma tissues and among them, 75%, 80% and 100% of patients were observed to contain the same mutations in their corresponding stool samples. In HPs, mutations of BRAF and K-fas were detected in the tumor DNA of 2 (11.1%) and 8 (33.3%) of 18 patients respectively, all of whom had identical alterations in their stools. Taken together, the three genetic markers detected 15 (60%) of 25 adenomas and 8 (44.4%) of 18 HPs. The sensitivity of stool detection was 80% for adenomas and 100% for HPs with an overall specificity of 92% for adenomas and 100% for HPs. CONCLUSION: BRAF, K-ras and BAT26 genes have the potential to be molecular markers for colorectal adenomas and HPs, and can be used as non-invasive screening markers for colorectal polyps.

  19. The IAA RAS Correlator First Results

    Science.gov (United States)

    Surkis, Igor; Melnikov, Alexey; Shantyr, Violet; Zimovsky, Vladimir

    2010-01-01

    In 2009 the national Russian VLBI observations were processed by the new correlator ARC (Astrometric Radiointerferometric Correlator). The ARC is a VSI-H correlator and equipped with Mark 5B playback terminals. During 2009 ARC was used to process a series of VLBI sessions, observed on stations Svetloe, Zelenchukskaya, and Badary. NGS files were formed, and EOP parameters were obtained by IAA RAS Analysis Center. The accuracies of the pole coordinates and UT1-UTC were 1-2 mas and 0.07-0.1 ms, respectively.

  20. Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

    Science.gov (United States)

    Crucian, Brian E.; Sams, Clarence F.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing

  1. NF-Y activates genes of metabolic pathways altered in cancer cells.

    Science.gov (United States)

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-12

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

  2. Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation

    DEFF Research Database (Denmark)

    Mathiasen, D.P.; Egebjerg, C.; Andersen, S.H.;

    2012-01-01

    Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is...... essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that...

  3. Prenatal stress is a vulnerability factor for altered morphology and biological activity of microglia cells.

    Directory of Open Access Journals (Sweden)

    Joanna eŚlusarczyk

    2015-03-01

    Full Text Available Several lines of evidence suggest that the dysregulation of the immune system is an important factor in the development of depression. Microglia are the resident macrophages of the central nervous system and a key player in innate immunity of the brain. We hypothesized that prenatal stress (an animal model of depression as a priming factor could affect microglial cells and might lead to depressive-like disturbances in adult male rat offspring. We investigated the behavioral changes (sucrose preference test, Porsolt test, the expression of C1q and CD40 mRNA and the level of microglia (Iba1 positive in 3 month old control and prenatally stressed male offspring rats. In addition, we characterized the morphological and biochemical parameters of potentially harmful (NO, iNOS, IL-1β, IL-18, IL-6, TNF-α, CCL2, CXCL12, CCR2, CXCR4 and beneficial (IGF-1, BDNF phenotypes in cultures of microglia obtained from the cortices of 1-2 days old control and prenatally stressed pups. The adult prenatally stressed rats showed behavioral (anhedonic- and depression-like disturbances, enhanced expression of microglial activation markers and an increased number of Iba1-immunopositive cells in the hippocampus and frontal cortex. The morphology of glia was altered in cultures from prenatally stressed rats, as demonstrated by immunofluorescence microscopy. Moreover, in these cultures, we observed enhanced expression of CD40 and MHC II and release of pro-inflammatory cytokines, including IL-1β, IL-18, TNF-α and IL-6. Prenatal stress significantly up-regulated levels of the chemokines CCL2, CXCL12 and altered expression of their receptors, CCR2 and CXCR4 while IGF-1 production was suppressed in cultures of microglia from prenatally stressed rats.Our results suggest that prenatal stress may lead to excessive microglia activation and contribute to the behavioral changes observed in depression in adulthood.

  4. Altered Insula Activity during Visceral Interoception in Weight-Restored Patients with Anorexia Nervosa.

    Science.gov (United States)

    Kerr, Kara L; Moseman, Scott E; Avery, Jason A; Bodurka, Jerzy; Zucker, Nancy L; Simmons, W Kyle

    2016-01-01

    Anorexia nervosa (AN) is a devastating psychiatric illness that is associated with significant morbidity and mortality. Aberrant visceral interoceptive processing within the insula has been hypothesized to be an important mechanism in AN's pathophysiology due to the theoretical link between interoception and emotional experience. We therefore utilized functional magnetic resonance imaging (fMRI) to examine whether altered insula functioning underlies visceral interoception in AN. Fifteen females with restricting-type AN and 15 healthy control females underwent fMRI while performing an interoceptive attention task during which they focused on sensations in their heart, stomach, and bladder. Participants also performed an anxious rumination task while in the scanner. AN participants were weight-restored and free of psychotropic medications. Two distinct regions of the insula-anterior insula and dorsal mid-insula-exhibited a significant (p<0.05) interaction between group and interoceptive modality. The post hoc analyses revealed that in the dorsal mid-insula the interaction was driven by group differences during stomach interoception (p=0.002, Bonferroni corrected), whereas in the anterior insula the interaction was driven by group differences during heart interoception (p=0.03, Bonferroni corrected). In addition, individuals with AN displayed increased activation during anxious rumination in the dorsal mid-insula, and activation in this region during stomach interoception was correlated with measures of anxiety and psychopathology. This relationship between altered visceral interoception and clinical symptoms in AN suggests an important mechanism for the disorder. Additional research is needed to examine whether interventions targeting visceral interoception may increase the efficacy of treatments for AN. PMID:26084229

  5. SCD1 Expression is dispensable for hepatocarcinogenesis induced by AKT and Ras oncogenes in mice.

    Directory of Open Access Journals (Sweden)

    Lei Li

    Full Text Available Increased de novo lipogenesis is one of the major metabolic events in cancer. In human hepatocellular carcinoma (HCC, de novo lipogenesis has been found to be increased and associated with the activation of AKT/mTOR signaling. In mice, overexpression of an activated form of AKT results in increased lipogenesis and hepatic steatosis, ultimately leading to liver tumor development. Hepatocarcinogenesis is dramatically accelerated when AKT is co-expressed with an oncogenic form of N-Ras. SCD1, the major isoform of stearoyl-CoA desaturases, catalyzing the conversion of saturated fatty acids (SFA into monounsaturated fatty acids (MUFA, is a key enzyme involved in de novo lipogenesis. While many studies demonstrated the requirement of SCD1 for tumor cell growth in vitro, whether SCD1 is necessary for tumor development in vivo has not been previously investigated. Here, we show that genetic ablation of SCD1 neither inhibits lipogenesis and hepatic steatosis in AKT-overexpressing mice nor affects liver tumor development in mice co-expressing AKT and Ras oncogenes. Molecular analysis showed that SCD2 was strongly upregulated in liver tumors from AKT/Ras injected SCD1(-/- mice. Noticeably, concomitant silencing of SCD1 and SCD2 genes was highly detrimental for the growth of AKT/Ras cells in vitro. Altogether, our study provides the evidence, for the first time, that SCD1 expression is dispensable for AKT/mTOR-dependent hepatic steatosis and AKT/Ras-induced hepatocarcinogenesis in mice. Complete inhibition of stearoyl-CoA desaturase activity may be required to efficiently suppress liver tumor development.

  6. Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells.

    Directory of Open Access Journals (Sweden)

    Rikio Suzuki

    Full Text Available Heat shock protein (HSP90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

  7. Combination of a Selective HSP90α/β Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells.

    Science.gov (United States)

    Suzuki, Rikio; Kikuchi, Shohei; Harada, Takeshi; Mimura, Naoya; Minami, Jiro; Ohguchi, Hiroto; Yoshida, Yasuhiro; Sagawa, Morihiko; Gorgun, Gullu; Cirstea, Diana; Cottini, Francesca; Jakubikova, Jana; Tai, Yu-Tzu; Chauhan, Dharminder; Richardson, Paul G; Munshi, Nikhil; Ando, Kiyoshi; Utsugi, Teruhiro; Hideshima, Teru; Anderson, Kenneth C

    2015-01-01

    Heat shock protein (HSP)90 inhibitors have shown significant anti-tumor activities in preclinical settings in both solid and hematological tumors. We previously reported that the novel, orally available HSP90α/β inhibitor TAS-116 shows significant anti-MM activities. In this study, we further examined the combination effect of TAS-116 with a RAS-RAF-MEK-ERK signaling pathway inhibitor in RAS- or BRAF-mutated MM cell lines. TAS-116 monotherapy significantly inhibited growth of RAS-mutated MM cell lines and was associated with decreased expression of downstream target proteins of the RAS-RAF-MEK-ERK signaling pathway. Moreover, TAS-116 showed synergistic growth inhibitory effects with the farnesyltransferase inhibitor tipifarnib, the BRAF inhibitor dabrafenib, and the MEK inhibitor selumetinib. Importantly, treatment with these inhibitors paradoxically enhanced p-C-Raf, p-MEK, and p-ERK activity, which was abrogated by TAS-116. TAS-116 also enhanced dabrafenib-induced MM cytotoxicity associated with mitochondrial damage-induced apoptosis, even in the BRAF-mutated U266 MM cell line. This enhanced apoptosis in RAS-mutated MM triggered by combination treatment was observed even in the presence of bone marrow stromal cells. Taken together, our results provide the rationale for novel combination treatment with HSP90α/β inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM.

  8. Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1).

    Science.gov (United States)

    Fang, Zhenhao; Marshall, Christopher B; Yin, Jiani C; Mazhab-Jafari, Mohammad T; Gasmi-Seabrook, Geneviève M C; Smith, Matthew J; Nishikawa, Tadateru; Xu, Yang; Neel, Benjamin G; Ikura, Mitsuhiko

    2016-07-22

    RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes. PMID:27226556

  9. Exogenous Alpha-Synuclein Alters Pre- and Post-Synaptic Activity by Fragmenting Lipid Rafts.

    Science.gov (United States)

    Emanuele, Marco; Esposito, Alessandro; Camerini, Serena; Antonucci, Flavia; Ferrara, Silvia; Seghezza, Silvia; Catelani, Tiziano; Crescenzi, Marco; Marotta, Roberto; Canale, Claudio; Matteoli, Michela; Menna, Elisabetta; Chieregatti, Evelina

    2016-05-01

    Alpha-synuclein (αSyn) interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages. At the post-synaptic terminal, an acute increase in glutamatergic transmission, with increased density of PSD-95 puncta, is followed by disruption of the interaction between N-methyl-d-aspartate receptor (NMDAR) and PSD-95 with ensuing decrease of long term potentiation. While cholesterol loading prevents the acute effect of αSyn at the presynapse; inhibition of casein kinase 2, which appears activated by reduction of cholesterol, restores the correct localization and clustering of NMDARs.

  10. Interleukin-15 modulates adipose tissue by altering mitochondrial mass and activity.

    Directory of Open Access Journals (Sweden)

    Nicole G Barra

    Full Text Available Interleukin-15 (IL-15 is an immunomodulatory cytokine that affects body mass regulation independent of lymphocytes; however, the underlying mechanism(s involved remains unknown. In an effort to investigate these mechanisms, we performed metabolic cage studies, assessed intestinal bacterial diversity and macronutrient absorption, and examined adipose mitochondrial activity in cultured adipocytes and in lean IL-15 transgenic (IL-15tg, overweight IL-15 deficient (IL-15-/-, and control C57Bl/6 (B6 mice. Here we show that differences in body weight are not the result of differential activity level, food intake, or respiratory exchange ratio. Although intestinal microbiota differences between obese and lean individuals are known to impact macronutrient absorption, differing gut bacteria profiles in these murine strains does not translate to differences in body weight in colonized germ free animals and macronutrient absorption. Due to its contribution to body weight variation, we examined mitochondrial factors and found that IL-15 treatment in cultured adipocytes resulted in increased mitochondrial membrane potential and decreased lipid deposition. Lastly, IL-15tg mice have significantly elevated mitochondrial activity and mass in adipose tissue compared to B6 and IL-15-/- mice. Altogether, these results suggest that IL-15 is involved in adipose tissue regulation and linked to altered mitochondrial function.

  11. Exogenous Alpha-Synuclein Alters Pre- and Post-Synaptic Activity by Fragmenting Lipid Rafts

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    Marco Emanuele

    2016-05-01

    Full Text Available Alpha-synuclein (αSyn interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages. At the post-synaptic terminal, an acute increase in glutamatergic transmission, with increased density of PSD-95 puncta, is followed by disruption of the interaction between N-methyl-d-aspartate receptor (NMDAR and PSD-95 with ensuing decrease of long term potentiation. While cholesterol loading prevents the acute effect of αSyn at the presynapse; inhibition of casein kinase 2, which appears activated by reduction of cholesterol, restores the correct localization and clustering of NMDARs.

  12. Spaceflight alters expression of microRNA during T-cell activation.

    Science.gov (United States)

    Hughes-Fulford, Millie; Chang, Tammy T; Martinez, Emily M; Li, Chai-Fei

    2015-12-01

    Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21. PMID:26276131

  13. Ablation of p120-Catenin Altering the Activity of Small GTPase in Human Lung Cancer Cells

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    Nan LIU

    2009-05-01

    Full Text Available Background and objective p120-catenin (p120ctn, a member of the Armadillo gene family, has emerged as an important modulator of small GTPase activities. Therefore, it plays novel roles in tumor malignant phenotype, such as invasion and metastasis, whose mechanism are not well clarified yet. The aim of this study is to explore the roles of p120ctn on the regulation of small GTP family members in lung cancer and the effects to lung cancer invasions andmetastasis. Methods After p120ctn was knocked down by siRNA, in vivo and in vitro analysis was applied to investigate the role and possible mechanism of p120ctn in lung cancer, such as Western Blot, pull-down analysis, and nude mice models. Results p120ctn depletion inactivated RhoA, with the the activity of Cdc42 and Rac1 increased, the invasiveness of lung cancer cells was promoted both in vitro and in vivo . Conclusion p120ctn gene knockdown enhances the metastasis of lung cancer cells, probably by altering expression of small GTPase, such as inactivation of RhoA and activation of Cdc42/Rac1.

  14. The adipose renin-angiotensin system modulates sysemic markers of insulin sensitivity activates the intrarenal renin-angiotensin system

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Suyeon [University of Tennessee, Knoxville (UTK); Soltani-Bejnood, Morvarid [University of Tennessee, Knoxville (UTK); Quignard-Boulange, Annie [Centre Biomedical des Cordeliers, Paris, France; Massiera, Florence [Centre de Biochimie, Nice, France; Teboul, Michele [Centre de Biochimie, Nice, France; Ailhaud, Gerard [Centre de Biochimie, Nice, France; Kim, Jung [University of Tennessee, Knoxville (UTK); Moustaid-Moussa, Naima [University of Tennessee, Knoxville (UTK); Voy, Brynn H [ORNL

    2006-07-01

    BACKGROUND: A growing body of data provides increasing evidence that the adipose tissue renin-angiotensin system (RAS) contributes to regulation of fat mass. Beyond its paracrine actions within adipose tissue, adipocyte-derived angiotensin II (Ang II) may also impact systemic functions such as blood pressure and metabolism. METHODS AND RESULTS: We used a genetic approach to manipulate adipose RAS activity in mice and then study the consequences on metabolic parameters and on feedback regulation of the RAS. The models included deletion of the angiotensinogen (Agt) gene (Agt-KO), its expression solely in adipose tissue under the control of an adipocyte-specific promoter (aP2-Agt/ Agt-KO), and overexpression in adipose tissue of wild type mice (aP2-Agt). Total body weight, epididymal fat pad weight, and circulating levels of leptin, insulin and resistin were significantly decreased in Agt-KO mice, while plasma adiponectin levels were increased. Overexpression of Agt in adipose tissue resulted in increased adiposity and plasma leptin and insulin levels compared to wild type (WT) controls. Angiotensinogen and type I Ang II receptor protein levels were also markedly elevated in kidney of aP2-Agt mice, suggesting that hypertension in these animals may be in part due to stimulation of the intrarenal RAS. CONCLUSIONS: Taken together, the results from this study demonstrate that alterations in adipose RAS activity significantly alter both local and systemic physiology in a way that may contribute to the detrimental health effects of obesity.

  15. Stabilization of dendritic spine clusters and hyperactive Ras-MAPK signaling predict enhanced motor learning in an autistic savant mouse model

    Directory of Open Access Journals (Sweden)

    Ryan Thomas Ash

    2014-03-01

    Full Text Available That both prominent behavioral inflexibility and exceptional learning abilities are seen occasionally in autistic patients is a mystery. We hypothesize that these altered patterns of learning and memory can arise from a pathological imbalance between the stability and plasticity of internal neural representations. We evaluated this hypothesis in the mouse model of MECP2 duplication syndrome, which demonstrates enhanced motor learning, stereotyped behaviors, and social avoidance. Learning-associated structural plasticity was measured in the motor cortex of MECP2 duplication mice by 2-photon imaging (Fig. 1A. An increased stabilization rate of learning-associated dendritic spines was observed in mutants, and this correlated with rotarod performance. Analysis of the spatial distribution of stabilized spines revealed that the mutant’s increased spine stabilization was due to a specific increase in the stability of spines jointly formed in ~9-micron clusters. Clustered spine stabilization but not isolated spine stabilization predicted enhanced motor performance in MECP2 duplication mice (Fig. 1B. Biochemical assays of Ras-MAPK and mTOR pathway activation demonstrated profound hyperphosphorylation of MAPK in the motor cortex of MECP2 duplication mice with motor training (Fig. 1C. Taken together these data suggest that a pathological bias towards hyperstability of learning-associated dendritic spine clusters driven by hyperactive Ras-MAPK signaling could contribute to neurobehavioral phenotypes in this form of syndromic autism.

  16. Field Trip Guide to Serpentinite, Silica-Carbonate Alteration, and Related Hydrothermal Activity in the Clear Lake Region, California

    Energy Technology Data Exchange (ETDEWEB)

    Fraser Goff; George Guthrie

    1999-06-01

    This guide is designed to familiarize scientists with the geology, structure, alteration, and fluids typical of California serpentinites for purposes of carbon dioxide sequestration (Lackner et al., 1995). Goff et al. (1997) and Goff and Lackner (1998) describe the geology and geochemistry of some of the serpentinites from this area. Mechanisms of silica-carbonate alteration were outlined by Barnes et al. (1973). Donnelly-Nolan et al. (1993) most recently reviewed relations between regional hydrothermal alteration and Quarternary volcanic activity. Stanley et al. (1998) summarized geophysical characteristics of the region.

  17. Altered frontocingulate activation during aversive interoceptive processing in young adults transitioning to problem stimulant use

    Directory of Open Access Journals (Sweden)

    Jennifer Lorraine Stewart

    2013-11-01

    Full Text Available Problems associated with stimulant use have been linked to frontocingulate, insular, and thalamic dysfunction during decision-making and alterations in interoceptive processing. However, little is known about how interoception and decision-making interact and contribute to dysfunctions that promote the transition from recreational drug use to abuse or dependence. Here, we investigate brain activation in response to reward, punishment, and uncertainty during an aversive interoceptive challenge in current and former stimulant (cocaine and amphetamine users using functional magnetic resonance imaging (fMRI. Young adults previously identified as recreational users (n=184 were followed up three years later. Of these, 18 individuals progressed to problem stimulant use (PSU, whereas 15 desisted stimulant use (DSU. PSU, DSU, and 14 healthy comparison subjects (CTL performed a two-choice prediction task at three fixed error rates (20%=reward, 50%=uncertainty, 80%=punishment during which they anticipated and experienced episodes of inspiratory breathing load. Although groups did not differ in insula activation or subjective breathing load ratings, PSU exhibited lower right inferior frontal gyrus (IFG and bilateral anterior cingulate (ACC activation than DSU and CTL during aversive interoceptive processing as well as lower right IFG in response to decision making involving uncertainty. However, PSU exhibited greater bilateral IFG activation than DSU and CTL while making choices within the context of punishing feedback, and both PSU and DSU showed lower thalamic activation during breathing load than CTL. Findings suggest that frontocingulate attenuation, reflecting reduced resources devoted to goal maintenance and action selection in the presence of uncertainty and interoceptive perturbations, may be a biomarker for susceptibility to problem stimulant use.

  18. Parental exposure to medications and hydrocarbons and ras mutations in children with acute lymphoblastic leukemia: A report from the Children's Oncology Group

    Energy Technology Data Exchange (ETDEWEB)

    Shu, X.O.; Perentesis, J.P.; Wen, W.Q.; Buckley, J.D.; Boyle, E.; Ross, J.A.; Robison, L.L. [Childrens Oncology Group, Arcadia, CA (United States)

    2004-07-01

    Using data from a large case-control study of childhood acute lymphoblastic leukemia (ALL; age < 15 years), we used a case-case comparison approach to examine whether reported parental exposure to hydrocarbons at work or use of specific medications are related to ras gene mutations in the leukemia cells of children with ALL. We examined mutations in K-ras and N-ras genes atcodons 12, 13, and 61 by PCR and allele-specific oligonucleotide hybridization and confirmed them by DNA sequencing. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived from logistic regression to examine the association of parental exposures with ras mutations. A total of 127 (15.2%) cases had ras mutations (K-ras 4.7% and N-ras 10.68%). Both maternal (OR 3.2,95% CI 1.7-6.1) and paternal (OR 2.0, 95% CI 1.1-3.7) reported use of mind-altering drugs were associated with N-ras mutations. Paternal use of amphetamines or diet pills was associated with N-ras mutations (OR 4.1, 95% CI 1.1-15.0). Maternal exposure to solvents (OR 3.1, 95% CI 1.0-9.7) and plastic materials (OR 6.9, 95% CI 1.2-39.7) during pregnancy and plastic materials after pregnancy (OR 8.3, 95% CI 1.4-48.8) were related to K-ras mutation. Maternal over exposure to oil and coal products before case diagnosis (OR 2.3, 95% CI 1.1-4.8) and during the postnatal period (OR 2.2, 95% CI 1.0-5.5) and paternal exposure to plastic materials before index pregnancy (OR 2.4, 95% CI 1.1-5.1) and other hydrocarbons during the postnatal period (OR 1.8, 95% CI 1.0-1.3) were associated with N-ras mutations. This study suggests that parental exposure to specific chemicals may be associated with distinct ras mutations in children who develop ALL.

  19. Alteration of phospholipase D activity in the rat tissues by irradiation

    International Nuclear Information System (INIS)

    Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. Recently, PLD has been drawing much attentions and considered to be associated with cancer process since it is involved in cellular signal transduction. In this experiment, oleate-PLD activities were measured in various tissues of the living rats after whole body irradiation. The reaction mixture for the PLD assay contained 0.1μCi 1,2-di[1-14C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50mM HEPES buffer(pH 6.5), 10mM CaCl2, and 25mM KF. phosphatidic acid, the reaction product, was separated by TLC and its radioactivity was measured with a scintillation counter. The whole body irradiation was given to the female Wistar rats via Cobalt 60 Teletherapy with field size of 10cm x 10cm and an exposure of 2.7Gy per minute to the total doses of 10Gy and 25Gy. Among the tissues examined, PLD activity in lung was the highest one and was followed by kidney, skeletal muscle, brain, spleen, bone marrow, thymus, and liver. Upon irradiation, alteration of PLD activity was observed in thymus, spleen, lung, and bone marrow. Especially PLD activities of the spleen and thymus revealed the highest sensitivity toward γ-ray with more than two times amplification in their activities. In contrast, the PLD activity of bone marrow appears to be reduced to nearly 30%. Irradiation effect was hardly detected in liver which showed the lowest PLD activity. The PLD activities affected most sensitively by the whole-body irradiation seem to be associated with organs involved in immunity and hematopoiesis. This observation strongly indicates that the PLD is closely related to the physiological function of these organs. Furthermore, radiation stress could offer an important means to explore the phenomena covering from cell proliferation to cell death on these organs. (author)

  20. Alteration of phospholipase D activity in the rat tissues by irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, M. S. [Korea Univ., Seoul (Korea, Republic of). Coll. of Medicine; Cho, Y. J. [Hanyang Univ., Seoul (Korea, Republic of). Coll. of Medicine; Choi, M. U. [Seoul National Univ. (Korea, Republic of). Coll. of Natural Sciences

    1997-09-01

    Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. Recently, PLD has been drawing much attentions and considered to be associated with cancer process since it is involved in cellular signal transduction. In this experiment, oleate-PLD activities were measured in various tissues of the living rats after whole body irradiation. The reaction mixture for the PLD assay contained 0.1{mu}Ci 1,2-di[1-{sup 14}C]palmitoyl phosphatidylcholine, 0.5mM phosphatidylcholine, 5mM sodium oleate, 0.2% taurodeoxycholate, 50mM HEPES buffer(pH 6.5), 10mM CaCl{sub 2}, and 25mM KF. phosphatidic acid, the reaction product, was separated by TLC and its radioactivity was measured with a scintillation counter. The whole body irradiation was given to the female Wistar rats via Cobalt 60 Teletherapy with field size of 10cm x 10cm and an exposure of 2.7Gy per minute to the total doses of 10Gy and 25Gy. Among the tissues examined, PLD activity in lung was the highest one and was followed by kidney, skeletal muscle, brain, spleen, bone marrow, thymus, and liver. Upon irradiation, alteration of PLD activity was observed in thymus, spleen, lung, and bone marrow. Especially PLD activities of the spleen and thymus revealed the highest sensitivity toward {gamma}-ray with more than two times amplification in their activities. In contrast, the PLD activity of bone marrow appears to be reduced to nearly 30%. Irradiation effect was hardly detected in liver which showed the lowest PLD activity. The PLD activities affected most sensitively by the whole-body irradiation seem to be associated with organs involved in immunity and hematopoiesis. This observation strongly indicates that the PLD is closely related to the physiological function of these organs. Furthermore, radiation stress could offer an important means to explore the phenomena covering from cell proliferation to cell death on these organs. (author).

  1. Altered left ventricular performance in aging physically active mice with an ankle sprain injury.

    Science.gov (United States)

    Turner, Michael J; Guderian, Sophie; Wikstrom, Erik A; Huot, Joshua R; Peck, Bailey D; Arthur, Susan T; Marino, Joseph S; Hubbard-Turner, Tricia

    2016-02-01

    We assessed the impact of differing physical activity levels throughout the lifespan, using a musculoskeletal injury model, on the age-related changes in left ventricular (LV) parameters in active mice. Forty male mice (CBA/J) were randomly placed into one of three running wheel groups (transected CFL group, transected ATFL/CFL group, SHAM group) or a SHAM Sedentary group (SHAMSED). Before surgery and every 6 weeks after surgery, LV parameters were measured under 2.5 % isoflurane inhalation. Group effects for daily distance run was significantly greater for the SHAM and lesser for the ATLF/CFL mice (p = 0.013) with distance run decreasing with age for all mice (p < 0.0001). Beginning at 6 months of age, interaction (group × age) was noted with LV posterior wall thickness-to-radius ratios (h/r) where h/r increased with age in the ATFL/CFL and SHAMSED mice while the SHAM and CFL mice exhibited decreased h/r with age (p = 0.0002). Passive filling velocity (E wave) was significantly greater in the SHAM mice and lowest for the ATFL/CFL and SHAMSED mice (p < 0.0001) beginning at 9 months of age. Active filling velocity (A wave) was not different between groups (p = 0.10). Passive-to-active filling velocity ratio (E/A ratio) was different between groups (p < 0.0001), with higher ratios for the SHAM mice and lower ratios for the ATFL/CFL and SHAMSED mice in response to physical activity beginning at 9 months of age. Passive-to-active filling velocity ratio decreased with age (p < 0.0001). Regular physical activity throughout the lifespan improved LV structure, passive filling velocity, and E/A ratio by 6 to 9 months of age and attenuated any negative alterations throughout the second half of life. The diastolic filling differences were found to be significantly related to the amount of activity performed by 9 months and at the end of the lifespan. PMID:26803818

  2. Alterations of T cell activation signalling and cytokine production by postmenopausal estrogen levels

    Directory of Open Access Journals (Sweden)

    Taylor Douglas D

    2009-03-01

    Full Text Available Abstract Background Immunosenescence is an age-associated disorder occurring primarily in T cell compartments, including altered subset composition, functions, and activation. In women, evidence implicates diminished estrogen in the postmenopausal period as a contributing factor to diminished T cell responsiveness. Since hypoestrogenism is present in postmenopausal women, our objective focused on whether T cell activation, defined as signalling molecule expressions and activation, and function, identified as IL-2 production, were affected by low estrogen. Methods Using Jurkat 6.1 T cells, consequences of 4 pg/ml (corresponding to postmenopausal levels or 40 pg/ml (premenopausal levels of estradiol (E2 were analyzed on signalling proteins, CD3-zeta, JAK2, and JAK3, determined by Western immunoblotting. These consequences were correlated with corresponding gene expressions, quantified by real time-polymerase chain reaction. Tyrosine phosphorylation of CD3-zeta was defined by immunoprecipitation and western immunoblotting following activation by T cell receptor (TcR cross-linking. CD3-zeta expression and modulation was also confirmed in T cells from pre- and postmenopausal women. To assess functional consequences, IL-2 production, induced by PMA and ionomycin, was determined using enzyme-linked immunosorbent spot assay (ELISpot. Results At 40 pg/ml E2, the level of signalling protein CD3-zeta was elevated 1.57-fold, compared with cells exposed to 4 pg/ml E2. The CD3-zeta proteins also exhibited altered levels of activation-induced phosphorylation in the presence of 40 pg/ml E2 versus 4 pg/ml: 23 kD phosphorylated form increased 2.64-fold and the 21 kD form was elevated 2.95-fold. Examination of kinases associated with activation signalling also demonstrated that, in the presence of 40 pg/ml E2, JAK2 protein expression was increased 1.64-fold (p 2 (2.39, 2.01, and 2.21 fold, respectively versus 4 pg/ml. These findings were confirmed in vivo, since T

  3. Comparison of the Conformations of KRAS Isoforms, K-Ras4A and K-Ras4B, Points to Similarities and Significant Differences.

    Science.gov (United States)

    Chakrabarti, Mayukh; Jang, Hyunbum; Nussinov, Ruth

    2016-02-01

    Human HRAS, KRAS, and NRAS genes encode four isoforms of Ras, a p21 GTPase. Mutations in KRAS account for the majority of RAS-driven cancers. The KRAS has two splice variants, K-Ras4A and K-Ras4B. Due to their reversible palmitoylation, K-Ras4A and N-Ras have bimodal signaling states. K-Ras4A and K-Ras4B differ in four catalytic domain residues (G151R/D153E/K165Q/H166Y) and in their disordered C-terminal hypervariable region (HVR). In K-Ras4A, the HVR is not as strongly positively charged as in K-Ras4B (+6e vs +9e). Here, we performed all-atom molecular dynamics simulations to elucidate isoform-specific differences between the two splice variants. We observe that the catalytic domain of GDP-bound K-Ras4A has a more exposed nucleotide binding pocket than K-Ras4B, and the dynamic fluctuations in switch I and II regions also differ; both factors may influence guanine-nucleotide exchange. We further observe that like K-Kas4B, full-length K-Ras4A exhibits nucleotide-dependent HVR fluctuations; however, these fluctuations differ between the GDP-bound forms of K-Ras4A and K-Ras4B. Unlike K-Ras4B where the HVR tends to cover the effector binding region, in K-Ras4A, autoinhibited states are unstable. With lesser charge, the K-Ras4A HVR collapses on itself, making it less available for binding the catalytic domain. Since the HVRs of N- and H-Ras are weakly charged (+1e and +2e, respectively), autoinhibition may be a unique feature of K-Ras4B.

  4. Reproductive experience alters neural and behavioural responses to acute oestrogen receptor α activation.

    Science.gov (United States)

    Byrnes, E M; Casey, K; Carini, L M; Bridges, R S

    2013-12-01

    demonstrate that reproductive experience alters the behavioural response to acute ERα activation. Moreover, the findings suggest that central regulation of the hypothalamic-adrenal-pituitary axis is modified as a consequence of reproductive experience.

  5. Altered polymorphonuclear leukocyte Fc gamma R expression contributes to decreased candicidal activity during intraabdominal sepsis

    International Nuclear Information System (INIS)

    We investigated the effects of untreated intraabdominal sepsis on polymorphonuclear leukocyte (PMN) candicidal activity. Two groups of swine were studied. Group I (n=6) underwent sham laparotomy, group II (n=7) underwent cecal ligation and incision. Untreated intraabdominal sepsis resulted in a progressive decrease in PMN candicidal activity. Concomitant rosetting and phagocytosis assays demonstrated a decrease in both the attachment and phagocytosis of Candida albicans opsonized with both normal and septic swine serum by PMNs in group II. Iodine 125-labeled swine immunoglobulin G (IgG) and fluorescein isothioalanate (FITC)-labeled swine IgG were used to investigate Fc gamma receptor ligand interactions. Scatchard analyses demonstrated a progressive decline in both the binding affinity constant and number of IgG molecules bound per PMN. Stimulation of the oxidative burst markedly reduced 125I-labeled IgG binding in both group I and group II, with a greater decrement being seen in animals with intraabdominal sepsis. Further, in group II, PMN recycling of the Fc gamma receptor to the cell surface after generation of the oxidative burst was reduced by postoperative day 4. Binding of monoclonal antibodies to Fc gamma receptor II, but not Fc gamma receptor I/III markedly reduced intracellular candicidal activity. Immunofluorescence studies revealed a homogeneous pattern of FITC-IgG uptake by nearly all group I PMNs, whereas by postoperative day 8 a substantial number of PMNs from group II failed to internalize the FITC-IgG. These studies suggest that untreated intraabdominal sepsis reduces PMN candicidal activity and that this is due, in part, to altered PMN Fc gamma receptor ligand interactions

  6. Altered polymorphonuclear leukocyte Fc gamma R expression contributes to decreased candicidal activity during intraabdominal sepsis

    Energy Technology Data Exchange (ETDEWEB)

    Simms, H.H.; D' Amico, R.; Monfils, P.; Burchard, K.W. (Rhode Island Hospital, Providence (USA))

    1991-03-01

    We investigated the effects of untreated intraabdominal sepsis on polymorphonuclear leukocyte (PMN) candicidal activity. Two groups of swine were studied. Group I (n=6) underwent sham laparotomy, group II (n=7) underwent cecal ligation and incision. Untreated intraabdominal sepsis resulted in a progressive decrease in PMN candicidal activity. Concomitant rosetting and phagocytosis assays demonstrated a decrease in both the attachment and phagocytosis of Candida albicans opsonized with both normal and septic swine serum by PMNs in group II. Iodine 125-labeled swine immunoglobulin G (IgG) and fluorescein isothioalanate (FITC)-labeled swine IgG were used to investigate Fc gamma receptor ligand interactions. Scatchard analyses demonstrated a progressive decline in both the binding affinity constant and number of IgG molecules bound per PMN. Stimulation of the oxidative burst markedly reduced 125I-labeled IgG binding in both group I and group II, with a greater decrement being seen in animals with intraabdominal sepsis. Further, in group II, PMN recycling of the Fc gamma receptor to the cell surface after generation of the oxidative burst was reduced by postoperative day 4. Binding of monoclonal antibodies to Fc gamma receptor II, but not Fc gamma receptor I/III markedly reduced intracellular candicidal activity. Immunofluorescence studies revealed a homogeneous pattern of FITC-IgG uptake by nearly all group I PMNs, whereas by postoperative day 8 a substantial number of PMNs from group II failed to internalize the FITC-IgG. These studies suggest that untreated intraabdominal sepsis reduces PMN candicidal activity and that this is due, in part, to altered PMN Fc gamma receptor ligand interactions.

  7. Lingual muscle activity across sleep-wake states in rats with surgically altered upper airway

    Directory of Open Access Journals (Sweden)

    Irma eRukhadze

    2014-04-01

    Full Text Available Obstructive sleep apnea (OSA patients have increased upper airway muscle activity, including such lingual muscles as the genioglossus (GG, geniohyoid (GH and hyoglossus (HG. This adaptation partially protects their upper airway against obstructions. Rodents are used to study the central neural control of sleep and breathing but they do not naturally exhibit OSA. We investigated whether, in chronically instrumented, behaving rats, disconnecting the GH and HG muscles from the hyoid (H apparatus would result in a compensatory increase of other upper airway muscle activity (EMG and/or other signs of upper airway instability. We first determined that, in intact rats, lingual (GG and intrinsic muscles maintained stable activity levels when quantified based on 2 h-long recordings conducted on days 6 through 22 after instrumentation. We then studied 5 rats in which the tendons connecting the GH and HG muscles to the H apparatus were experimentally severed. When quantified across all recording days, lingual EMG during SWS was modestly but significantly increased in rats with surgically altered upper airway (8.6% ±0.7(SE vs. 6.2% ±0.7 of the mean during wakefulness; p=0.012. Respiratory modulation of lingual EMG occurred mainly during SWS and was similarly infrequent in both groups, and the incidence of sighs and central apneas also was similar. Thus, a weakened action of selected lingual muscles did not produce sleep-disordered breathing but resulted in a relatively elevated activity in other lingual muscles during SWS. These results encourage more extensive surgical manipulations with the aim to obtain a rodent model with collapsible upper airway.

  8. Mechanism of E1A-mediated escape from ras-induced senescence in human fibraobIasts%E1A阻断ras诱导人成纤维细胞衰老机制的研究

    Institute of Scientific and Technical Information of China (English)

    李亦蕾; 余乐

    2011-01-01

    Objective To study the effect of binding activities of the NH2 terminus of E1A to the proteins regulating cell growth on ras-induced cell senescence and explore the mechanism of ElA-mediated escape from ras-induced senescence by E1A in human fibroblast. Methods In primary human fibroblasts, the proteins regulating cell growth in association with E1A NH2 terminus, including the Rb family proteins, p300/CBP, and p400, were inactivated or interfered. The effect of alterations in the binding activities of these proteins on cell senescence bypass mediated by E1A was evaluated by cell growth curve. Results Inactivation of Rb family proteins alone was not sufficient to rescue ras-induced cell senescence, whereas inactivation of both the Rb proteins and p300/CBP blocked ras-induced senescence of human fibroblasts. Conclusion Rb and p300/CBP binding activities are both required for E1A to bypass ras-induced senescence in human fibroblasts.%目的 通过研究ElA氨基端细胞生长调节蛋白结合活性对其阻断ras诱导的细胞衰老的影响,以明确在人类成纤维细胞ElA阻断ras诱导的细胞衰老的机制.方法 采用原代培养的人类成纤维细胞,通过灭活或干扰与ElA氨基端相关的细胞生长调节蛋白,包括Rb家族蛋白、p300/CBP、p400,利用细胞生长曲线确定这些蛋白结合活性对于ElA阻断ras诱导的细胞衰老的作用.结果 单纯灭活Rb家族蛋白不能阻断ras诱导的细胞衰老,而同时灭活Rb和p300/CBP即可阻断r弱诱导的细胞衰老.结论 Rb和p300/CBP的结合活性均是EIA阻断ras诱导的人成纤维细胞细胞衰老所必需的.

  9. The impact of initiation: Early onset marijuana smokers demonstrate altered Stroop performance and brain activation

    Directory of Open Access Journals (Sweden)

    K.A. Sagar

    2015-12-01

    Full Text Available Marijuana (MJ use is on the rise, particularly among teens and emerging adults. This poses serious public health concern, given the potential deleterious effects of MJ on the developing brain. We examined 50 chronic MJ smokers divided into early onset (regular MJ use prior to age 16; n = 24 and late onset (age 16 or later; n = 26, and 34 healthy control participants (HCs. All completed a modified Stroop Color Word Test during fMRI. Results demonstrated that MJ smokers exhibited significantly poorer performance on the Interference subtest of the Stroop, as well as altered patterns of activation in the cingulate cortex relative to HCs. Further, early onset MJ smokers exhibited significantly poorer performance relative to both HCs and late onset smokers. Additionally, earlier age of MJ onset as well as increased frequency and magnitude (grams/week of MJ use were predictive of poorer Stroop performance. fMRI results revealed that while late onset smokers demonstrated a more similar pattern of activation to the control group, a different pattern was evident in the early onset group. These findings underscore the importance of assessing age of onset and patterns of MJ use and support the need for widespread education and intervention efforts among youth.

  10. Altered protoxin activation by midgut enzymes from a Bacillus thuringiensis resistant strain of Plodia interpunctella.

    Science.gov (United States)

    Oppert, B; Kramer, K J; Johnson, D E; MacIntosh, S C; McGaughey, W H

    1994-02-15

    Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.

  11. Theory of mind network activity is altered in subjects with familial liability for schizophrenia.

    Science.gov (United States)

    Mohnke, Sebastian; Erk, Susanne; Schnell, Knut; Romanczuk-Seiferth, Nina; Schmierer, Phöbe; Romund, Lydia; Garbusow, Maria; Wackerhagen, Carolin; Ripke, Stephan; Grimm, Oliver; Haller, Leila; Witt, Stephanie H; Degenhardt, Franziska; Tost, Heike; Heinz, Andreas; Meyer-Lindenberg, Andreas; Walter, Henrik

    2016-02-01

    As evidenced by a multitude of studies, abnormalities in Theory of Mind (ToM) and its neural processing might constitute an intermediate phenotype of schizophrenia. If so, neural alterations during ToM should be observable in unaffected relatives of patients as well, since they share a considerable amount of genetic risk. While behaviorally, impaired ToM function is confirmed meta-analytically in relatives, evidence on aberrant function of the neural ToM network is sparse and inconclusive. The present study therefore aimed to further explore the neural correlates of ToM in relatives of schizophrenia. About 297 controls and 63 unaffected first-degree relatives of patients with schizophrenia performed a ToM task during functional magnetic resonance imaging. Consistent with the literature relatives exhibited decreased activity of the medial prefrontal cortex. Additionally, increased recruitment of the right middle temporal gyrus and posterior cingulate cortex was found, which was related to subclinical paranoid symptoms in relatives. These results further support decreased medial prefrontal activation during ToM as an intermediate phenotype of genetic risk for schizophrenia. Enhanced recruitment of posterior ToM areas in relatives might indicate inefficiency mechanisms in the presence of genetic risk. PMID:26341902

  12. Enhanced carotid body chemosensory activity and the cardiovascular alterations induced by intermittent hypoxia

    Directory of Open Access Journals (Sweden)

    Rodrigo eIturriaga

    2014-12-01

    Full Text Available The carotid body (CB plays a main role in the maintenance of the oxygen homeostasis. The hypoxic stimulation of the CB increases the chemosensory discharge, which in turn elicits reflex sympathetic, cardiovascular and ventilatory adjustments. An exacerbate carotid chemosensory activity has been associated with human sympathetic-mediated diseases such as hypertension, insulin resistance, heart failure and obstructive sleep apnea (OSA. Indeed, the CB chemosensory discharge becomes tonically hypereactive in experimental models of OSA and heart failure. Chronic intermittent hypoxia (CIH, a main feature of OSA, enhances CB chemosensory baseline discharges in normoxia and in response to hypoxia, inducing sympathetic overactivity and hypertension. Oxidative stress, increased levels of ET-1, Angiotensin II and pro-inflammatory cytokines, along with a reduced production of NO in the CB, have been associated with the enhanced carotid chemosensory activity. In this review, we will discuss new evidence supporting a main role for the CB chemoreceptor in the autonomic and cardiorespiratory alterations induced by intermittent hypoxia, as well as the molecular mechanisms involved in the CB chemosensory potentiation.

  13. Paracetamol Supplementation Does Not Alter The Antitumor Activity and Lung Toxicity of Bleomycin

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    Ghada M. Suddek

    2014-01-01

    Full Text Available Bleomycin (BLM is well known by its antitumor activity both in vitro and in vivo. However, pulmonary fibrosis has been considered the dose limiting toxicity of the drug. Hyperpyrexia following injection of BLM was reported thus, paracetamol is sometimes administered with BLM as antipyretic drug. Actually, paracetamol was found to interfere with cytotoxicity of some drugs. This study was conducted to investigate the effect of paracetamol administration on the antitumor and lung toxicity of BLM. The antitumor activity was evaluated both in vitro and in vivo using Ehrlich ascites carcinoma (EAC cells. Paracetamol did not alter the antitumor effect of BLM in vitro or in vivo. The lung toxicity of BLM was evidenced by decrease in the body weight, increase in the lung/body weight ratio, decrease in the response of pulmonary arterial rings to 5-hydroxytryptamine (5-HT and increase in the contractility of tracheal smooth muscles induced by acetylcholine (ACh. The toxicity was also confirmed biochemically by marked increases in hydroxyproline and lipid peroxidation in rat lung and the decrease in reduced glutathione (GSH level. Pretreatment with paracetamol did not significantly change lipid peroxidation, GSH level, percent survival of rats or the response of pulmonary arterial rings and tracheal smooth muscles to 5-HT and ACh respectively. The results of the present study indicated that paracetamol neither modified the antitumor effect of BLM nor changed drug-induced lung toxicity.

  14. Altered regional and circuit resting-state activity associated with unilateral hearing loss.

    Directory of Open Access Journals (Sweden)

    Xingchao Wang

    Full Text Available The deprivation of sensory input after hearing damage results in functional reorganization of the brain including cross-modal plasticity in the sensory cortex and changes in cognitive processing. However, it remains unclear whether partial deprivation from unilateral auditory loss (UHL would similarly affect the neural circuitry of cognitive processes in addition to the functional organization of sensory cortex. Here, we used resting-state functional magnetic resonance imaging to investigate intrinsic activity in 34 participants with UHL from acoustic neuroma in comparison with 22 matched normal controls. In sensory regions, we found decreased regional homogeneity (ReHo in the bilateral calcarine cortices in UHL. However, there was an increase of ReHo in the right anterior insular cortex (rAI, the key node of cognitive control network (CCN and multimodal sensory integration, as well as in the left parahippocampal cortex (lPHC, a key node in the default mode network (DMN. Moreover, seed-based resting-state functional connectivity analysis showed an enhanced relationship between rAI and several key regions of the DMN. Meanwhile, lPHC showed more negative relationship with components in the CCN and greater positive relationship in the DMN. Such reorganizations of functional connectivity within the DMN and between the DMN and CCN were confirmed by a graph theory analysis. These results suggest that unilateral sensory input damage not only alters the activity of the sensory areas but also reshapes the regional and circuit functional organization of the cognitive control network.

  15. 3'-Azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta

    International Nuclear Information System (INIS)

    The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, β-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, β-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier

  16. Cambios en el porcentaje de sodio intercambiable (PSI) y la relación de absorción de sodio (RAS) de un suelo y su influencia en la actividad y biomasa microbiana Changes specific absortion rate (SAR) and exchange sodium percentaje (ESP) of a soil and its influence on microbial activity and biomass

    Directory of Open Access Journals (Sweden)

    Cesar A Gasca

    2011-01-01

    Full Text Available Con el objetivo de evaluar los cambios en el PSI, la RAS y su influencia en la actividad y biomasa microbiana del suelo, se aplicaron diversas concentraciones de vinaza como enmienda procedente de la industria de alcohol carburante, sobre un suelo afectado por sodicidad con severas limitaciones en las condiciones físicas, químicas y biológicas. Se aplicó un diseño en bloques completos al azar que incluye cuatro tratamientos y tres repeticiones, y muestreos de suelo al inicio y final del proceso a tres profundidades (0-20, 20-40 y 40-60 cm), cuyas variables de respuesta a medir fueron la respiración, C- biomasa microbiana, MO%, pH, CIC, CE, RAS y PSI. La actividad biológica (CO2) y el C-biomasa microbiana mostraron incrementos significativos en el rango ideal para el establecimento del cultivo de caña.To evaluate changes in ESP, SAR and its influence on the activity and soil microbial biomass, different concentrations of vinasse from the fuel ethanol industry as an amendment were applied on a soil affected by sodicity with strong physical, chemical and biological limitations. A randomized complete block design was used involving four treatments and three replications, which included soil sampling at the beginning and the end of the process at three different depths (0-20, 20-40 and 40-60 cm). Variables measured were respiration, microbial biomass C, OM%, pH, CIC, EC, SAR and ESP. Biological activity (CO2) and microbial biomass, C showed a significant increase in the ideal range for planting of sugar cane crop.

  17. Craniofacial and Dental Development in Cardio-facio-cutaneous Syndrome: The Importance of Ras Signaling Homeostasis

    OpenAIRE

    Goodwin, Alice F.; Oberoi, Snehlata; Landan, Maya; Charles, Cyril; Groth, Jessica; Martinez, Anna; Fairley, Cecilia; Weiss, Lauren A.; Tidyman, William E.; Klein, Ophir D.; Rauen, Katherine A.

    2012-01-01

    Cardio-facio-cutaneous syndrome (CFC) is a RASopathy that is characterized by craniofacial, dermatologic, gastrointestinal, ocular, cardiac, and neurologic anomalies. CFC is caused by activating mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathway that lies downstream of receptor tyrosine kinase (RTK) signaling. RTK signaling is known to play a central role in craniofacial and dental development, but to date, no studies have systematically examined individuals with C...

  18. Leukocyte activity is altered in a ground based murine model of microgravity and proton radiation exposure.

    Directory of Open Access Journals (Sweden)

    Jenine K Sanzari

    Full Text Available Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (2 Gy dose and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC, lymphocyte, neutrophil, and monocyte counts are reduced by approximately 65%, 70%, 55%, and 70%, respectively, compared to the non-treated control group at 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared to the non-treated control group and cell proliferation was suppressed by approximately 50%, compared to the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure result in decreased leukocyte numbers and function, which could contribute to immune system dysfunction in crew members. This investigation is one of the first to report on combined proton radiation and

  19. The time course of altered brain activity during 7-day simulated microgravity

    Directory of Open Access Journals (Sweden)

    Yang eLiao

    2015-05-01

    Full Text Available Microgravity causes multiple changes in physical and mental levels in humans, which can induce performance deficiency among astronauts. Studying the variations in brain activity that occur during microgravity would help astronauts to deal with these changes. In the current study, resting-state functional magnetic resonance imaging (rs-fMRI was used to observe the variations in brain activity during a 7-day head down tilt (HDT bed rest, which is a common and reliable model for simulated microgravity. The amplitudes of low frequency fluctuation (ALFF of twenty subjects were recorded pre-head down tilt (pre-HDT, during a bed rest period (HDT0, and then each day in the HDT period (HDT1–HDT7. One-way analysis of variance of the ALFF values over these 8 days was used to test the variation across time period (P<0.05, corrected. Compared to HDT0, subjects presented lower ALFF values in the posterior cingulate cortex and higher ALFF values in the anterior cingulate cortex during the HDT period, which may partially account for the lack of cognitive flexibility and alterations in autonomic nervous system seen among astronauts in microgravity. Additionally, the observed improvement in function in CPL during the HDT period may play a compensatory role to the functional decline in the paracentral lobule to sustain normal levels of fine motor control for astronauts in a microgravity environment. Above all, those floating brain activities during 7 days of simulated microgravity may indicate that the brain self-adapts to help astronauts adjust to the multiple negative stressors encountered in a microgravity environment.

  20. Regulation of calpain activity in rat brain with altered Ca2+ homeostasis.

    Science.gov (United States)

    Averna, Monica; Stifanese, Roberto; De Tullio, Roberta; Passalacqua, Mario; Defranchi, Enrico; Salamino, Franca; Melloni, Edon; Pontremoli, Sandro

    2007-01-26

    Activation of calpain occurs as an early event in correlation with an increase in [Ca2+]i induced in rat brain upon treatment with a high salt diet for a prolonged period of time. The resulting sequential events have been monitored in the brain of normal and hypertensive rats of the Milan strain, diverging for a constitutive alteration in the level of [Ca2+]i found to be present in nerve cells of hypertensive animals. After 2 weeks of treatment, the levels of the plasma membrane Ca2+-ATPase and of native calpastatin are profoundly decreased. These degradative processes, more pronounced in the brain of hypertensive rats, are progressively and efficiently compensated in the brain of both rat strains by different incoming mechanisms. Along with calpastatin degradation, 15-kDa still-active inhibitory fragments are accumulated, capable of efficiently replacing the loss of native inhibitor molecules. A partial return to a more efficient control of Ca2+ homeostasis occurs in parallel, assured by an early increase in the expression of Ca2+-ATPase and of calpastatin, both producing, after 12 weeks of a high salt (sodium) diet, the restoration of almost original levels of the Ca2+ pump and of significant amounts of native inhibitor molecules. Thus, conservative calpastatin fragmentation, associated with an increased expression of Ca2+-ATPase and of the calpain natural inhibitor, has been demonstrated to occur in vivo in rat brain. This represents a sequential adaptive response capable of overcoming the effects of calpain activation induced by a moderate long term elevation of [Ca2+]i.

  1. Frequency of climbing behavior as a predictor of altered motor activity in rat forced swimming test.

    Science.gov (United States)

    Vieira, Cíntia; De Lima, Thereza C M; Carobrez, Antonio de Pádua; Lino-de-Oliveira, Cilene

    2008-11-14

    Previous work has shown that the frequency of climbing behavior in rats submitted to the forced swimming test (FST) correlated to the section's crosses in the open field test, which suggest it might be taken as a predictor of motor activity in rat FST. To investigate this proposal, the frequency, duration, as well as the ratio duration/frequency for each behavior expressed in the FST (immobility, swimming and climbing) were compared in animals treated with a motor stimulant, caffeine (CAF), and the antidepressant, clomipramine (CLM). Male Wistar rats were submitted to 15min of forced swimming (pre-test) and 24h later received saline (SAL, 1ml/kg, i.p.) or CAF (6.5mg/kg, i.p.) 30min prior a 5-min session (test) of FST. To validate experimental procedures, an additional group of rats received three injections of SAL (1ml/kg, i.p.) or clomipramine (CLM, 10mg/kg, i.p.) between the pre-test and test sessions. The results of the present study showed that both drugs, CLM and CAF, significantly reduced the duration of immobility and significantly increased the duration of swimming. In addition, CAF significantly decreased the ratio of immobility, and CLM significantly increased the ratio of swimming and climbing. Moreover, CLM significantly increased the duration of climbing but only CAF increased the frequency of climbing. Thus, it seems that the frequency of climbing could be a predictor of altered motor activity scored directly in the FST. Further, we believe that this parameter could be useful for fast and reliable discrimination between antidepressant drugs and stimulants of motor activity.

  2. Removal of visual feedback alters muscle activity and reduces force variability during constant isometric contractions.

    Science.gov (United States)

    Baweja, Harsimran S; Patel, Bhavini K; Martinkewiz, Julie D; Vu, Julie; Christou, Evangelos A

    2009-07-01

    The purpose of this study was to compare force accuracy, force variability and muscle activity during constant isometric contractions at different force levels with and without visual feedback and at different feedback gains. In experiment 1, subjects were instructed to accurately match the target force at 2, 15, 30, 50, and 70% of their maximal isometric force with abduction of the index finger and maintain their force even in the absence of visual feedback. Each trial lasted 22 s and visual feedback was removed from 8-12 to 16-20 s. Each subject performed 6 trials at each target force, half with visual gain of 51.2 pixels/N and the rest with a visual gain of 12.8 pixels/N. Force error was calculated as the root mean square error of the force trace from the target line. Force variability was quantified as the standard deviation and coefficient of variation (CVF) of the force trace. The EMG activity of the agonist (first dorsal interosseus; FDI) was measured with bipolar surface electrodes placed distal to the innervation zone. Independent of visual gain and force level, subjects exhibited lower force error with the visual feedback condition (2.53 +/- 2.95 vs. 2.71 +/- 2.97 N; P FDI muscle was higher during the visual feedback condition and this difference increased especially at higher force levels (70%: 370 +/- 149 vs. 350 +/- 143 microV, P high (0.057 +/- 0.03 vs. 0.095 +/- 0.05 N). However, force variability was not affected by the vastly distinct feedback gains at this force, which supported and extended the findings from experiment 1. Our findings demonstrate that although removal of visual feedback amplifies force error, it can reduce force variability during constant isometric contractions due to an altered activation of the primary agonist muscle most likely at moderate force levels in young adults.

  3. Differential dynamics of RAS isoforms in GDP- and GTP-bound states.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2015-06-01

    RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP- (total of 3.06 μs) and GTP-bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25-40) and SwitchII (Residues 59-75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP-bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild-type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF-assisted nucleotide exchange.

  4. RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

    Science.gov (United States)

    Saito, Suguru; Kawamura, Toshihiko; Higuchi, Masaya; Kobayashi, Takahiro; Yoshita-Takahashi, Manami; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Kanda, Yasuhiro; Kawamura, Hiroki; Jiang, Shuying; Naito, Makoto; Yoshizaki, Takumi; Takahashi, Masahiko; Fujii, Masahiro

    2015-05-01

    Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases.

  5. The NPM-ALK tyrosine kinase mimics TCR signalling pathways, inducing NFAT and AP-1 by RAS-dependent mechanisms.

    Science.gov (United States)

    Turner, Suzanne D; Yeung, Debra; Hadfield, Kathryn; Cook, Simon J; Alexander, Denis R

    2007-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expression is associated with the lymphoid malignancy anaplastic large cell lymphoma (ALCL) and results from a t(2;5) chromosomal translocation. We show that NPM-ALK induces Ras activation and phosphorylation of the ERK MAP Kinase consistent with activation of the Ras-MAP Kinase pathway. Furthermore, we demonstrate that activation of Ras is necessary for inducing transcription via NFAT/AP-1 composite transcriptional binding sites. This activity is dependent on NPM-ALK forming complexes with proteins that bind to autophosphorylated tyrosine residues at positions 156, 567 and 664, associated with binding to IRS-1, Shc and PLCgamma, respectively. Specifically, NPM-ALK activates transcription from the TRE promoter element, an AP-1 binding region, an activity dependent on both Ras and Shc activity. Our results show that NPM-ALK mimics activated T-cell receptor signalling by inducing pathways associated with the activation of NFAT/AP-1 transcription factors that bind to promoter elements found in a broad array of cytokine genes.

  6. The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways.

    Science.gov (United States)

    Vasseur, Romain; Skrypek, Nicolas; Duchêne, Belinda; Renaud, Florence; Martínez-Maqueda, Daniel; Vincent, Audrey; Porchet, Nicole; Van Seuningen, Isabelle; Jonckheere, Nicolas

    2015-12-01

    The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

  7. Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

    DEFF Research Database (Denmark)

    Willumsen, B M; Papageorge, A G; Hubbert, N;

    1985-01-01

    that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...

  8. K-Ras mutation detection in liquid biopsy and tumor tissue as prognostic biomarker in patients with pancreatic cancer: a systematic review with meta-analysis.

    Science.gov (United States)

    Li, Tao; Zheng, Yuanting; Sun, Hong; Zhuang, Rongyuan; Liu, Jing; Liu, Tianshu; Cai, Weimin

    2016-07-01

    K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95 % confidence interval (CI) 1.36-1.90; p K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95 % CI 1.20-1.57; p K-Ras mutations were detected in fresh frozen samples (HR = 2.01, 95 % CI 1.28-3.16, p = 0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR = 1.29, 95 % CI 1.12-1.49, p K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples.

  9. A rare myelin protein zero (MPZ variant alters enhancer activity in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Anthony Antonellis

    Full Text Available BACKGROUND: Myelin protein zero (MPZ is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants that affect MPZ expression. METHODOLOGY: We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants. PRINCIPAL FINDINGS: Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish. CONCLUSIONS: This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function.

  10. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks

    Science.gov (United States)

    Canals, Isaac; Soriano, Jordi; Orlandi, Javier G.; Torrent, Roger; Richaud-Patin, Yvonne; Jiménez-Delgado, Senda; Merlin, Simone; Follenzi, Antonia; Consiglio, Antonella; Vilageliu, Lluïsa; Grinberg, Daniel; Raya, Angel

    2015-01-01

    Summary Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration. PMID:26411903

  11. Mild blast events alter anxiety, memory, and neural activity patterns in the anterior cingulate cortex.

    Directory of Open Access Journals (Sweden)

    Kun Xie

    Full Text Available There is a general interest in understanding of whether and how exposure to emotionally traumatizing events can alter memory function and anxiety behaviors. Here we have developed a novel laboratory-version of mild blast exposure comprised of high decibel bomb explosion sound coupled with strong air blast to mice. This model allows us to isolate the effects of emotionally fearful components from those of traumatic brain injury or bodily injury typical associated with bomb blasts. We demonstrate that this mild blast exposure is capable of impairing object recognition memory, increasing anxiety in elevated O-maze test, and resulting contextual generalization. Our in vivo neural ensemble recording reveal that such mild blast exposures produced diverse firing changes in the anterior cingulate cortex, a region processing emotional memory and inhibitory control. Moreover, we show that these real-time neural ensemble patterns underwent post-event reverberations, indicating rapid consolidation of those fearful experiences. Identification of blast-induced neural activity changes in the frontal brain may allow us to better understand how mild blast experiences result in abnormal changes in memory functions and excessive fear generalization related to post-traumatic stress disorder.

  12. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks

    Directory of Open Access Journals (Sweden)

    Isaac Canals

    2015-10-01

    Full Text Available Induced pluripotent stem cell (iPSC technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.

  13. Alteration of transcriptional networks in the entorhinal cortex after maternal immune activation and adolescent cannabinoid exposure.

    Science.gov (United States)

    Hollins, Sharon L; Zavitsanou, Katerina; Walker, Frederick Rohan; Cairns, Murray J

    2016-08-01

    Maternal immune activation (MIA) and adolescent cannabinoid exposure (ACE) have both been identified as major environmental risk factors for schizophrenia. We examined the effects of these two risk factors alone, and in combination, on gene expression during late adolescence. Pregnant rats were exposed to the viral infection mimic polyriboinosinic-polyribocytidylic acid (poly I:C) on gestational day (GD) 15. Adolescent offspring received daily injections of the cannabinoid HU210 for 14days starting on postnatal day (PND) 35. Gene expression was examined in the left entorhinal cortex (EC) using mRNA microarrays. We found prenatal treatment with poly I:C alone, or HU210 alone, produced relatively minor changes in gene expression. However, following combined treatments, offspring displayed significant changes in transcription. This dramatic and persistent alteration of transcriptional networks enriched with genes involved in neurotransmission, cellular signalling and schizophrenia, was associated with a corresponding perturbation in the expression of small non-coding microRNA (miRNA). These results suggest that a combination of environmental exposures during development leads to significant genomic remodeling that disrupts maturation of the EC and its associated circuitry with important implications as the potential antecedents of memory and learning deficits in schizophrenia and other neuropsychiatric disorders. PMID:26923065

  14. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks.

    Science.gov (United States)

    Canals, Isaac; Soriano, Jordi; Orlandi, Javier G; Torrent, Roger; Richaud-Patin, Yvonne; Jiménez-Delgado, Senda; Merlin, Simone; Follenzi, Antonia; Consiglio, Antonella; Vilageliu, Lluïsa; Grinberg, Daniel; Raya, Angel

    2015-10-13

    Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.

  15. Nerve growth factor alters the sensitivity of rat masseter muscle mechanoreceptors to NMDA receptor activation.

    Science.gov (United States)

    Wong, Hayes; Dong, Xu-Dong; Cairns, Brian E

    2014-11-01

    Intramuscular injection of nerve growth factor (NGF) into rat masseter muscle induces a local mechanical sensitization that is greater in female than in male rats. The duration of NGF-induced sensitization in male and female rats was associated with an increase in peripheral N-methyl-d-aspartate (NMDA) receptor expression by masseter muscle afferent fibers that began 3 days postinjection. Here, we investigated the functional consequences of increased NMDA expression on the response properties of masseter muscle mechanoreceptors. In vivo extracellular single-unit electrophysiological recordings of trigeminal ganglion neurons innervating the masseter muscle were performed in anesthetized rats 3 days after NGF injection (25 μg/ml, 10 μl) into the masseter muscle. Mechanical activation threshold was assessed before and after intramuscular injection of NMDA. NMDA injection induced mechanical sensitization in both sexes that was increased significantly following NGF injection in the male rats but not in the female rats. However, in female but not male rats, further examination found that preadministration of NGF induced a greater sensitization in slow Aδ-fibers (2-7 m/s) than fast Aδ-fibers (7-12 m/s). This suggests that preadministration of NGF had a different effect on slowly conducting mechanoreceptors in the female rats compared with the male rats. Although previous studies have found an association between estrogenic tone and NMDA activity, no correlation was observed between NMDA-evoked mechanical sensitization and plasma estrogen level. This study suggests NGF alters NMDA-induced mechanical sensitization in the peripheral endings of masseter mechanoreceptors in a sexually dimorphic manner.

  16. Spectrum of K ras mutations in Pakistani colorectal cancer patients

    Energy Technology Data Exchange (ETDEWEB)

    Murtaza, B.N.; Bibi, A. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan); Rashid, M.U.; Khan, Y.I. [Shaukat Khanum Memorial Cancer Hospital and Research Centre, Johar Town, Lahore (Pakistan); Chaudri, M.S. [Services Institute of Medical Sciences, Lahore (Pakistan); Shakoori, A.R. [School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore (Pakistan)

    2013-11-29

    The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

  17. Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene

    International Nuclear Information System (INIS)

    Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that the authors examined. However, cells isolated from foci were incapable of growth in soft agar. They then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and the activated form of Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed

  18. Andrographolide Sensitizes Ras-Transformed Cells to Radiation in vitro and in vivo

    International Nuclear Information System (INIS)

    Purpose: Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization. Methods and Materials: An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-κB) and Akt in andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting. Results: Concurrent andrographolide treatment (10 μM, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 ± 2.5 days) compared with radiation alone (22 ± 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-κB activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-κB activity. Conclusion: Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-κB activity. These observations indicate that andrographolide is a novel radiosensitizing agent

  19. Does changing from a first generation antipsychotic (perphenazin) to a second generation antipsychotic (risperidone) alter brain activation and motor activity? A case report

    OpenAIRE

    Berle, Jan Øystein; Løberg, Else-Marie; Fasmer, Ole Bernt

    2013-01-01

    Background: In patients with schizophrenia, altered brain activation and motor activity levels are central features, reflecting cognitive impairments and negative symptoms, respectively. Newer studies using nonlinear methods have addressed the severe disturbances in neurocognitive functioning that is regarded as one of the core features of schizophrenia. Our aim was to compare brain activation and motor activity in a patient during pharmacological treatment that was switched fr...

  20. Upregulated Ras/Raf/ERK1/2 signaling pathway: a new hope in the repair of spinal cord injury

    Directory of Open Access Journals (Sweden)

    Tao Liu

    2015-01-01

    Full Text Available An increasing number of studies report that the Ras/Raf/extracellular signal-regulated kinase 1/2 (ERK1/2 signaling pathway has a death-promoting apoptotic function in neural cells. We hypothesized that the Ras/Raf/ERK1/2 signaling pathway may be abnormally regulated in rat injured spinal cord models. The weight drop method was used to establish rat spinal cord injury at T 9 . Western blot analysis and immunohistochemical staining revealed Ras expression was dramatically elevated, and the phosphorylations of A-Raf, B-Raf and C-Raf were all upregulated in the injured spinal cord. Both mitogen-activated protein kinase kinase 1/2 and ERK1/2, which belong to the Ras/Raf signaling kinases, were upregulated. These results indicate that Ras/Raf/ERK1/2 signaling may be upregulated in injured spinal cord and are involved in recovery after spinal cord injury.

  1. Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.

    Directory of Open Access Journals (Sweden)

    Mona Meyer

    Full Text Available Acute myeloid leukemia (AML is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.

  2. In Silico Screening of Mutated K-Ras Inhibitors from Malaysian Typhonium flagelliforme for Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ayesha Fatima

    2014-01-01

    Full Text Available K-ras is an oncogenic GTPase responsible for at least 15–25% of all non-small cell lung cancer cases worldwide. Lung cancer of both types is increasing with an alarming rate due to smoking habits in Malaysia among men and women. Natural products always offer alternate treatment therapies that are safe and effective. Typhonium flagelliforme or Keladi Tikus is a local plant known to possess anticancer properties. The whole extract is considered more potent than individual constituents. Since K-ras is the key protein in lung cancer, our aim was to identify the constituents of the plant that could target the mutated K-ras. Using docking strategies, reported potentially active compounds of Typhonium flagelliforme were docked into the allosteric surface pockets and switch regions of the K-ras protein to identify possible inhibitors. The selected ligands were found to have a high binding affinity for the switch II and the interphase region of the ras-SOS binding surface.

  3. Diagnostic RAS mutation analysis by polymerase chain reaction (PCR

    Directory of Open Access Journals (Sweden)

    Ian A. Cree

    2016-06-01

    Full Text Available RAS mutation analysis is an important companion diagnostic test. Treatment of colorectal cancer with anti-Epidermal Growth Factor Receptor (EGFR therapy requires demonstration of RAS mutation status (both KRAS and NRAS, and it is good practice to include BRAF. In Non-Small Cell Lung Cancer (NSCLC and melanoma, assessment of RAS mutation status can be helpful in triaging patient samples for more extensive testing. This mini-review will discuss the role of PCR methods in providing rapid diagnostic information for cancer patients.

  4. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations | Office of Cancer Genomics

    Science.gov (United States)

    The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway.

  5. Diet, lifestyle and risk of K-ras mutation-positive and -negative colorectal adenomas.

    NARCIS (Netherlands)

    Wark, P.A.; Kuil, W. van der; Ploemacher, J.; Muijen, G.N.P. van; Mulder, C.J.J.; Weijenberg, M.P.; Kok, F.J.; Kampman, E.

    2006-01-01

    K-ras mutation-positive (K-ras+) and -negative (K-ras-) colorectal adenomas may differ clinically and pathologically. As environmental compounds may cause mutations in the growth-related K-ras oncogene or affect clonal selection depending on mutational status, we evaluated whether the aetiology of K

  6. Diet, Lifestyle and risk of K-ras mutation-positive and -negative colorectal adenomas

    NARCIS (Netherlands)

    Wark, P.A.; Kuil, van der W.; Ploemacher, J.; Muijen, van G.N.P.; Mulder, Ch.J.J.; Weijenberg, M.P.; Kok, F.J.; Kampman, E.

    2006-01-01

    K-ras mutation-positive (K-ras+) and -negative (K-ras-) colorectal adenomas may differ clinically and pathologically. As environmental compounds may cause mutations in the growth-related K-ras oncogene or affect clonal selection depending on mutational status, we evaluated whether the aetiology of K

  7. Altered cardiovascular autonomic regulation in overweight children engaged in regular physical activity.

    Science.gov (United States)

    Lucini, Daniela; de Giacomi, Gaia; Tosi, Fabio; Malacarne, Mara; Respizzi, Stefano; Pagani, Massimo

    2013-03-01

    Overweight (OW) and obesity in children are important forerunners of cardiovascular risk, possibly through autonomic nervous system (ANS) dysregulation, while physical exercise exerts a beneficial influence. In this observational study we hypothesise that OW might influence ANS profile even in a population performing high volume of supervised exercise. We study 103 young soccer players, homogeneous in terms of gender (all male), cultural background, school, age (11.2 ± 1 years) and exercise routine, since they all belong to the same soccer club, thus guaranteeing equality of supervised training and similar levels of competitiveness. ANS is evaluated by autoregressive spectral analysis of heart rate and systolic arterial pressure (SAP) variabilities. We estimate also the accumulated weekly Metabolic Equivalents and time spent in sedentary activities. We subdivide the entire population in two subgroups (normal weight and OW) based on the International Obesity Task Force criteria. In OW soccer players (10.7% of total group) we observe an altered profile of autonomic cardiovascular regulation, characterised by higher values of SAP (113 ± 4 vs 100 ± 1 mm Hg, 39.7 ± 3 vs 66.2 ± 10%), higher Low Frequency variability power of SAP (an index of vasomotor sympathetic regulation) (12 ± 3 vs 4.5 mm Hg(2)) and smaller spontaneous baroreflex gain (an index of cardiac vagal regulation) (19 ± 3 vs 33 ± 3 ms/mm Hg) (all (p < 0.02)). Moreover Correlation analysis on the entire study population shows a significant link between anthropometric and autonomic indices. These data show that OW is associated to a clear autonomic impairment even in children subjected to an intense aerobic training. PMID:23086975

  8. EGFR-Ras-Raf Signaling in Epidermal Stem Cells: Roles in Hair Follicle Development, Regeneration, Tissue Remodeling and Epidermal Cancers

    Directory of Open Access Journals (Sweden)

    Manuela Baccarini

    2013-09-01

    Full Text Available The mammalian skin is the largest organ of the body and its outermost layer, the epidermis, undergoes dynamic lifetime renewal through the activity of somatic stem cell populations. The EGFR-Ras-Raf pathway has a well-described role in skin development and tumor formation. While research mainly focuses on its role in cutaneous tumor initiation and maintenance, much less is known about Ras signaling in the epidermal stem cells, which are the main targets of skin carcinogenesis. In this review, we briefly discuss the properties of the epidermal stem cells and review the role of EGFR-Ras-Raf signaling in keratinocyte stem cells during homeostatic and pathological conditions.

  9. Gut Taste Stimulants Alter Brain Activity in Areas Related to Working Memory: a Pilot Study

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    Anne Christin Meyer-Gerspach

    2016-07-01

    Full Text Available Background/Aims: Taste perception is one of the most important primary oral reinforcers, driving nutrient and energy intake as well as toxin avoidance. Taste receptors in the gastrointestinal tract might as well impact appetitive or aversive behavior and thus influence learning tasks and a close relation of neural taste processing and working memory networks seems plausible. Methods: In the present pilot study, we determined the effects of five taste qualities “bitter” (quinine, “sweet” (glucose, “sour” (citric acid, “salty” (NaCl and “umami” (monosodium glutamate, MSG on working memory processing using functional MRI and their effect on plasma insulin and glucose levels. On six separate occasions, subjects received one of the following test substances dissolved in 200 mL tap water via a nasogastric tube (to circumvent the oral cavity: 1 2g citric acid corresponding to 52 mM, 2 2g NaCl; 171 mM, 3 0.017g quinine; 0.26 mM, 4 1g monosodium glutamate; 30 mM, 5 25g glucose; 694 mM and 6 200 mL tap water (placebo. Results: The taste qualities “bitter” and “umami” significantly altered brain activation patterns in the primary gustatory cortex as well as in subcortical structures, previously reported to be involved in emotional learning and memory. In contrast, glucose did not reveal any statistically significant brain activation difference. Working memory performance was not different over the six treatments. Plasma insulin and glucose levels were not affected by the different taste substances (MSG, quinine, NaCl and citric acid. Conclusions: in this pilot trial, we demonstrate that acute intragastric administration of different taste substances does not affect working memory performance in humans. However, “umami” and “bitter” have effects on brain areas involved in neural working memory, overpowering the effects of “sweet”, “salty” and “sour” reception.

  10. Ras- ERK signaling in behavior: old questions and new perspectives

    Directory of Open Access Journals (Sweden)

    Stefania eFasano

    2011-11-01

    Full Text Available The role of Ras-ERK signaling in behavioral plasticity is well established. Inhibition studies using the blood-brain barrier permeable drug SL327 have conclusively demonstrated that this neuronal cell signaling cascade is a crucial component of the synaptic machinery implicated in the formation of various forms of long-term memory, from spatial learning to fear and operant conditioning. However, abnormal Ras-ERK signaling has also been linked to a number of neuropsychiatric conditions, including mental retardation syndromes (RASopathies, drug addiction and L-DOPA induced Dyskinesia (LID. The work recently done on these brain disorders has pointed to previously underappreciated roles of Ras-ERK in specific subsets of neurons, like GABAergic interneurons of the hippocampus or the cortex, as well as in the medium spiny neurons of the striatum. Here we will highlight the open questions related to Ras-ERK signaling in these behavioral manifestations and propose crucial experiments for the future.

  11. Methoxychlor induces atresia by altering Bcl2 factors and inducing caspase activity in mouse ovarian antral follicles in vitro.

    Science.gov (United States)

    Basavarajappa, Mallikarjuna S; Karman, Bethany N; Wang, Wei; Gupta, Rupesh K; Flaws, Jodi A

    2012-12-01

    Methoxychlor (MXC) is an organochlorine pesticide widely used in many countries against various species of insects that attack crops and domestic animals. MXC reduces fertility by increasing atresia (death) of antral follicles in vivo. MXC also induces atresia of antral follicles after 96 h in vitro. The current work tested the hypothesis that MXC induces morphological atresia at early time points (24 and 48 h) by altering pro-apoptotic (Bax, Bok, Casp3, and caspase activity) and anti-apoptotic (Bcl2 and Bcl-xL) factors in the follicles. The results indicate that at 24 h, MXC increased Bcl-xL and Bax mRNA levels and increased the ratio of Bax/Bcl2. At 48-96 h, MXC induced morphological atresia. At 24-96 h, MXC increased caspase activities. These data suggest that MXC may induce atresia by altering Bcl2 factors and inducing caspase activities in antral follicles.

  12. Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4.

    OpenAIRE

    Satoh, T.; Nakafuku, M; Miyajima, A; Kaziro, Y

    1991-01-01

    The protooncogene ras acts as a component of signal-transduction networks in many kinds of cells. The ras gene product (p21) is a GTP-binding protein, and the activity of the protein is regulated by bound GDP/GTP. Recent studies have shown that a certain class of growth factors stimulates the formation of active p21-GTP complexes in fibroblasts and that oncogene products with enhanced tyrosine kinase activities have a similar effect on ras p21. We have measured the ratio of active GTP-bound p...

  13. Novel determinants of H-Ras plasma membrane localization and transformation

    DEFF Research Database (Denmark)

    Willumsen, B M; Cox, A D; Solski, P A;

    1996-01-01

    cysteine did not abolish palmitoylation. However, despite continued lipid modification the mutant proteins failed to bind to plasma membranes and instead accumulated on internal membranes and, importantly, were not transforming. Addition of an N-terminal myristoylation signal to these defective mutants....... However, in this native state, the C-terminus appears to provide a combination of lipids and a previously unrecognized signal for specific plasma membrane targeting that are essential for the correct localization and biological function of H-Ras......., or to proteins entirely lacking the C-terminal 25 residues restored both plasma membrane association and transforming activity. Thus, H-Ras does not absolutely require prenylation or palmitoylation nor indeed its hypervariable domain in order to interact with effectors that ultimately cause transformation...

  14. Epidermal growth factor and ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways

    Energy Technology Data Exchange (ETDEWEB)

    Pickett, C.A.; Gutierrez-Hartmann, A. [Univ. of Colorado Health Sciences Center, Denver, CO (United States)

    1995-12-01

    This report discusses the role of the epidermal growth factor (EGF) in promoting activation of the rat prolactin promoter in neuroendocrine cells via a Ras-independent mechanism. It also discusses the role of phosphotransferases in mediating EGF response. 32 refs., 8 figs., 1 tab.

  15. Activation of protein kinase A alters subnuclear distribution pattern of human steroidogenic factor 1 in living cells

    Institute of Scientific and Technical Information of China (English)

    LIU Wei刘伟; FAN Wu-qiang范吴强; Toshihiko Yanase; Masayuki Saitoh; WU Yin吴茵

    2004-01-01

    Background The aim of this study was to identify the subnuclear distribution pattern of human orphan nuclear receptor steroidogenic factor 1 (SF-1) in living cells with and without the activation of protein kinase A (PKA) signal pathway, and thus try to explain the unknown mechanism by which PKA potentiates SF-1 transactivation. Methods Full-length cDNAs of wild type and a naturally occurring mutant (G35E) human SF-1 were cloned and fused with green fluorescent protein (GFP). Subcellular distribution pattern of human SF-1 in living cells, whose PKA signaling was either activated or not, was studied by laser confocal microscopy after the validity of the gene sequence was confirmed.Results The transactivation ability of the GFP-SF-1 chimeric protein was highly conserved. Wild type human SF-1 diffused homogeneously within the nuclei of cells when PKA was not active, and converged to clear foci when PKA was activated. Mutant SF-1 diffused within the nuclei even in the presence of PKA activation, surprisingly aggregating as fluorescent dots inside the nucleoli, a phenomenon not altered by PKA.Conclusions Activation of PKA causes wild type, but not mutant SF-1 to alter its subnuclear distribution pattern to a transactivationally active form (foci formation). This finding may throw new light on the mechanism by which PKA activates the orphan nuclear receptor.

  16. The study on the correlation of activation of the Hedgehog signaling pathway and K-ras gene mutations in pancreatic cancer cell lines%胰腺癌细胞株Hedgehog信号通路活化与K-ras基因突变的相关性研究

    Institute of Scientific and Technical Information of China (English)

    胡佳佳; 吴红玉; 金晶; 朱春平; 李兆申; 高军; 李淑德

    2013-01-01

    Objective To investigate the correlation of K-ras gene mutations and activation of Hedgehog signaling pathway in pancreatic cancer cell lines.Methods Real-time PCR was used to detect K-ras gene mutations at codon 12 and 13 in pancreatic cancer cell lines of SW1990,PaTu8988,CFPAC-1,PANC1,AsPC-1,Capanc-2,BxPC-3 and the mRNA expression of Glil,Smo in these cell lines.Results The K-ras mutation of PANC1,CFPAC-1,AsPC-1,PaTu8988 was at codon 12,while SW1990 was at codon 13,BxPC-3,Capanc-2 were wild type.The expressions of Glil mRNA of AsPC-1,CFPAC-1,PANC1,PaTu8988,SW1990,BxPC-3,Capanc-2 were 7.84 ± 8.92,1.82 ± 3.45,1.00± 0.00,0.07 ± 0.10,0.88 ± 1.48,0.52 ± 0.98,0.15 ± 0.19,and the expressions of Smo mRNA were 144.00 ± 58.33,3.48 ±3.77,1.00 ±0.00,81.68 ±28.26,0.72 ±0.87,0.34 ±0.60,0.02 ±0.03.Glil,Smo mRNA expressions of cells with wild tpye K-ras were significantly lower than those with mutant type,and wild type BxPC-3's Glil,Smo mRNA expressions were significantly lower than that of mutant AxPC-1 (P < 0.05).Conclusions Activation of Hedgehog signaling pathway may be related to K-ras gene mutations.%目的 探讨胰腺癌细胞株中Hedgehog信号通路活化与K-ras基因突变的相关性.方法 采用实时定量PCR法检测人胰腺癌细胞株SW1990、PaTu8988、CFPAC-1、PANC1、AsPC-1、Capanc-2、BxPC-3的K-ras基因突变状况及Hedgehog信号通路主要成员Glil、Smo mRNA的相对表达量.结果 PANC1、CFPAC-1、AsPC-1、PaTu8988细胞为12密码子突变型,SW1990细胞为13密码子突变型,BxPC-3、Capanc-2细胞为纯野生型.AsPC-1、CFPAC-1、PANC1、PaTu8988、SW1990、BxPC-3、Capanc-2的Glil mRNA 相对表达量分别为7.84±8.92、1.82±3.45、1.00±0.00、0.07±0.10、0.88±1.48、0.52±0.98、0.15±0.19;Smo mRNA的相对表达量分别为144.00±58.33、3.48±3.77、1.00±0.00、81.68±28.26、0.72±0.87、0.34±0.60、0.02±0.03.K-ras基因野生型胰腺癌细胞Gli1、Smo mRNA的表达水平均较突变型胰腺癌细

  17. Superoxide Inhibits Guanine Nucleotide Exchange Factor (GEF) Action on Ras, but not on Rho, through Desensitization of Ras to GEF

    OpenAIRE

    Wey, Michael; Phan, Vinh; Yepez, Gerardo; Heo, Jongyun

    2014-01-01

    Ras and Rho GTPases are molecular switches for various vital cellular signaling pathways. Overactivation of these GTPases often causes development of cancer. Guanine nucleotide exchange factors (GEFs) and oxidants function to upregulate these GTPases through facilitation of guanine nucleotide exchange (GNE) of these GTPases. However, the effect of oxidants on GEF functions, or vice versa, has not been known. We show that, via targeting Ras Cys51, an oxidant inhibits the catalytic action of Cd...

  18. Exposure to Forced Swim Stress Alters Local Circuit Activity and Plasticity in the Dentate Gyrus of the Hippocampus

    Directory of Open Access Journals (Sweden)

    Mouna Maroun

    2008-02-01

    Full Text Available Studies have shown that, depending on its severity and context, stress can affect neural plasticity. Most related studies focused on synaptic plasticity and long-term potentiation (LTP of principle cells. However, evidence suggests that following high-frequency stimulation, which induces LTP in principal cells, modifications also take place at the level of complex interactions with interneurons within the dentate gyrus, that is, at the local circuit level. So far, the possible effects of stress on local circuit activity and plasticity were not studied. Therefore, we set out to examine the possible alterations in local circuit activity and plasticity following exposure to stress. Local circuit activity and plasticity were measured by using frequency dependant inhibition (FDI and commissural modulation protocols following exposure to a 15 minute-forced swim trial. Exposure to stress did not alter FDI. The application of theta-burst stimulation (TBS reduced FDI in both control and stressed rats, but this type of plasticity was greater in stressed rats. Commissural-induced inhibition was significantly higher in stressed rats both before and after applying theta-burst stimulation. These findings indicate that the exposure to acute stress affects aspects of local circuit activity and plasticity in the dentate gyrus. It is possible that these alterations underlie some of the behavioral consequences of the stress experience.

  19. Exposure to forced swim stress alters local circuit activity and plasticity in the dentate gyrus of the hippocampus.

    Science.gov (United States)

    Yarom, Orli; Maroun, Mouna; Richter-Levin, Gal

    2008-01-01

    Studies have shown that, depending on its severity and context, stress can affect neural plasticity. Most related studies focused on synaptic plasticity and long-term potentiation (LTP) of principle cells. However, evidence suggests that following high-frequency stimulation, which induces LTP in principal cells, modifications also take place at the level of complex interactions with interneurons within the dentate gyrus, that is, at the local circuit level. So far, the possible effects of stress on local circuit activity and plasticity were not studied. Therefore, we set out to examine the possible alterations in local circuit activity and plasticity following exposure to stress. Local circuit activity and plasticity were measured by using frequency dependant inhibition (FDI) and commissural modulation protocols following exposure to a 15 minute-forced swim trial. Exposure to stress did not alter FDI. The application of theta-burst stimulation (TBS) reduced FDI in both control and stressed rats, but this type of plasticity was greater in stressed rats. Commissural-induced inhibition was significantly higher in stressed rats both before and after applying theta-burst stimulation. These findings indicate that the exposure to acute stress affects aspects of local circuit activity and plasticity in the dentate gyrus. It is possible that these alterations underlie some of the behavioral consequences of the stress experience.

  20. Alteration of non-swelling clay minerals and magadiite by acid activation

    NARCIS (Netherlands)

    Steudel, A.; Batenburg, L.F.; Fischer, H.R.; Weidler, P.G.; Emmerich, K.

    2009-01-01

    The bulk material of three kaolins, a sepiolite, an illite and one magadiite were treated with 1, 5 and 10 M H2SO4 at 80 °C for several hours. The alteration of the non-swelling clay mineral structures was controlled by the individual character of each mineral (chemical composition and initial parti

  1. Aspafilioside B induces G2/M cell cycle arrest and apoptosis by up-regulating H-Ras and N-Ras via ERK and p38 MAPK signaling pathways in human hepatoma HepG2 cells.

    Science.gov (United States)

    Liu, Wei; Ning, Rui; Chen, Rui-Ni; Huang, Xue-Feng; Dai, Qin-Sheng; Hu, Jin-Hua; Wang, Yu-Wen; Wu, Li-Li; Xiong, Jing; Hu, Gang; Guo, Qing-Long; Yang, Jian; Wang, Hao

    2016-05-01

    We recently establish that aspafilioside B, a steroidal saponin extracted from Asparagus filicinus, is an active cytotoxic component. However, its antitumor activity is till unknown. In this study, the anticancer effect of aspafilioside B against HCC cells and the underlying mechanisms were investigated. Our results showed that aspafilioside B inhibited the growth and proliferation of HCC cell lines. Further study revealed that aspafilioside B could significantly induce G2 phase cell cycle arrest and apoptosis, accompanying the accumulation of reactive oxygen species (ROS), but blocking ROS generation with N-acetyl-l-cysteine (NAC) could not prevent G2/M arrest and apoptosis. Additionally, treatment with aspafilioside B induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase. Moreover, both ERK inhibitor PD98059 and p38 inhibitor SB203580 almost abolished the G2/M phase arrest and apoptosis induced by aspafilioside B, and reversed the expression of cell cycle- and apoptosis-related proteins. We also found that aspafilioside B treatment increased both Ras and Raf activation, and transfection of cells with H-Ras and N-Ras shRNA almost attenuated aspafilioside B-induced G2 phase arrest and apoptosis as well as the ERK and p38 activation. Finally, in vivo, aspafilioside B suppressed tumor growth in mouse xenograft models, and the mechanism was the same as in vitro study. Collectively, these findings indicated that aspafilioside B may up-regulate H-Ras and N-Ras, causing c-Raf phosphorylation, and lead to ERK and p38 activation, which consequently induced the G2 phase arrest and apoptosis. This study provides the evidence that aspafilioside B is a promising therapeutic agent against HCC. PMID:25683703

  2. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Lindblad, Maiken Marie; Jakobsen, Jannik E.;

    2015-01-01

    analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs......) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased...... expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early...

  3. Lack of SOD1 gene mutations and activity alterations in two Italian families with amyotrophic lateral sclerosis.

    Science.gov (United States)

    Gestri, D; Cecchi, C; Tedde, A; Latorraca, S; Orlacchio, A; Grassi, E; Massaro, A M; Liguri, G; St George-Hyslop, P H; Sorbi, S

    2000-08-11

    Amyotrophic lateral sclerosis (ALS) is a progressive fatal disorder, which results from the degeneration of motor neurons in the brain and spinal cord. Approximately 20% of the inherited autosomal dominant cases are due to mutations within the gene coding for Cu/Zn superoxide dismutase 1 (SOD1), a cytosolic homodimeric enzyme that catalyzes the dismutation of toxic superoxide anion. We investigated the presence of SOD1 gene mutations and activity alterations in two unrelated families of ALS patients from Elba, an island of central Italy. No mutation in SOD1 exon 1 to 5 and no activity alteration were observed in all members of the two analyzed ALS families (FALS). These data show an apparent heterogeneous distribution of ALS patients with SOD1 gene mutations among different populations and suggest that another genetic locus could be involved in the disease. PMID:10961653

  4. Differential Larval Toxicity and Oviposition Altering Activity of Some Indigenous Plant Extracts against Dengue and Chikungunya Vector Aedes albopictus.

    OpenAIRE

    Ruchi Yadav; Varun Tyagi; Tikar, Sachin N; Sharma, Ajay K.; Murlidhar J Mendki; Jain, Ashok K; Devanathan Sukumaran

    2014-01-01

    Background: Mosquitoes are well known as vectors of several disease causing pathogens. The extensive use of synthetic insecticides in the mosquito control strategies resulted to the development of pesticide resistance and fostered environmental deterioration. Hence in recent years plants become alternative source of mosquito control agents. The present study assessed the larvicidal and oviposition altering activity of six different plants species-Alstonia scholaris, Callistemon viminalis, Hyp...

  5. The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

    International Nuclear Information System (INIS)

    Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion

  6. The Interplay between ROS and Ras GTPases: Physiological and Pathological Implications

    Directory of Open Access Journals (Sweden)

    Elisa Ferro

    2012-01-01

    Full Text Available The members of the RasGTPase superfamily are involved in various signaling networks responsible for fundamental cellular processes. Their activity is determined by their guanine nucleotide-bound state. Recent evidence indicates that some of these proteins may be regulated by redox agents. Reactive oxygen species (ROSs and reactive nitrogen species (RNSs have been historically considered pathological agents which can react with and damage many biological macromolecules including DNA, proteins, and lipids. However, a growing number of reports have suggested that the intracellular production of ROS is tightly regulated and that these redox agents serve as signaling molecules being involved in a variety of cell signaling pathways. Numerous observations have suggested that some Ras GTPases appear to regulate ROS production and that oxidants function as effector molecules for the small GTPases, thus contributing to their overall biological function. Thus, redox agents may act both as upstream regulators and as downstream effectors of Ras GTPases. Here we discuss current understanding concerning mechanisms and physiopathological implications of the interplay between GTPases and redox agents.

  7. 1α, 25-Dihydroxyvitamin D regulates hypoxia-inducible factor-1α in untransformed and Harvey-ras transfected breast epithelial cells.

    Science.gov (United States)

    Jiang, Yan; Zheng, Wei; Teegarden, Dorothy

    2010-12-01

    The purpose of this study was to determine the mechanism by which 1α, 25-dihydroxyvitamin D (1,25(OH)(2)D) alters hypoxia-inducible factor-1α (HIF-1α) protein in untransformed and Harvey-ras (H-ras) oncogene transfected MCF10A breast epithelial cells. Treatment with 1,25(OH)(2)D (10nM) increased both mRNA (2.55±0.6-fold vs. vehicle, p=0.03) and protein levels (2.37±0.3-fold vs. vehicle, pMCF10A cells in 12h, which remained elevated at 24h. However, in H-ras transfected MCF10A cells, 1,25(OH)(2)D treatment increased HIF-1α protein level (2.08±0.38-fold vs. vehicle, p=0.05) at 12h, with no change in mRNA level and HIF-1α protein level returned to baseline after 24h. A transcription inhibitor prevented the 1,25(OH)(2)D induction of HIF-1α protein and mRNA levels in MCF10A cells, but failed to alter the induction of HIF-1α protein level in H-ras transfected MCF10A cells. On the other hand, inhibition of proteasomal degradation prevented the 1,25(OH)(2)D-induced HIF-1α protein level in H-ras transfected MCF10A but not in MCF10A cells. These results support that 1,25(OH)(2)D regulates HIF-1α protein level via transcriptional regulation in MCF10A cells in contrast to through proteosomal degradation with the presence of H-ras oncogene in MCF10A cells.

  8. Prenatal Immune Activation Induces Maturation-Dependent Alterations in the Prefrontal GABAergic Transcriptome

    OpenAIRE

    Richetto, J; Calabrese, F; M.A. RIVA; Meyer, U.

    2014-01-01

    Neuronal dysfunctions in the cortical GABAergic system have been widely documented in neuropsychiatric disorders with prenatal infectious etiologies, including schizophrenia. At least some of these abnormalities may stem from transcriptional impairments in the GABAergic transcriptome. However, the extent to which prenatal exposure to immune challenge can induce long-term alterations in GABAergic gene transcription remains largely elusive. Here, we use an established mouse model of prenatal im...

  9. The Activity of Major Faults and the Hydrothermal Alteration Zone at Tianchi Volcano of Changbaishan

    Institute of Scientific and Technical Information of China (English)

    Liu Mingjun; Gu Menglin; Sun Zhenguo; Wei Haiquan; Jin Bolu

    2004-01-01

    It is found by field investigation that the near horizontal top surface of the brown or brick-red hydrothermal alteration zone varies obviously in elevation at different sections of the same layer on the caldera's inner wall of Tianchi, with that at the north section near the Tianwen Peak about 110 m higher than that at the south near the Jiangjun Peak in Korea. The top surface of the hydrothermal alteration zone can be taken as key horizon to tectonic movement. The difference indicates that the total uplift height of the NW wall of the Liudaogou-TianchiJingfengshan fault, the principal fault trending NE at Tianchi, is bigger than that of the SE wall ever since the occurrence of hydrothermal alteration. This also explains why the topography in the northwest side of Tianchi is steeper and with more developed river system than in the southeast. The uplifting of the northeastern wall is bigger than that of the southwest along the principal NW-trend fault, namely, the Baishanzhen-Tianchi-Jince fault. It is observed from characters of hydrothermal alteration and the palaeoresiduum, that the recent vertical movement rate along the principal NE-trend fault is larger than that of the principal NW-trend fault. The two faults intersect at Tianchi, dividing the volcano into 4 blocks, with the uplift magnitudes decreasing successively in the order of the north, the west, the east and the south block. The biggest uplift of the north block corresponds well to the shallow magma batch in the north of Tianchi observed by DSS and telluric electromagnetic sounding, and etc.and they may be related with the causes.

  10. Alterations of metabolic activity in human osteoarthritic osteoblasts by lipid peroxidation end product 4-hydroxynonenal

    OpenAIRE

    Shi, Qin; Vaillancourt, France; Côté, Véronique; Fahmi, Hassan; Lavigne, Patrick; Afif, Hassan; Di Battista, John A.; Fernandes, Julio C; Benderdour, Mohamed

    2006-01-01

    4-Hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues, but its role in bone metabolism is ill-defined. In this study, we tested the hypothesis that alterations in OA osteoblast metabolism are attributed, in part, to increased levels of HNE. Our data showed that HNE/protein adduct levels were higher in OA osteoblasts compared to normal and when OA osteoblasts were treated with H2O2. Investigating osteoblast markers, we found t...

  11. Central muscarinic cholinergic activation alters interaction between splenic dendritic cell and CD4+CD25- T cells in experimental colitis.

    Directory of Open Access Journals (Sweden)

    Peris Munyaka

    Full Text Available BACKGROUND: The cholinergic anti-inflammatory pathway (CAP is based on vagus nerve (VN activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR signaling. Inflammatory bowel disease (IBD patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs and sequential CD4+/CD25-T cell activation in the context of experimental colitis. METHODS: The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25-T cell co-culture were determined. RESULTS: McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25-T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy. CONCLUSIONS: Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.

  12. RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer

    Science.gov (United States)

    Hrustanovic, Gorjan; Olivas, Victor; Pazarentzos, Evangelos; Tulpule, Asmin; Asthana, Saurabh; Blakely, Collin M; Okimoto, Ross A; Lin, Luping; Neel, Dana S; Sabnis, Amit; Flanagan, Jennifer; Chan, Elton; Varella-Garcia, Marileila; Aisner, Dara L; Vaishnavi, Aria; Ou, Sai-Hong I; Collisson, Eric A; Ichihara, Eiki; Mack, Philip C; Lovly, Christine M; Karachaliou, Niki; Rosell, Rafael; Riess, Jonathan W; Doebele, Robert C; Bivona, Trever G

    2016-01-01

    One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS–mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRASWT) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK–positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes. PMID:26301689

  13. STANDARDIZATION OF SURYA SHEKHARA RAS: A KUPI PAKWA RASAYANA

    Directory of Open Access Journals (Sweden)

    Neha Arya

    2013-10-01

    Full Text Available Demand and acceptance of rasa aushadhis would not increase until unless these mineral and metallic preparations, particularly those which contain heavy metals like Surya Shekhara Ras are standardized. Surya Shekhara Ras is a typical herbo-mineral Kupipakwa rasayana indicated for fever especially in viral fevers. The present work highlights the procedures and techniques adopted for the physico-chemical analysis and characterization of three different samples of Surya Shekhara Ras obtained by well defined process in Rasa Shastra text. Pharmaceutically prepared Surya Shekhara Rasa was subjected for physico-chemical analysis, X-ray diffraction (X-RD, Inductive Coupled Plasma with Optical Emission Spectrometry (I.C.P.O.E.S., Scanning Electron Microscopy (SEM, Energy Dispersive X-ray Analysis (EDAX and Fourier Transform Infrared Spectrometry (FT-IR with a view to standardize the drug through modern techniques. Values of Physico-chemical studies of all three samples were almost in the same range. HgS compound form was detected in X-RD study of 3 samples with Hexagonal crystal structure. I.C.P.O.E.S showed presence of 94 % Hg. FT-IR analysis reveals the presence of similar organic functional groups in three samples of Surya Shekhara Ras. As final results and quantity of elements in all three samples of Surya Shekhara Ras were in same range, it reveals the uniformity in quality of prepared medicine and purity of drug. Thus suggests that the adopted method of preparation can be taken as standard.

  14. Epidermal growth factor receptor (EGFR-RAS signaling pathway in penile squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Hong-Feng Gou

    Full Text Available Penile Squamous Cell Carcinoma (SCC is a rare cancer with poor prognosis and limited response to conventional chemotherapy. The genetic and epigenetic alterations of Epidermal Growth Factor Receptor (EGFR-RAS-RAF signaling in penile SCC are unclear. This study aims to investigate four key members of this pathway in penile SCC. We examined the expression of EGFR and RAS-association domain family 1 A (RASSF1A as well as the mutation status of K-RAS and BRAF in 150 cases of penile SCC. EGFR and RASSF1A expression was evaluated by immunohistochemistry. KRAS mutations at codons 12 and 13, and the BRAF mutation at codon 600 were analyzed on DNA isolated from formalin fixed paraffin embedded tissues by direct genomic sequencing. EGFR expression was positive in all specimens, and its over-expression rate was 92%. RASSF1A expression rate was only 3.42%. Significant correlation was not found between the expression of EGFR or RASSF1A and tumor grade, pT stage or lymph node metastases. The detection of KRAS and BRAF mutations analysis was performed in 94 and 83 tumor tissues, respectively. We found KRAS mutation in only one sample and found no BRAF V600E point mutation. In summary, we found over-expression of EGFR in the majority cases of penile SCC, but only rare expression of RASSF1A, rare KRAS mutation, and no BRAF mutation in penile SCC. These data suggest that anti-EGFR agents may be potentially considered as therapeutic options in penile SCC.

  15. APC and K-ras gene mutation in aberrant crypt foci of human colon

    Institute of Scientific and Technical Information of China (English)

    Ping Yuan; Meng Hong Sun; Jin Sheng Zhang; Xiong Zeng Zhu; Da Ren Shi

    2001-01-01

    AIM To study the genetic alteration in ACF andto define the possibility that ACF may be a veryearly morphological lesion with molecularchanges, and to explore the relationshipbetween ACF and colorectal adenoma evencarcinoma.METHODS DNA from 35 CRC, 15 adenomas, 34ACF and 10 normal mucus was isolated by meansof microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well asthe mutation cluster region (MCR) of APC genewas performed.RESULTS K-ras gene mutation frequency inACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/ 15), and 14.3% (5/ 35)respectively, showing no difference ( P > 0.05)in K-fas gene mutation among three pathologicprocedures. The K-ras gene mutation inadenoma, carcinoma and 4 ACF restricted incodon 12 (GGT→GAT), but the other 2 mutationsfrom ACF located in codon 13 (GGC→GAC). K-res gene mutation was found more frequently inolder patients and patients with polypoidcancer. No mutation in codon 61 was found in thethree tissue types. Mutation rate of APO gene inadenoma and carcinoma was 22.9% (8/35) and26.7% (4/ 15), which was higher than ACF(2.9%) (P < 0.05). APC gene mutation incarcinoma was not correlated with age ofpatients, location, size and differentiation oftumor.CONCLUSION ACF might be a very earlymorphological lesion in the tumorogenesis ofcolorectal tumor. The morphological feature andgene mutation status was different in ACF andadenoma. ACF is possibly putative"microadenoma" that might be the precursor ofadenoma. In addition, the development of asubgroup of colorectal carcinomas mightundergo a way of "normal epithelium→ ACF→carcinomas".

  16. miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras

    International Nuclear Information System (INIS)

    Research highlights: → MicroRNA-181a (miR-181a) was frequently downregulated in oral squamous cell carcinoma (OSCC). → Overexpression of miR-181a suppressed OSCC growth. → K-ras is a novel target of miR-181a. → Decreased miR-181a expression is attributed to its lower promoter activity in OSCC. -- Abstract: MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biological role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3'-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.

  17. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    Directory of Open Access Journals (Sweden)

    Vanessa J. Hall

    2015-10-01

    Full Text Available Animal models of familial juvenile onset of Alzheimer's disease (AD often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw. We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

  18. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

    Science.gov (United States)

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

  19. Alterations of cytochrome P450-dependent monooxygenase activities in Eriocheir japonicus in response to water pollution.

    OpenAIRE

    Ishizuka, M; Hoshi, H.; Minamoto, N; Masuda, M; Kazusaka, A; Fujita, S.

    1996-01-01

    Eriocheir japonicus, fresh-water crabs inhabiting rivers and estuaries in Japan, were investigated for cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activities to see if these activities reflect the river pollution gradient. From the laboratory dose-response experiments, we found that the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene induced total CYP contents, ethoxycoumarin O-deethylase activity, and bunitrolol 4-hydroxylase activity in crab hepatopancreas. In the fi...

  20. Altered neuronal activity in the pedunculopontine nucleus: An electrophysiological study in a rat model of Parkinson's disease.

    Science.gov (United States)

    Geng, Xiwen; Xie, Jinlu; Wang, Xuenan; Wang, Xiusong; Zhang, Xiao; Hou, Yabing; Lei, Chengdong; Li, Min; Qu, Qingyang; He, Tingting; Han, Hongyu; Yao, Xiaomeng; Wang, Min

    2016-05-15

    The pedunculopontine nucleus (PPN) is a new deep brain stimulation target for treating Parkinson's disease (PD). But the alterations of the PPN electrophysiological activities in PD are still debated. To investigate these potential alterations, extracellular single unit and local field potential (LFP) activities in the PPN were recorded in unilateral hemispheric 6-hydroxydopamine (6-OHDA) lesioned rats and in control rats, respectively. The spike activity results revealed two types of neurons (Type I and Type II) with distinct electrophysiological characteristics in the PPN. Both types of neurons had increased firing rate and changed firing pattern in lesioned rats when compared to control rats. Specifically, Type II neurons showed an increased firing rate when the rat state was switched from rest to locomotion. The LFP results demonstrated that lesioned rats had lower LFP power at 0.7-12Hz and higher power at 12-30Hz than did control animals in either resting or locomotor state. These findings provide a better understanding of the effects of 6-OHDA lesion on neuronal activities in the PPN and also provide a proof of the link between this structure and locomotion, which contributes to better understanding the mechanisms of the PPN functioning in the pathophysiology of PD. PMID:26924016

  1. The Protective Arm of the Renin Angiotensin System (RAS)

    DEFF Research Database (Denmark)

    The Protective Arm of the Renin Angiotensin System: Functional Aspects and Therapeutic Implications is the first comprehensive publication to signal the protective role of a distinct part of the renin-angiotensin system (RAS), providing readers with early insight into a complex system which...... will become of major medical importance in the near future. Focusing on recent research, The Protective Arm of the Renin Angiotensin System presents a host of new experimental studies on specific components of the RAS, namely angiotensin AT2 receptors (AT2R), the angiotensin (1-7) peptide with its receptor...... understanding of the protective side of the Renin Angiotensin System (RAS) involving angiotensin AT2 receptor, ACE2, and Ang(1-7)/Mas receptor Combines the knowledge of editors who pioneered research on the protective renin angiotensin system including; Dr. Thomas Unger, one of the founders of AT2 receptor...

  2. A Novel Ras Inhibitor (MDC-1016 Reduces Human Pancreatic Tumor Growth in Mice

    Directory of Open Access Journals (Sweden)

    Gerardo G Mackenzie

    2013-10-01

    Full Text Available Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its persistent resistance to chemotherapy. The currently limited treatment options for pancreatic cancer underscore the need for more efficient agents. Because activating Kras mutations initiate and maintain pancreatic cancer, inhibition of this pathway should have a major therapeutic impact. We synthesized phospho-farnesylthiosalicylic acid (PFTS; MDC-1016 and evaluated its efficacy, safety, and metabolism in preclinical models of pancreatic cancer. PFTS inhibited the growth of human pancreatic cancer cells in culture in a concentration- and time-dependent manner. In an MIA PaCa-2 xenograft mouse model, PFTS at a dose of 50 and 100 mg/kg significantly reduced tumor growth by 62% and 65% (P < .05 vs vehicle control. Furthermore, PFTS prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. PFTS appeared to be safe, with the animals showing no signs of toxicity during treatment. Following oral administration, PFTS was rapidly absorbed, metabolized to FTS and FTS glucuronide, and distributed through the blood to body organs. Mechanistically, PFTS inhibited Ras-GTP, the active form of Ras, both in vitro and in vivo, leading to the inhibition of downstream effector pathways c-RAF/mitogen-activated protein-extracellular signal-regulated kinase (ERK kinase (MEK/ERK1/2 kinase and phosphatidylinositol 3-kinase/AKT. In addition, PFTS proved to be a strong combination partner with phospho-valproic acid, a novel signal transducer and activator of transcription 3 (STAT3 inhibitor, displaying synergy in the inhibition of pancreatic cancer growth. In conclusion, PFTS, a direct Ras inhibitor, is an efficacious agent for the treatment of pancreatic cancer in preclinical models, deserving further evaluation.

  3. Altered Hub Functioning and Compensatory Activations in the Connectome: A Meta-Analysis of Functional Neuroimaging Studies in Schizophrenia

    Science.gov (United States)

    Crossley, Nicolas A.; Mechelli, Andrea; Ginestet, Cedric; Rubinov, Mikail; Bullmore, Edward T.; McGuire, Philip

    2016-01-01

    Background: Functional neuroimaging studies of schizophrenia have identified abnormal activations in many brain regions. In an effort to interpret these findings from a network perspective, we carried out a meta-analysis of this literature, mapping anatomical locations of under- and over-activation to the topology of a normative human functional connectome. Methods: We included 314 task-based functional neuroimaging studies including more than 5000 patients with schizophrenia and over 5000 controls. Coordinates of significant under- or over-activations in patients relative to controls were mapped to nodes of a normative connectome defined by a prior meta-analysis of 1641 functional neuroimaging studies of task-related activation in healthy volunteers. Results: Under-activations and over-activations were reported in a wide diversity of brain regions. Both under- and over-activations were significantly more likely to be located in hub nodes that constitute the “rich club” or core of the normative connectome. In a subset of 121 studies that reported both under- and over-activations in the same patients, we found that, in network terms, these abnormalities were located in close topological proximity to each other. Under-activation in a peripheral node was more frequently associated specifically with over-activation of core nodes than with over-activation of another peripheral node. Conclusions: Although schizophrenia is associated with altered brain functional activation in a wide variety of regions, abnormal responses are concentrated in hubs of the normative connectome. Task-specific under-activation in schizophrenia is accompanied by over-activation of topologically central, less functionally specialized network nodes, which may represent a compensatory response. PMID:26472684

  4. Supramammillary serotonin reduction alters place learning and concomitant hippocampal, septal, and supramammillar theta activity in a Morris water maze

    Science.gov (United States)

    Hernández-Pérez, J. Jesús; Gutiérrez-Guzmán, Blanca E.; López-Vázquez, Miguel Á.; Olvera-Cortés, María E.

    2015-01-01

    Hippocampal theta activity is related to spatial information processing, and high-frequency theta activity, in particular, has been linked to efficient spatial memory performance. Theta activity is regulated by the synchronizing ascending system (SAS), which includes mesencephalic and diencephalic relays. The supramamillary nucleus (SUMn) is located between the reticularis pontis oralis and the medial septum (MS), in close relation with the posterior hypothalamic nucleus (PHn), all of which are part of this ascending system. It has been proposed that the SUMn plays a role in the modulation of hippocampal theta-frequency; this could occur through direct connections between the SUMn and the hippocampus or through the influence of the SUMn on the MS. Serotonergic raphe neurons prominently innervate the hippocampus and several components of the SAS, including the SUMn. Serotonin desynchronizes hippocampal theta activity, and it has been proposed that serotonin may regulate learning through the modulation of hippocampal synchrony. In agreement with this hypothesis, serotonin depletion in the SUMn/PHn results in deficient spatial learning and alterations in CA1 theta activity-related learning in a Morris water maze. Because it has been reported that SUMn inactivation with lidocaine impairs the consolidation of reference memory, we asked whether changes in hippocampal theta activity related to learning would occur through serotonin depletion in the SUMn, together with deficiencies in memory. We infused 5,7-DHT bilaterally into the SUMn in rats and evaluated place learning in the standard Morris water maze task. Hippocampal (CA1 and dentate gyrus), septal and SUMn EEG were recorded during training of the test. The EEG power in each region and the coherence between the different regions were evaluated. Serotonin depletion in the SUMn induced deficient spatial learning and altered the expression of hippocampal high-frequency theta activity. These results provide evidence in

  5. Supplemental feeding for ecotourism reverses diel activity and alters movement patterns and spatial distribution of the southern stingray, Dasyatis americana.

    Directory of Open Access Journals (Sweden)

    Mark J Corcoran

    Full Text Available Southern stingrays, Dasyatis americana, have been provided supplemental food in ecotourism operations at Stingray City Sandbar (SCS, Grand Cayman since 1986, with this site becoming one of the world's most famous and heavily visited marine wildlife interaction venues. Given expansion of marine wildlife interactive tourism worldwide, there are questions about the effects of such activities on the focal species and their ecosystems. We used a combination of acoustic telemetry and tag-recapture efforts to test the hypothesis that human-sourced supplemental feeding has altered stingray activity patterns and habitat use at SCS relative to wild animals at control sites. Secondarily, we also qualitatively estimated the population size of stingrays supporting this major ecotourism venue. Tag-recapture data indicated that a population of at least 164 stingrays, over 80% female, utilized the small area at SCS for prolonged periods of time. Examination of comparative movements of mature female stingrays at SCS and control sites revealed strong differences between the two groups: The fed animals demonstrated a notable inversion of diel activity, being constantly active during the day with little movement at night compared to the nocturnally active wild stingrays; The fed stingrays utilized significantly (p<0.05 smaller 24 hour activity spaces compared to wild conspecifics, staying in close proximity to the ecotourism site; Fed stingrays showed a high degree of overlap in their core activity spaces compared to wild stingrays which were largely solitary in the spaces utilized (72% vs. 3% overlap respectively. Supplemental feeding has strikingly altered movement behavior and spatial distribution of the stingrays, and generated an atypically high density of animals at SCS which could have downstream fitness costs for individuals and potentially broader ecosystem effects. These findings should help environmental managers plan mitigating measures for existing

  6. K-rasG12V transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis

    Institute of Scientific and Technical Information of China (English)

    Yumin Hu; Helene Pelicano; Paul J Chiao; Michael J Keating; Guillermo Garcia-Manero; Peng Huang; Weiqin Lu; Gang Chen; Peng Wang; Zhao Chen; Yan Zhou; Marcia Ogasawara; Dunyaporn Trachootham; Li Feng

    2012-01-01

    Increased aerobic glycolysis and oxidative stress are important features of cancer cell metabolism,but the underlying biochemical and molecular mechanisms remain elusive.Using a tetracycline inducible model,we show that activation of K-rasG12V causes mitochondrial dysfunction,leading to decreased respiration,elevated glycolysis,and increased generation of reactive oxygen species.The K-RAS protein is associated with mitochondria,and induces a rapid suppression of respiratory chain complex-I and a decrease in mitochondrial transmembrane potential by affecting the cyclosporin-sensitive permeability transition pore.Furthermore,pre-induction of K-rasG12V expression in vitro to allow metabolic adaptation to high glycolytic metabolism enhances the ability of the transformed cells to form tumor in vivo.Our study suggests that induction of mitochondrial dysfunction is an important mechanism by which K-rasG12V causes metabolic changes and ROS stress in cancer cells,and promotes tumor development.

  7. The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway.

    Science.gov (United States)

    Park, Jong-In; Strock, Christopher J; Ball, Douglas W; Nelkin, Barry D

    2003-01-01

    Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.

  8. The Ras/Raf/MEK/Extracellular Signal-Regulated Kinase Pathway Induces Autocrine-Paracrine Growth Inhibition via the Leukemia Inhibitory Factor/JAK/STAT Pathway

    OpenAIRE

    Park, Jong-In; Strock, Christopher J.; Ball, Douglas W.; Nelkin, Barry D.

    2003-01-01

    Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF express...

  9. Short-term withdrawal from developmental exposure to cocaine activates the glucocorticoid receptor and alters spine dynamics.

    Science.gov (United States)

    Caffino, Lucia; Giannotti, Giuseppe; Malpighi, Chiara; Racagni, Giorgio; Fumagalli, Fabio

    2015-10-01

    Although glucocorticoid receptors (GRs) contribute to the action of cocaine, their role following developmental exposure to the psychostimulant is still unknown. To address this issue, we exposed adolescent male rats to cocaine (20mg/kg/day) from post-natal day (PND) 28 to PND 42 and sacrificed them at PND 45 or 90. We studied the medial prefrontal cortex (mPFC), a brain region that is still developing during adolescence. In PND 45 rats we found enhanced GR transcription and translation as well as increased trafficking toward the nucleus of the receptor, with no alteration in plasma corticosterone levels. We also showed reduced expression of the GR co-chaperone FKBP51, that normally keeps the receptor in the cytoplasm, and increased expression of Src1, which cooperates in the activation of GR transcriptional activity, revealing that short withdrawal alters the finely tuned mechanisms regulating GR action. Since activation of GRs regulate dendritic spine morphology, we next investigated spine dynamics in cocaine-withdrawn rats. We found that PSD95, cofilin and F-actin, molecules regulating spine actin network, are reduced in the mPFC of PND 45 rats suggesting reduced spine density, confirmed by confocal imaging. Further, formation of filopodia, i.e. the inactive spines, is enhanced suggesting the formation of non-functional spines. Of note, no changes were found in molecules related to GR machinery or spine dynamics following long-term abstinence, i.e. in adult rats (PND 90). These findings demonstrate that short withdrawal promotes plastic changes in the developing brain via the dysregulation of the GR system and alterations in the spine network. PMID:26004981

  10. Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain.

    Science.gov (United States)

    Guasti, Leonardo; Richardson, Denise; Jhaveri, Maulik; Eldeeb, Khalil; Barrett, David; Elphick, Maurice R; Alexander, Stephen P H; Kendall, David; Michael, Gregory J; Chapman, Victoria

    2009-07-01

    Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P pain states.