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Sample records for requires multiple pathways

  1. Multiple Pathways for All Students

    ERIC Educational Resources Information Center

    Stirling, Lee Anna

    2012-01-01

    Maine has been focusing on the importance of postsecondary training. Maine's Skowhegan Area High School (SAHS) and Somerset Career and Technical Center (SCTC) have partnered in a Multiple Pathways initiative (funded by the Nellie Mae Education Foundation) to increase students' high school completion rate and to increase enrollment in postsecondary…

  2. Multiple pathways regulate shoot branching

    PubMed Central

    Rameau, Catherine; Bertheloot, Jessica; Leduc, Nathalie; Andrieu, Bruno; Foucher, Fabrice; Sakr, Soulaiman

    2015-01-01

    Shoot branching patterns result from the spatio-temporal regulation of axillary bud outgrowth. Numerous endogenous, developmental and environmental factors are integrated at the bud and plant levels to determine numbers of growing shoots. Multiple pathways that converge to common integrators are most probably involved. We propose several pathways involving not only the classical hormones auxin, cytokinins and strigolactones, but also other signals with a strong influence on shoot branching such as gibberellins, sugars or molecular actors of plant phase transition. We also deal with recent findings about the molecular mechanisms and the pathway involved in the response to shade as an example of an environmental signal controlling branching. We propose the TEOSINTE BRANCHED1, CYCLOIDEA, PCF transcription factor TB1/BRC1 and the polar auxin transport stream in the stem as possible integrators of these pathways. We finally discuss how modeling can help to represent this highly dynamic system by articulating knowledges and hypothesis and calculating the phenotype properties they imply. PMID:25628627

  3. Destruction Complex Function in the Wnt Signaling Pathway of Drosophila Requires Multiple Interactions Between Adenomatous Polyposis Coli 2 and Armadillo

    PubMed Central

    Kunttas-Tatli, Ezgi; Zhou, Meng-Ning; Zimmerman, Sandra; Molinar, Olivia; Zhouzheng, Fangyuan; Carter, Krista; Kapur, Megha; Cheatle, Alys; Decal, Richard; McCartney, Brooke M.

    2012-01-01

    The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, ?-catenin (?cat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to ?cat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds ?cat and the affinity is significantly increased. These distinct APC–?cat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2’s destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for ?cat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex. PMID:22174073

  4. rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.

    PubMed Central

    Shamloula, Hoda K; Mbogho, Mkajuma P; Pimentel, Angel C; Chrzanowska-Lightowlers, Zosia M A; Hyatt, Vanneta; Okano, Hideyuki; Venkatesh, Tadmiri R

    2002-01-01

    In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways. PMID:12072466

  5. Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF

    SciTech Connect

    Chen Xiaoli; Matsumoto, Hideko; Hinck, Cynthia S.; Al-Hasani, Hadi; St-Denis, Jean-Francois; Whiteheart, Sidney W.; Cushman, Samuel W. . E-mail: sam_cushman@nih.gov

    2005-07-22

    In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by {approx}50% without affecting the GLUT4 fold-translocation response to insulin. However, high expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins.

  6. Beyond Tracking: Multiple Pathways to College, Career, and Civic Participation

    ERIC Educational Resources Information Center

    Oakes, Jeannie, Ed.; Saunders, Marisa, Ed.

    2008-01-01

    "Beyond Tracking" responds to the a sobering assessment of American high schools by delineating and promoting an innovative and well-defined notion of multiple pathways. The book's authors clearly distinguish their use of the term "multiple pathways" from any updated version of the tracking system that marked so many American high schools during…

  7. Moral enhancement requires multiple virtues.

    PubMed

    Hughes, James J

    2015-01-01

    Some of the debates around the concept of moral enhancement have focused on whether the improvement of a single trait, such as empathy or intelligence, would be a good in general, or in all circumstances. All virtue theories, however, both secular and religious, have articulated multiple virtues that temper and inform one another in the development of a mature moral character. The project of moral enhancement requires a reengagement with virtue ethics and contemporary moral psychology to develop an empirically grounded model of the virtues and a fuller model of character development. Each of these virtues may be manipulable with electronic, psychopharmaceutical, and genetic interventions. A set of interdependent virtues is proposed, along with some of the research pointing to ways such virtues could be enhanced. PMID:25473861

  8. Multiple Pathways Linking Racism to Health Outcomes

    PubMed Central

    Harrell, Camara Jules P.; Burford, Tanisha I.; Cage, Brandi N.; Nelson, Travette McNair; Shearon, Sheronda; Thompson, Adrian; Green, Steven

    2012-01-01

    This commentary discusses advances in the conceptual understanding of racism and selected research findings in the social neurosciences. The traditional stress and coping model holds that racism constitutes a source of aversive experiences that, when perceived by the individual, eventually lead to poor health outcomes. Current evidence points to additional psychophysiological pathways linking facets of racist environments with physiological reactions that contribute to disease. The alternative pathways emphasize prenatal experiences, subcortical emotional neural circuits, conscious and preconscious emotion regulation, perseverative cognitions, and negative affective states stemming from racist cognitive schemata. Recognition of these pathways challenges change agents to use an array of cognitive and self-controlling interventions in mitigating racism’s impact. Additionally, it charges policy makers to develop strategies that eliminate deep-seated structural aspects of racism in society. PMID:22518195

  9. Multiple pathways of commodity crop expansion in tropical forest landscapes

    NASA Astrophysics Data System (ADS)

    Meyfroidt, Patrick; Carlson, Kimberly M.; Fagan, Matthew E.; Gutiérrez-Vélez, Victor H.; Macedo, Marcia N.; Curran, Lisa M.; DeFries, Ruth S.; Dyer, George A.; Gibbs, Holly K.; Lambin, Eric F.; Morton, Douglas C.; Robiglio, Valentina

    2014-07-01

    Commodity crop expansion, for both global and domestic urban markets, follows multiple land change pathways entailing direct and indirect deforestation, and results in various social and environmental impacts. Here we compare six published case studies of rapid commodity crop expansion within forested tropical regions. Across cases, between 1.7% and 89.5% of new commodity cropland was sourced from forestlands. Four main factors controlled pathways of commodity crop expansion: (i) the availability of suitable forestland, which is determined by forest area, agroecological or accessibility constraints, and land use policies, (ii) economic and technical characteristics of agricultural systems, (iii) differences in constraints and strategies between small-scale and large-scale actors, and (iv) variable costs and benefits of forest clearing. When remaining forests were unsuitable for agriculture and/or policies restricted forest encroachment, a larger share of commodity crop expansion occurred by conversion of existing agricultural lands, and land use displacement was smaller. Expansion strategies of large-scale actors emerge from context-specific balances between the search for suitable lands; transaction costs or conflicts associated with expanding into forests or other state-owned lands versus smallholder lands; net benefits of forest clearing; and greater access to infrastructure in already-cleared lands. We propose five hypotheses to be tested in further studies: (i) land availability mediates expansion pathways and the likelihood that land use is displaced to distant, rather than to local places; (ii) use of already-cleared lands is favored when commodity crops require access to infrastructure; (iii) in proportion to total agricultural expansion, large-scale actors generate more clearing of mature forests than smallholders; (iv) property rights and land tenure security influence the actors participating in commodity crop expansion, the form of land use displacement, and livelihood outcomes; (v) intensive commodity crops may fail to spare land when inducing displacement. We conclude that understanding pathways of commodity crop expansion is essential to improve land use governance.

  10. Multiple Pathways of Commodity Crop Expansion in Tropical Forest Landscapes

    NASA Technical Reports Server (NTRS)

    Meyfroidt, Patrick; Carlson, Kimberly M.; Fagan, Matthew E.; Gutierrez-Velez, Victor H.; Macedo, Marcia N.; Curran, Lisa M.; DeFries, Ruth S.; Dyer, George A.; Gibbs, Holly K.; Lambin, Eric F.; Morton, Douglas C.; Robiglio, Valentina

    2014-01-01

    Commodity crop expansion, for both global and domestic urban markets, follows multiple land change pathways entailing direct and indirect deforestation, and results in various social and environmental impacts. Here we compare six published case studies of rapid commodity crop expansion within forested tropical regions. Across cases, between 1.7% and 89.5% of new commodity cropland was sourced from forestlands. Four main factors controlled pathways of commodity crop expansion: (i) the availability of suitable forestland, which is determined by forest area, agroecological or accessibility constraints, and land use policies, (ii) economic and technical characteristics of agricultural systems, (iii) differences in constraints and strategies between small-scale and large-scale actors, and (iv) variable costs and benefits of forest clearing. When remaining forests were unsuitable for agriculture and/or policies restricted forest encroachment, a larger share of commodity crop expansion occurred by conversion of existing agricultural lands, and land use displacement was smaller. Expansion strategies of large-scale actors emerge from context-specific balances between the search for suitable lands; transaction costs or conflicts associated with expanding into forests or other state-owned lands versus smallholder lands; net benefits of forest clearing; and greater access to infrastructure in already cleared lands. We propose five hypotheses to be tested in further studies: (i) land availability mediates expansion pathways and the likelihood that land use is displaced to distant, rather than to local places; (ii) use of already-cleared lands is favored when commodity crops require access to infrastructure; (iii) in proportion to total agricultural expansion, large-scale actors generate more clearing of mature forests than smallholders; (iv) property rights and land tenure security influence the actors participating in commodity crop expansion, the form of land use displacement, and livelihood outcomes; (v) intensive commodity crops may fail to spare land when inducing displacement. We conclude that understanding pathways of commodity crop expansion is essential to improve land use governance.

  11. Costs of California Multiple Pathway Programs. Policy Report

    ERIC Educational Resources Information Center

    Parsi, Ace; Plank, David; Stern, David

    2010-01-01

    There is widespread agreement that many of California's high schools are doing a poor job of preparing their students for college and careers. The James Irvine Foundation is sponsoring a major initiative to develop "Multiple Pathways"--now called the Linked Learning approach--as a strategy for improving the performance of California high schools.…

  12. Developing Teacher Leadership in Singapore: Multiple Pathways for Differentiated Journeys

    ERIC Educational Resources Information Center

    Goodwin, A. Lin; Low, Ee Ling; Ng, Pak Tee

    2015-01-01

    In this article, we examine quality teachers through teacher leadership development. Using Singapore as an illustrative case, we describe the redefinition of the teaching profession to include deliberate structures and multiple pathways designed to nurture teacher leaders, and the role of teacher leaders in supporting education reform. We go on to…

  13. Multiple pathways to cellular senescence: role of telomerase repressors.

    PubMed

    Oshimura, M; Barrett, J C

    1997-04-01

    Telomeres progressively shorten with age in somatic cells in culture and in vivo because DNA replication results in the loss of sequences at the 5' ends of double-stranded DNA. Whereas somatic cells do not express the enzyme, telomerase, which adds repeated telomere sequences to chromosome ends, telomerase activity is detected in immortalised and tumour cells in vitro and in primary tumour tissues. This represents an important difference between normal cells and cancer cells, suggesting that telomere shortening causes cellular senescence. Hybrids between immortal cells and normal cells senesce, indicating that immortal cells have lost, mutated or inactivated genes that are required for the programme of senescence in normal cells. Genes involved in the senescence programme have been mapped to over ten different genetic loci using microcell fusion to introduce human chromosomes and restore the senescence programme. Multiple pathways of cellular senescence have also been demonstrated by chromosome transfer, indicating that the functions of the mapped senescence genes are probably different. One possibility is that one or more of these senescence genes may suppress telomerase activity in immortal cells, resulting in telomere shortening and cellular senescence. To test this hypothesis, telomerase activity and the length of terminal restriction fragments (TRFs) have been examined in microcell hybrids. Re-introduction of a normal chromosome 3 into the renal cell carcinoma cell line RCC23, which has the short arm of chromosome 3, restored cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells containing the introduced chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to the parental RCC23. Thus, restoration of cellular senescence by chromosome 3 is associated with repression of telomerase function in RCC cells. This evidence suggests that telomerase suppression is one of several pathways involved in immortalisation. PMID:9282109

  14. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  15. EVIDENCE FOR MULTIPLE PATHWAYS TO DEUTERIUM ENHANCEMENTS IN PROTOPLANETARY DISKS

    SciTech Connect

    Oeberg, Karin I.; Qi, Chunhua; Wilner, David J.; Hogerheijde, Michiel R.

    2012-04-20

    The distributions of deuterated molecules in protoplanetary disks are expected to depend on the molecular formation pathways. We use observations of spatially resolved DCN emission from the disk around TW Hya, acquired during ALMA science verification with a {approx}3'' synthesized beam, together with comparable DCO{sup +} observations from the Submillimeter Array, to investigate differences in the radial distributions of these species and hence differences in their formation chemistry. In contrast to DCO{sup +}, which shows an increasing column density with radius, DCN is better fit by a model that is centrally peaked. We infer that DCN forms at a smaller radii and thus at higher temperatures than DCO{sup +}. This is consistent with chemical network model predictions of DCO{sup +} formation from H{sub 2}D{sup +} at T < 30 K and DCN formation from additional pathways involving CH{sub 2}D{sup +} at higher temperatures. We estimate a DCN/HCN abundance ratio of {approx}0.017, similar to the DCO{sup +}/HCO{sup +} abundance ratio. Deuterium fractionation appears to be efficient at a range of temperatures in this protoplanetary disk. These results suggest caution in interpreting the range of deuterium fractions observed in solar system bodies, as multiple formation pathways should be taken into account.

  16. Multiple sweet receptors and transduction pathways revealed in knockout mice by temperature dependence and gurmarin sensitivity.

    PubMed

    Ohkuri, Tadahiro; Yasumatsu, Keiko; Horio, Nao; Jyotaki, Masafumi; Margolskee, Robert F; Ninomiya, Yuzo

    2009-04-01

    Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's Galpha subunit (Galphagust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, Galphagust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In Galphagust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for Galphagust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue. PMID:19211717

  17. Germline Mutations in Oncogene-Induced Senescence Pathways are Associated with Multiple Sessile Serrated Adenomas

    PubMed Central

    Gala, Manish K.; Mizukami, Yusuke; Le, Long P.; Moriichi, Kentaro; Austin, Thomas; Yamamoto, Masayoshi; Lauwers, Gregory Y.; Bardeesy, Nabeel; Chung, Daniel C.

    2014-01-01

    Background & Aims Little is known about the genetic factors that contribute to development of sessile serrated adenomas (SSAs). SSAs contain somatic mutations in BRAF or KRAS early in development. However, evidence from humans and mouse models indicates that these mutations result in oncogene-induced senescence (OIS) of intestinal crypt cells. Progression to serrated neoplasia requires cells to escape OIS, via inactivation of tumor suppressor pathways. We investigated whether individuals with multiple SSAs carry germline loss-of-function mutations (nonsense and splice-site) in genes that regulate OIS – the p16–Rb and ATM–ATR DNA damage response pathways. Methods Through bioinformatic analysis of the literature, we identified a set of genes that function at main nodes of the p16–Rb and ATM–ATR DNA damage response pathways. We performed whole-exome sequencing of 20 unrelated individuals with multiple SSAs; most had features of serrated polyposis. We compared sequences with those from 4300 individuals, matched for ethnicity (controls). We also used an integrative genomics approach to identify additional genes involved in senescence mechanisms. Results We identified mutations in genes that regulate senescence (ATM, PIF1, TELO2, XAF1, and RBL1) in 5/20 individuals with multiple SSAs (odds ratio [OR]=3.0; 95% confidence interval [CI], 0.9–8.9; P=.04). In 2 individuals, we found nonsense mutations in RNF43, indicating that it is also associated with multiple serrated polyps (OR=460; 95% CI, 23.1– 16384; P=6.8×10?5). In knockdown experiments with pancreatic duct cells exposed to ultraviolet light, RNF43 appeared to function as a regulator of ATM–ATR DNA damage response. Conclusions We associated germline loss-of-function variants in genes that regulate senescence pathways with the development of multiple SSAs. We identified RNF43 as a regulator of the DNA damage response, and associated nonsense variants in this gene with high risk of developing SSAs. PMID:24512911

  18. Multiple Molecular Pathways in Melanomagenesis: Characterization of Therapeutic Targets

    PubMed Central

    Palmieri, Giuseppe; Ombra, MariaNeve; Colombino, Maria; Casula, Milena; Sini, MariaCristina; Manca, Antonella; Paliogiannis, Panagiotis; Ascierto, Paolo Antonio; Cossu, Antonio

    2015-01-01

    Molecular mechanisms involved in pathogenesis of malignant melanoma have been widely studied and novel therapeutic treatments developed in recent past years. Molecular targets for therapy have mostly been recognized in the RAS–RAF–MEK–ERK and PI3K–AKT signaling pathways; small-molecule inhibitors were drawn to specifically target key kinases. Unfortunately, these targeted drugs may display intrinsic or acquired resistance and various evidences suggest that inhibition of a single effector of the signal transduction cascades involved in melanoma pathogenesis may be ineffective in blocking the tumor growth. In this sense, a wider comprehension of the multiple molecular alterations accounting for either response or resistance to treatments with targeted inhibitors may be helpful in assessing, which is the most effective combination of such therapies. In the present review, we summarize the known molecular mechanisms underlying either intrinsic and acquired drug resistance either alternative roads to melanoma pathogenesis, which may become targets for innovative anticancer approaches. PMID:26322273

  19. Estrogen Signaling Multiple Pathways to Impact Gene Transcription

    PubMed Central

    Marino, Maria; Galluzzo, Paola; Ascenzi, Paolo

    2006-01-01

    Steroid hormones exert profound effects on cell growth, development, differentiation, and homeostasis. Their effects are mediated through specific intracellular steroid receptors that act via multiple mechanisms. Among others, the action mechanism starting upon 17?-estradiol (E2) binds to its receptors (ER) is considered a paradigmatic example of how steroid hormones function. Ligand-activated ER dimerizes and translocates in the nucleus where it recognizes specific hormone response elements located in or near promoter DNA regions of target genes. Behind the classical genomic mechanism shared with other steroid hormones, E2 also modulates gene expression by a second indirect mechanism that involves the interaction of ER with other transcription factors which, in turn, bind their cognate DNA elements. In this case, ER modulates the activities of transcription factors such as the activator protein (AP)-1, nuclear factor-?B (NF-?B) and stimulating protein-1 (Sp-1), by stabilizing DNA-protein complexes and/or recruiting co-activators. In addition, E2 binding to ER may also exert rapid actions that start with the activation of a variety of signal transduction pathways (e.g. ERK/MAPK, p38/MAPK, PI3K/AKT, PLC/PKC). The debate about the contribution of different ER-mediated signaling pathways to coordinate the expression of specific sets of genes is still open. This review will focus on the recent knowledge about the mechanism by which ERs regulate the expression of target genes and the emerging field of integration of membrane and nuclear receptor signaling, giving examples of the ways by which the genomic and non-genomic actions of ERs on target genes converge. PMID:18369406

  20. Multiple Reaction Pathways during Radiolytic Oxidation of Pyrite

    NASA Astrophysics Data System (ADS)

    Lefticariu, L.; Pratt, L.; Laverne, J. A.; Ripley, E. M.

    2005-12-01

    Passage of ionizing radiation through groundwater produces a complex mixture of short-lived ions, free radicals, and excited molecules that participate in a wide range of chemical reactions and accelerate water-rock interaction. Radiolysis of groundwater in contact with sulfide minerals or elemental sulfur can produce plumes of partially to fully oxidized sulfur species, thereby stimulating microbial metabolism in unexpected subsurface environments. In order to study fractionation of sulfur isotopes during radiolysis, initial experiments were performed using sealed quartz tubes that contained pyrite and millimolar solutions of hydrogen peroxide (H2O2) that were reacted at temperatures from 4 to 150 C over time periods of days to week. Mineralogical, chemical, and stable isotopic date from H2O2 experiments reveal multiple pathways for pyrite oxidation and distinct assemblages of products at difference temperatures. Sulfur isotopic signatures of oxidized products are enriched in 32S by 0.5 to 2 per mil compared to source sulfate. Radiation experiments were carried out using a 60Co gamma sources at the Radiation Laboratory of the University of Notre Dame. Sealed quartz tubes that contained pyrite and deoxygenated DI water were irradiated from 1 to 14 hours with a dose rate of 11.3 krad/min (113 Gy/min). Initial experiments produced oxidized sulfur as gaseous (e.g., SO2) and aqueous (e.g, SO4) species at concentrations directly correlated to the volume of water and total irradiation dose. Radiolysis proves to be an effective mechanism for the production of oxidizing species in geologically long-lived oxidizing systems. Iron sulfide minerals are decomposed and iron oxide/hydroxide minerals and sulfate ions are produced. Recognizing geochemical signatures of radiolytic oxidation is particularly important for understanding biotic and abiotic reaction pathways in environments where molecular oxygen is negligible and for assessing potential sources of chemical energy for microbial metabolism in the deep subsurface of Earth and Mars.

  1. Multiple-camera tracking: UK government requirements

    NASA Astrophysics Data System (ADS)

    Hosmer, Paul

    2007-10-01

    The Imagery Library for Intelligent Detection Systems (i-LIDS) is the UK government's new standard for Video Based Detection Systems (VBDS). The standard was launched in November 2006 and evaluations against it began in July 2007. With the first four i-LIDS scenarios completed, the Home Office Scientific development Branch (HOSDB) are looking toward the future of intelligent vision in the security surveillance market by adding a fifth scenario to the standard. The fifth i-LIDS scenario will concentrate on the development, testing and evaluation of systems for the tracking of people across multiple cameras. HOSDB and the Centre for the Protection of National Infrastructure (CPNI) identified a requirement to track targets across a network of CCTV cameras using both live and post event imagery. The Detection and Vision Systems group at HOSDB were asked to determine the current state of the market and develop an in-depth Operational Requirement (OR) based on government end user requirements. Using this OR the i-LIDS team will develop a full i-LIDS scenario to aid the machine vision community in its development of multi-camera tracking systems. By defining a requirement for multi-camera tracking and building this into the i-LIDS standard the UK government will provide a widely available tool that developers can use to help them turn theory and conceptual demonstrators into front line application. This paper will briefly describe the i-LIDS project and then detail the work conducted in building the new tracking aspect of the standard.

  2. Multiple Pathways to Graduation: New Routes to High School Completion. CRPE Working Paper #2010_2

    ERIC Educational Resources Information Center

    Marsh, Shannon

    2010-01-01

    Concerned about the persistently high dropout rates from big-city secondary schools, education leaders are trying a new approach to increasing the graduation rate--multiple pathways to graduation. Multiple pathways initiatives are relatively new and far from proven. Even the most advanced examples face significant issues, i.e., the need to…

  3. Targeting Apoptosis and Multiple Signaling Pathways with Icariside II in Cancer Cells

    PubMed Central

    Khan, Muhammad; Maryam, Amara; Qazi, Javed Iqbal; Ma, Tonghui

    2015-01-01

    Cancer is the second leading cause of deaths worldwide. Despite concerted efforts to improve the current therapies, the prognosis of cancer remains dismal. Highly selective or specific blocking of only one of the signaling pathways has been associated with limited or sporadic responses. Using targeted agents to inhibit multiple signaling pathways has emerged as a new paradigm for anticancer treatment. Icariside II, a flavonol glycoside, is one of the major components of Traditional Chinese Medicine Herba epimedii and possesses multiple biological and pharmacological properties including anti-inflammatory, anti-osteoporosis, anti-oxidant, anti-aging, and anticancer activities. Recently, the anticancer activity of Icariside II has been extensively investigated. Here, in this review, our aim is to give our perspective on the current status of Icariside II, and discuss its natural sources, anticancer activity, molecular targets and the mechanisms of action with specific emphasis on apoptosis pathways which may help the further design and conduct of preclinical and clinical trials. Icariside II has been found to induce apoptosis in various human cancer cell lines of different origin by targeting multiple signaling pathways including STAT3, PI3K/AKT, MAPK/ERK, COX-2/PGE2 and ?-Catenin which are frequently deregulated in cancers, suggesting that this collective activity rather than just a single effect may play an important role in developing Icariside II into a potential lead compound for anticancer therapy. This review suggests that Icariside II provides a novel opportunity for treatment of cancers, but additional investigations and clinical trials are still required to fully understand the mechanism of therapeutic effects to further validate it in anti-tumor therapy. PMID:26221076

  4. The Cbln Family of Proteins Interact with Multiple Signaling Pathways

    PubMed Central

    Wei, Peng; Pattarini, Roberto; Rong, Yongqi; Guo, Hong; Bansal, Parmil K; Kusnoor, Sheila V; Deutch, Ariel Y; Parris, Jennifer; Morgan, James I

    2012-01-01

    Cbln1 is essential for synapse integrity in cerebellum through assembly into complexes that bridge presynaptic ?-neurexins (Nrxn) to postsynaptic GluR?2. However, GluR?2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluR?2 and Nrxns1–3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, DCC (deleted in colorectal cancer) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln1-null mice. Thus Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less-well characterized role of netrin/DCC in adult brain. PMID:22220752

  5. Rho/Rock signal transduction pathway is required for MSC tenogenic differentiation

    PubMed Central

    Maharam, Edward; Yaport, Miguel; Villanueva, Nathaniel L; Akinyibi, Takintope; Laudier, Damien; He, Zhiyong; Leong, Daniel J; Sun, Hui B

    2015-01-01

    Mesenchymal stem cell (MSC)-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical stimuli, including into tenocytes. Cell elongation and cytoskeletal tension have been shown to be instrumental to the process of MSC differentiation. Previous studies have shown that inhibition of stress fiber formation leads MSCs to default toward an adipogenic lineage, which suggests that stress fibers are required for MSCs to sense the environmental factors that can induce differentiation into tenocytes. As the Rho/ROCK signal transduction pathway plays a critical role in both stress fiber formation and in cell sensation, we examined whether the activation of this pathway was required when inducing MSC tendon differentiation using rope-like silk scaffolds. To accomplish this, we employed a loss-of-function approach by knocking out ROCK, actin and myosin (two other components of the pathway) using the specific inhibitors Y-27632, Latrunculin A and blebbistatin, respectively. We demonstrated that independently disrupting the cytoskeleton and the Rho/ROCK pathway abolished the expression of tendon differentiation markers and led to a loss of spindle morphology. Together, these studies suggest that the tension that is generated by MSC elongation is essential for MSC teno-differentiation and that the Rho/ROCK pathway is a critical mediator of tendon differentiation on rope-like silk scaffolds. PMID:26509098

  6. Pathway-based network analysis of myeloma tumors: monoclonal gammopathy of unknown significance, smoldering multiple myeloma, and multiple myeloma.

    PubMed

    Dong, L; Chen, C Y; Ning, B; Xu, D L; Gao, J H; Wang, L L; Yan, S Y; Cheng, S

    2015-01-01

    Although many studies have been carried out on monoclonal gammopathy of unknown significances (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma (MM), their classification and underlying pathogenesis are far from elucidated. To discover the relationships among MGUS, SMM, and MM at the transcriptome level, differentially expressed genes in MGUS, SMM, and MM were identified by the rank product method, and then co-expression networks were constructed by integrating the data. Finally, a pathway-network was constructed based on Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and the relationships between the pathways were identified. The results indicated that there were 55, 78, and 138 pathways involved in the myeloma tumor developmental stages of MGUS, SMM, and MM, respectively. The biological processes identified therein were found to have a close relationship with the immune system. Processes and pathways related to the abnormal activity of DNA and RNA were also present in SMM and MM. Six common pathways were found in the whole process of myeloma tumor development. Nine pathways were shown to participate in the progression of MGUS to SMM, and prostate cancer was the sole pathway that was involved only in MGUS and MM. Pathway-network analysis might provide a new indicator for the developmental stage diagnosis of myeloma tumors. PMID:26345890

  7. Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

    E-print Network

    Siegrist, M. Sloan

    The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation ...

  8. ANGUSTIFOLIA mediates one of the multiple SCRAMBLED signaling pathways regulating cell growth pattern in Arabidopsis thaliana.

    PubMed

    Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

    2015-09-25

    In Arabidopsis thaliana, an atypical leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), is required for multiple developmental processes including root epidermal cell fate determination, silique dehiscence, inflorescence growth, ovule morphogenesis, and tissue morphology. Previous work suggested that SCM regulates these multiple pathways using distinct mechanisms via interactions with specific downstream factors. ANGUSTIFOLIA (AN) is known to regulate cell and tissue morphogenesis by influencing cortical microtubule arrangement, and recently, the AN protein was reported to interact with the SCM protein. Therefore, we examined whether AN might be responsible for mediating some of the SCM-dependent phenotypes. We discovered that both scm and an mutant lines cause an abnormal spiral or twisting growth of roots, but only the scm mutant affected root epidermal patterning. The siliques of the an and scm mutants also exhibited spiral growth, as previously reported, but only the scm mutant altered silique dehiscence. Interestingly, we discovered that the spiral growth of roots and siliques of the scm mutant is rescued by a truncated SCM protein that lacks its kinase domain, and that a juxtamembrane domain of SCM was sufficient for AN binding in the yeast two-hybrid analysis. These results suggest that the AN protein is one of the critical downstream factors of SCM pathways specifically responsible for mediating its effects on cell/tissue morphogenesis through cortical microtubule arrangement. PMID:26296462

  9. Cinnamon polyphenols regulate multiple metabolic pathways involved in intestinal lipid metabolism of primary small intestinal enterocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing evidence suggests that dietary factors may affect the expression of multiple genes and signaling pathways including those that regulate intestinal lipoprotein metabolism. The small intestine is actively involved in the regulation of dietary lipid absorption, intracellular transport and me...

  10. Reliable pre-eclampsia pathways based on multiple independent microarray data sets.

    PubMed

    Kawasaki, Kaoru; Kondoh, Eiji; Chigusa, Yoshitsugu; Ujita, Mari; Murakami, Ryusuke; Mogami, Haruta; Brown, J B; Okuno, Yasushi; Konishi, Ikuo

    2015-02-01

    Pre-eclampsia is a multifactorial disorder characterized by heterogeneous clinical manifestations. Gene expression profiling of preeclamptic placenta have provided different and even opposite results, partly due to data compromised by various experimental artefacts. Here we aimed to identify reliable pre-eclampsia-specific pathways using multiple independent microarray data sets. Gene expression data of control and preeclamptic placentas were obtained from Gene Expression Omnibus. Single-sample gene-set enrichment analysis was performed to generate gene-set activation scores of 9707 pathways obtained from the Molecular Signatures Database. Candidate pathways were identified by t-test-based screening using data sets, GSE10588, GSE14722 and GSE25906. Additionally, recursive feature elimination was applied to arrive at a further reduced set of pathways. To assess the validity of the pre-eclampsia pathways, a statistically-validated protocol was executed using five data sets including two independent other validation data sets, GSE30186, GSE44711. Quantitative real-time PCR was performed for genes in a panel of potential pre-eclampsia pathways using placentas of 20 women with normal or severe preeclamptic singleton pregnancies (n = 10, respectively). A panel of ten pathways were found to discriminate women with pre-eclampsia from controls with high accuracy. Among these were pathways not previously associated with pre-eclampsia, such as the GABA receptor pathway, as well as pathways that have already been linked to pre-eclampsia, such as the glutathione and CDKN1C pathways. mRNA expression of GABRA3 (GABA receptor pathway), GCLC and GCLM (glutathione metabolic pathway), and CDKN1C was significantly reduced in the preeclamptic placentas. In conclusion, ten accurate and reliable pre-eclampsia pathways were identified based on multiple independent microarray data sets. A pathway-based classification may be a worthwhile approach to elucidate the pathogenesis of pre-eclampsia. PMID:25323968

  11. Biochemical Genetic Pathways that Modulate Aging in Multiple Species.

    PubMed

    Bitto, Alessandro; Wang, Adrienne M; Bennett, Christopher F; Kaeberlein, Matt

    2015-01-01

    The mechanisms underlying biological aging have been extensively studied in the past 20 years with the avail of mainly four model organisms: the budding yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fruitfly Drosophila melanogaster, and the domestic mouse Mus musculus. Extensive research in these four model organisms has identified a few conserved genetic pathways that affect longevity as well as metabolism and development. Here, we review how the mechanistic target of rapamycin (mTOR), sirtuins, adenosine monophosphate-activated protein kinase (AMPK), growth hormone/insulin-like growth factor 1 (IGF-1), and mitochondrial stress-signaling pathways influence aging and life span in the aforementioned models and their possible implications for delaying aging in humans. We also draw some connections between these biochemical pathways and comment on what new developments aging research will likely bring in the near future. PMID:26525455

  12. Piceatannol exhibits selective toxicity to multiple myeloma cells and influences the Wnt/ beta-catenin pathway.

    PubMed

    Schmeel, Frederic Carsten; Schmeel, Leonard Christopher; Kim, Young; Schmidt-Wolf, Ingo G H

    2014-12-01

    Aberrant activation of Wnt/?-catenin signaling promotes development and progression of various malignant neoplasms. Recent studies observed that the Wnt pathway is constitutively active in myeloma cells and promotes an exaggerated proliferation. Thus, the Wnt signaling pathway might be an attractive therapeutic target for multiple myeloma. In this study, we identified piceatannol as an inhibitor of the Wnt/?-catenin pathway and as a potent inducer of apoptosis in myeloma cells. Interestingly, healthy cells remained mainly unaffected. These results reveal a significant selective induction of apoptosis by piceatannol and suggest a significant in vivo effect against multiple myeloma. PMID:24470348

  13. Cytoplasmic Phospholipase A2 Antagonists Inhibit Multiple Endocytic Membrane Trafficking Pathways

    PubMed Central

    Doody, Anne M.; Antosh, Amy L.; Brown, William J.

    2009-01-01

    Previous studies have suggested a role for cytosolic Ca2+-independent phospholipase A2 (PLA2) activity in the formation of endosome membrane tubules that participate in the export of transferrin (Tf) and transferrin receptors (TfR) from sorting endosomes (SEs) and the endocytic recycling compartment (ERC). Here we show that the PLA2 requirement is a general feature of endocytic trafficking. The reversible cytoplasmic PLA2-antagonist ONO-RS-082 (ONO) produced a concentration-dependent, differential block in the endocytic recycling of both low-density lipoprotein receptor (LDLR) and TfRs, and in the degradative pathways of LDL and epidermal growth factor (EGF). These results are consistent with the model that a cytoplasmic PLA2 plays a general role in the export of cargo from multiple endocytic compartments by mediating the formation of membrane tubules. PMID:19695219

  14. Hedgehog Pathway Modulation by Multiple Lipid Binding Sites on the Smoothened Effector of Signal Response

    PubMed Central

    Myers, Benjamin R.; Sever, Navdar; Chong, Yong Chun; Kim, James; Belani, Jitendra D.; Rychnovsky, Scott; Bazan, J. Fernando; Beachy, Philip A.

    2014-01-01

    Summary Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo), by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs. PMID:23954590

  15. Ferritin Is Required in Multiple Tissues during Drosophila melanogaster Development

    PubMed Central

    Blowes, Liisa M.; Missirlis, Fanis; Riesgo-Escovar, Juan R.

    2015-01-01

    In Drosophila melanogaster, iron is stored in the cellular endomembrane system inside a protein cage formed by 24 ferritin subunits of two types (Fer1HCH and Fer2LCH) in a 1:1 stoichiometry. In larvae, ferritin accumulates in the midgut, hemolymph, garland, pericardial cells and in the nervous system. Here we present analyses of embryonic phenotypes for mutations in Fer1HCH, Fer2LCH and in both genes simultaneously. Mutations in either gene or deletion of both genes results in a similar set of cuticular embryonic phenotypes, ranging from non-deposition of cuticle to defects associated with germ band retraction, dorsal closure and head involution. A fraction of ferritin mutants have embryonic nervous systems with ventral nerve cord disruptions, misguided axonal projections and brain malformations. Ferritin mutants die with ectopic apoptotic events. Furthermore, we show that ferritin maternal contribution, which varies reflecting the mother’s iron stores, is used in early development. We also evaluated phenotypes arising from the blockage of COPII transport from the endoplasmic reticulum to the Golgi apparatus, feeding the secretory pathway, plus analysis of ectopically expressed and fluorescently marked Fer1HCH and Fer2LCH. Overall, our results are consistent with insect ferritin combining three functions: iron storage, intercellular iron transport, and protection from iron-induced oxidative stress. These functions are required in multiple tissues during Drosophila embryonic development. PMID:26192321

  16. Bayesian Joint Selection of Genes and Pathways: Applications in Multiple Myeloma Genomics

    PubMed Central

    Zhang, Lin; Morris, Jeffrey S; Zhang, Jiexin; Orlowski, Robert Z; Baladandayuthapani, Veerabhadran

    2014-01-01

    It is well-established that the development of a disease, especially cancer, is a complex process that results from the joint effects of multiple genes involved in various molecular signaling pathways. In this article, we propose methods to discover genes and molecular pathways significantly associated with clinical outcomes in cancer samples. We exploit the natural hierarchal structure of genes related to a given pathway as a group of interacting genes to conduct selection of both pathways and genes. We posit the problem in a hierarchical structured variable selection (HSVS) framework to analyze the corresponding gene expression data. HSVS methods conduct simultaneous variable selection at the pathway (group level) and the gene (within-group) level. To adapt to the overlapping group structure present in the pathway–gene hierarchy of the data, we developed an overlap-HSVS method that introduces latent partial effect variables that partition the marginal effect of the covariates and corresponding weights for a proportional shrinkage of the partial effects. Combining gene expression data with prior pathway information from the KEGG databases, we identified several gene–pathway combinations that are significantly associated with clinical outcomes of multiple myeloma. Biological discoveries support this relationship for the pathways and the corresponding genes we identified. PMID:25520554

  17. Multiple pathways in the oxidation of a NADH analogue.

    PubMed

    Song, Na; Zhang, Ming-Tian; Binstead, Robert A; Fang, Zhen; Meyer, Thomas J

    2014-04-21

    Oxidation of the NADH analogue, N-benzyl-1,4-dihydronicotinamide (BNAH), by the 1e(-) acceptor, [Os(dmb)3](3+), and 2e(-)/2H(+) acceptor, benzoquinone (Q), has been investigated in aqueous solutions over extended pH and buffer concentration ranges by application of a double-mixing stopped-flow technique in order to explore the redox pathways available to this important redox cofactor. Our results indicate that oxidation by quinone is dominated by hydride transfer, and a pathway appears with added acids involving concerted hydride-proton transfer (HPT) in which synchronous transfer of hydride to one O-atom at Q and proton transfer to the second occurs driven by the formation of the stable H2Q product. Oxidation by [Os(dmb)3](3+) occurs by outer-sphere electron transfer including a pathway involving ion-pair preassociation of HPO4(2-) with the complex that may also involve a concerted proton transfer. PMID:24716437

  18. Multiple Pathways Involved in the Biosynthesis of Anandamide

    PubMed Central

    Liu, Jie; Wang, Lei; Harvey-White, Judith; Huang, Bill X.; Kim, Hee-Yong; Luquet, Serge; Palmiter, Richard D.; Krystal, Gerald; Rai, Ravi; Mahadevan, Anu; Razdan, Raj K.; Kunos, George

    2008-01-01

    Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by ?,?-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5? phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD?/? mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer incubations (60 min). In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway. PMID:17631919

  19. Romidepsin targets multiple survival signaling pathways in malignant T cells

    PubMed Central

    Valdez, B C; Brammer, J E; Li, Y; Murray, D; Liu, Y; Hosing, C; Nieto, Y; Champlin, R E; Andersson, B S

    2015-01-01

    Romidepsin is a cyclic molecule that inhibits histone deacetylases. It is Food and Drug Administration-approved for treatment of cutaneous and peripheral T-cell lymphoma, but its precise mechanism of action against malignant T cells is unknown. To better understand the biological effects of romidepsin in these cells, we exposed PEER and SUPT1 T-cell lines, and a primary sample from T-cell lymphoma patient (Patient J) to romidepsin. We then examined the consequences in some key oncogenic signaling pathways. Romidepsin displayed IC50 values of 10.8, 7.9 and 7.0?nm in PEER, SUPT1 and Patient J cells, respectively. Strong inhibition of histone deacetylases and demethylases, increased production of reactive oxygen species and decreased mitochondrial membrane potential were observed, which may contribute to the observed DNA-damage response and apoptosis. The stress-activated protein kinase/c-Jun N-terminal kinase signaling pathway and unfolded protein response in the endoplasmic reticulum were activated, whereas the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and ?-catenin pro-survival pathways were inhibited. The decreased level of ?-catenin correlated with the upregulation of its inhibitor SFRP1 through romidepsin-mediated hypomethylation of its gene promoter. Our results provide new insights into how romidepsin invokes malignant T-cell killing, show evidence of its associated DNA hypomethylating activity and offer a rationale for the development of romidepsin-containing combination therapies. PMID:26473529

  20. Directed evolution of a cellobiose utilization pathway in Saccharomyces cerevisiae by simultaneously engineering multiple proteins

    PubMed Central

    2013-01-01

    Background The optimization of metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals. One such significant pathway is the cellobiose utilization pathway, identified as a promising route in biomass utilization. Here we describe the optimization of cellobiose consumption and ethanol productivity by simultaneously engineering both proteins of the pathway, the ?-glucosidase (gh1-1) and the cellodextrin transporter (cdt-1), in an example of pathway engineering through directed evolution. Results The improved pathway was assessed based on the strain specific growth rate on cellobiose, with the final mutant exhibiting a 47% increase over the wild-type pathway. Metabolite analysis of the engineered pathway identified a 49% increase in cellobiose consumption (1.78 to 2.65 g cellobiose/(L?·?h)) and a 64% increase in ethanol productivity (0.611 to 1.00 g ethanol/(L?·?h)). Conclusions By simultaneously engineering multiple proteins in the pathway, cellobiose utilization in S. cerevisiae was improved. This optimization can be generally applied to other metabolic pathways, provided a selection/screening method is available for the desired phenotype. The improved in vivo cellobiose utilization demonstrated here could help to decrease the in vitro enzyme load in biomass pretreatment, ultimately contributing to a reduction in the high cost of biofuel production. PMID:23802545

  1. Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes

    PubMed Central

    Alsford, Sam; Horn, David; Field, Mark C.

    2015-01-01

    Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes. PMID:26492041

  2. 10 CFR 63.115 - Requirements for multiple barriers.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Technical Criteria Postclosure Performance Assessment § 63.115 Requirements for multiple barriers....

  3. Patient-Specific Pathway Analysis Using PARADIGM Identifies Key Activities in Multiple Cancers - Josh Stuart, TCGA Scientific Symposium 2011

    Cancer.gov

    Home News and Events Multimedia Library Videos Patient-Specific Pathway Analysis Using PARADIGM Identifies Key Activities in Cancers - Josh Stuart Patient-Specific Pathway Analysis Using PARADIGM Identifies Key Activities in Multiple Cancers - Josh

  4. Retinoic acid activates two pathways required for meiosis in mice.

    PubMed

    Koubova, Jana; Hu, Yueh-Chiang; Bhattacharyya, Tanmoy; Soh, Y Q Shirleen; Gill, Mark E; Goodheart, Mary L; Hogarth, Cathryn A; Griswold, Michael D; Page, David C

    2014-08-01

    In all sexually reproducing organisms, cells of the germ line must transition from mitosis to meiosis. In mice, retinoic acid (RA), the extrinsic signal for meiotic initiation, activates transcription of Stra8, which is required for meiotic DNA replication and the subsequent processes of meiotic prophase. Here we report that RA also activates transcription of Rec8, which encodes a component of the cohesin complex that accumulates during meiotic S phase, and which is essential for chromosome synapsis and segregation. This RA induction of Rec8 occurs in parallel with the induction of Stra8, and independently of Stra8 function, and it is conserved between the sexes. Further, RA induction of Rec8, like that of Stra8, requires the germ-cell-intrinsic competence factor Dazl. Our findings strengthen the importance of RA and Dazl in the meiotic transition, provide important details about the Stra8 pathway, and open avenues to investigate early meiosis through analysis of Rec8 induction and function. PMID:25102060

  5. Requirements for innate immune pathways in environmentally induced autoimmunity

    PubMed Central

    2013-01-01

    There is substantial evidence that environmental triggers in combination with genetic and stochastic factors play an important role in spontaneous autoimmune disease. Although the specific environmental agents and how they promote autoimmunity remain largely unknown, in part because of diverse etiologies, environmentally induced autoimmune models can provide insights into potential mechanisms. Studies of idiopathic and environmentally induced systemic autoimmunity show that they are mediated by common adaptive immune response genes. By contrast, although the innate immune system is indispensable for autoimmunity, there are clear differences in the molecular and cellular innate components that mediate specific systemic autoimmune diseases, suggesting distinct autoimmune-promoting pathways. Some of these differences may be related to the bifurcation of toll-like receptor signaling that distinguishes interferon regulatory factor 7-mediated type I interferon production from nuclear factor-?B-driven proinflammatory cytokine expression. Accordingly, idiopathic and pristane-induced systemic autoimmunity require both type I interferon and proinflammatory cytokines whereas the less aggressive mercury-induced autoimmunity, although dependent on nucleic acid-binding toll-like receptors, does not require type I interferon but needs proinflammatory cytokines. Scavenger receptors and the inflammasome may contribute to silica-induced autoimmunity. Greater understanding of the innate mechanisms responsible for idiopathic and environmentally induced autoimmunity should yield new information into the processes that instigate and drive systemic autoimmunity. PMID:23557436

  6. Epigenetic silencing of multiple interferon pathway genes after cellular immortalization.

    PubMed

    Kulaeva, Olga I; Draghici, Sorin; Tang, Lin; Kraniak, Janice M; Land, Susan J; Tainsky, Michael A

    2003-06-26

    Abrogating cellular senescence is a necessary step in the formation of a cancer cell. Promoter hypermethylation is an epigenetic mechanism of gene regulation known to silence gene expression in carcinogenesis. Treatment of spontaneously immortal Li-Fraumeni fibroblasts with 5-aza-2'-deoxycytidine (5AZA-dC), an inhibitor of DNA methyltransferase (DNMT), induces a senescence-like state. We used microarrays containing 12 558 genes to determine the gene expression profile associated with cellular immortalization and also regulated by 5AZA-dC. Remarkably, among 85 genes with methylation-dependent downregulation (silencing) after immortalization, 39 (46%) are known to be regulated during interferon signaling, a known growth-suppressive pathway. This work indicates that gene silencing may be associated with an early event in carcinogenesis, cellular immortalization. PMID:12821946

  7. Network-Based Identification of Biomarkers Coexpressed with Multiple Pathways

    PubMed Central

    Guo, Nancy Lan; Wan, Ying-Wooi

    2014-01-01

    Unraveling complex molecular interactions and networks and incorporating clinical information in modeling will present a paradigm shift in molecular medicine. Embedding biological relevance via modeling molecular networks and pathways has become increasingly important for biomarker identification in cancer susceptibility and metastasis studies. Here, we give a comprehensive overview of computational methods used for biomarker identification, and provide a performance comparison of several network models used in studies of cancer susceptibility, disease progression, and prognostication. Specifically, we evaluated implication networks, Boolean networks, Bayesian networks, and Pearson’s correlation networks in constructing gene coexpression networks for identifying lung cancer diagnostic and prognostic biomarkers. The results show that implication networks, implemented in Genet package, identified sets of biomarkers that generated an accurate prediction of lung cancer risk and metastases; meanwhile, implication networks revealed more biologically relevant molecular interactions than Boolean networks, Bayesian networks, and Pearson’s correlation networks when evaluated with MSigDB database. PMID:25392692

  8. Stochastic robustness and relative stability of multiple pathways in biological networks

    E-print Network

    Guo, Yongyi; Qian, Min; Ge, Hao

    2015-01-01

    Multiple dynamic pathways always exist in biological networks, but their robustness against internal fluctuations and relative stability have not been well recognized and carefully analyzed yet. Here we try to address these issues through an illustrative example, namely the Siah-1/beta-catenin/p14/19 ARF loop of protein p53 dynamics. Its deterministic Boolean network model predicts that two parallel pathways with comparable magnitudes of attractive basins should exist after the protein p53 is activated when a cell becomes harmfully disturbed. Once the low but non-neglectable intrinsic fluctuations are incorporated into the model, we show that a phase transition phenomenon is emerged: in one parameter region the probability weights of the normal pathway, reported in experimental literature, are comparable with the other pathway which is seemingly abnormal with the unknown functions, whereas, in some other parameter regions, the probability weight of the abnormal pathway can even dominate and become globally at...

  9. ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways

    PubMed Central

    Benseddik, Khedidja; Sen Nkwe, Nadine; Daou, Pascale; Verdier-Pinard, Pascal; Badache, Ali

    2013-01-01

    Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLC?) also contributed to cell guidance. We further showed that PLC? signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis. PMID:23383112

  10. Microgravity gradiometry measurement schemes with multiple-pathway atom interferometers

    NASA Astrophysics Data System (ADS)

    Ashwood, E.; Edwards, M.; Clark, C. W.

    2015-05-01

    We propose a new atom-interferometric scheme for measuring the value and derivatives of the gravitational field in the microgravity environment found in the Cold-Atom Laboratory to be deployed to the International Space Station. The operation of the proposed atom interferometer consists of splitting a harmonically confined Bose-Einstein condensate into multiple pieces using a sequence of laser pulses. In a perfect harmonic oscillator potential all of the condensate pieces will come to rest at the same time. At this point, the harmonic trap is turned off. The nearly motionless condensate clouds then accumulate different phases due to their respective accelerations at different points in space. The trap is then turned back on bringing all of the clouds together at the same time at which point they are again split producing multiple interference patterns. We have simulated some of these interferometric schemes using a Lagrangian variational approximation to the 3D time-dependent Gross-Pitaevskii equation. We have used this method to facilitate rapid interferometer design and to understand how these interference patterns can be used to measure the gravitational field and its derivatives. We also compare the sensitivity of the different interferometric schemes. Supported by NSF grants PHY-1068761 and ARO Atomtronics MURI.

  11. Involvement of Multiple Gene-Silencing Pathways in a Paramutation-like Phenomenon in Arabidopsis.

    PubMed

    Zheng, Zhimin; Yu, Hasi; Miki, Daisuke; Jin, Dan; Zhang, Qingzhu; Ren, Zhonghai; Gong, Zhizhong; Zhang, Heng; Zhu, Jian-Kang

    2015-05-26

    Paramutation is an epigenetic phenomenon that has been observed in a number of multicellular organisms. The epigenetically silenced state of paramutated alleles is not only meiotically stable but also "infectious" to active homologous alleles. The molecular mechanism of paramutation remains unclear, but components involved in RNA-directed DNA methylation (RdDM) are required. Here, we report a multi-copy pRD29A-LUC transgene in Arabidopsis thaliana that behaves like a paramutation locus. The silent state of LUC is induced by mutations in the DNA glycosylase gene ROS1. The silent alleles of LUC are not only meiotically stable but also able to transform active LUC alleles into silent ones, in the absence of ros1 mutations. Maintaining silencing at the LUC gene requires action of multiple pathways besides RdDM. Our study identified specific factors that are involved in the paramutation-like phenomenon and established a model system for the study of paramutation in Arabidopsis. PMID:25981044

  12. Multiple pathways leading to genomic instability and tumorigenesis

    SciTech Connect

    Sweezy, M.A.; Fishel, R.

    1994-12-31

    Genome instability is a hallmark of many cancers and is thought to play a role in tumorigenesis. Several types of genomic alterations have been described in various tumor cell lines, including expansion and contraction of microsatellite sequences and gross chromosomal rearrangements such as translocations, deletions, and gene amplification events. Several lines of evidence implicate a role for genome instability in the development of neoplasias as well as tumor progression. In hereditary nonpolyposis colon cancer (HNPCC), the instability of microsatellite sequences throughout the genome of tumors has been observed. Furthermore, microsatellite instability has been observed in 20-30% of ovarian, gastric, pancreatic, prostate, and lung tumors, and occurs at a very early stage in the development of sporadic endometrial tumors. Chromosomal translocations have been implicated in oncogene activation in several lymphomas. Barrett`s esophagus is a condition in which the progression into a cancerous state appears to proceed through a loss of cell cycle regulation, to genetic instability, and finally to the production of a solid tumor. In addition, other cancer-prone syndromes such as ataxia telangiectasia display an increase in chromosomal translocations and a loss of cell cycle regulation. The biochemical activities involved in the generation of these alterations are poorly understood and the subject of a great deal of debate. Defects in several aspects of DNA metabolism and cell cycle regulation have been proposed to be involved in the alteration of chromosomal structure. Here we discuss the possibility that several pathways exist that could lead to the development of genome instability, a higher than normal mutation rate, and the development of tumor cells.

  13. SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway.

    PubMed

    Wu, Ching-Shyi; Ouyang, Jian; Mori, Eiichiro; Nguyen, Hai Dang; Maréchal, Alexandre; Hallet, Alexander; Chen, David J; Zou, Lee

    2014-07-01

    The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway. PMID:24990965

  14. SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

    PubMed Central

    Wu, Ching-Shyi; Ouyang, Jian; Mori, Eiichiro; Nguyen, Hai Dang; Maréchal, Alexandre; Hallet, Alexander; Chen, David J.; Zou, Lee

    2014-01-01

    The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR–Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11–RAD50–NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway. PMID:24990965

  15. Creating Multiple Pathways in the Arts: A New York City Case Study

    ERIC Educational Resources Information Center

    Maguire, Cindy; Mishook, Jacob; Garcia, Ivonne; de Gaillande, Genevieve

    2013-01-01

    Increasingly, education policy makers understand the importance of students and families having access to a range of high quality educational opportunities inside and outside of school, 365 days a year. This paper explores the concept of multiple pathways in arts education to further conceptualize and build upon such opportunities, inside and…

  16. Use of multiple dispersal pathways facilitates amphibian persistence in stream networks

    E-print Network

    Fagan, William

    Use of multiple dispersal pathways facilitates amphibian persistence in stream networks Evan H. Campbell Granta,b,1 , James D. Nicholsa , Winsor H. Lowec , and William F. Fagand a Northeast Amphibian) Although populations of amphibians are declining worldwide, there is no evidence that salamanders occupying

  17. Multiple Developmental Pathways Leading to a Single Morph: Monosulcate Pollen (Examples From the Asparagales)

    E-print Network

    Multiple Developmental Pathways Leading to a Single Morph: Monosulcate Pollen (Examples From to play a role in pollen morphology: variation in cytokinesis type, cell wall formation, tetrad shape and aperture polarity are responsible for pollen aperture patterning. Despite the existence of other

  18. Functional replacement of a primary metabolic pathway via multiple independent eukaryote-to-eukaryote gene transfers

    E-print Network

    Nedelcu, Aurora M.

    Functional replacement of a primary metabolic pathway via multiple independent eukaryote-to-eukaryote, the contribution of LGT (especially eukaryote-to-eukaryote transfer) to the evolution and diversification of eukaryotic lineages is less understood (Andersson, 2005; Keeling & Palmer, 2008). Laterally acquired genes

  19. Direct Observation of Multiple Pathways of Single-Stranded DNA Stretching

    NASA Astrophysics Data System (ADS)

    Chen, Wuen-Shiu; Chen, Wei-Hung; Chen, Zephan; Gooding, Ashton A.; Lin, Kuan-Jiuh; Kiang, Ching-Hwa

    2010-11-01

    We observed multiple pathways of stretching single-stranded polydeoxynucleotides, poly(dA). Poly(dA) has been shown to undergo unique transitions under mechanical force, and such transitions were attributed to the stacking characteristics of poly(dA). Using single-molecule manipulation studies, we found that poly(dA) has two stretching pathways at high forces. The previously observed pathway has a free energy that is less than what is expected of single-stranded DNA with a random sequence, indicating the existence of a novel conformation of poly(dA) at large extensions. We also observed stepwise transitions between the two pathways by pulling the molecule with constant force, and found that the transitions are cooperative. These results suggest that the unique mechanical property of poly(dA) may play an important role in biological processes such as gene expression.

  20. Genetic and Epigenetic Events Generate Multiple Pathways in Colorectal Cancer Progression

    PubMed Central

    Pancione, Massimo; Remo, Andrea; Colantuoni, Vittorio

    2012-01-01

    Colorectal cancer (CRC) is one of the most common causes of death, despite decades of research. Initially considered as a disease due to genetic mutations, it is now viewed as a complex malignancy because of the involvement of epigenetic abnormalities. A functional equivalence between genetic and epigenetic mechanisms has been suggested in CRC initiation and progression. A hallmark of CRC is its pathogenetic heterogeneity attained through at least three distinct pathways: a traditional (adenoma-carcinoma sequence), an alternative, and more recently the so-called serrated pathway. While the alternative pathway is more heterogeneous and less characterized, the traditional and serrated pathways appear to be more homogeneous and clearly distinct. One unsolved question in colon cancer biology concerns the cells of origin and from which crypt compartment the different pathways originate. Based on molecular and pathological evidences, we propose that the traditional and serrated pathways originate from different crypt compartments explaining their genetic/epigenetic and clinicopathological differences. In this paper, we will discuss the current knowledge of CRC pathogenesis and, specifically, summarize the role of genetic/epigenetic changes in the origin and progression of the multiple CRC pathways. Elucidation of the link between the molecular and clinico-pathological aspects of CRC would improve our understanding of its etiology and impact both prevention and treatment. PMID:22888469

  1. The PKC/NF-?B Signaling Pathway Induces APOBEC3B Expression in Multiple Human Cancers.

    PubMed

    Leonard, Brandon; McCann, Jennifer L; Starrett, Gabriel J; Kosyakovsky, Leah; Luengas, Elizabeth M; Molan, Amy M; Burns, Michael B; McDougle, Rebecca M; Parker, Peter J; Brown, William L; Harris, Reuben S

    2015-11-01

    Overexpression of the antiviral DNA cytosine deaminase APOBEC3B has been linked to somatic mutagenesis in many cancers. Human papillomavirus infection accounts for APOBEC3B upregulation in cervical and head/neck cancers, but the mechanisms underlying nonviral malignancies are unclear. In this study, we investigated the signal transduction pathways responsible for APOBEC3B upregulation. Activation of protein kinase C (PKC) by the diacylglycerol mimic phorbol-myristic acid resulted in specific and dose-responsive increases in APOBEC3B expression and activity, which could then be strongly suppressed by PKC or NF-?B inhibition. PKC activation caused the recruitment of RELB, but not RELA, to the APOBEC3B promoter, implicating noncanonical NF-?B signaling. Notably, PKC was required for APOBEC3B upregulation in cancer cell lines derived from multiple tumor types. By revealing how APOBEC3B is upregulated in many cancers, our findings suggest that PKC and NF-?B inhibitors may be repositioned to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes, such as drug resistance and metastasis. Cancer Res; 75(21); 4538-47. ©2015 AACR. PMID:26420215

  2. 10 CFR 63.115 - Requirements for multiple barriers.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false Requirements for multiple barriers. 63.115 Section 63.115 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Technical Criteria Postclosure Performance Assessment §...

  3. 10 CFR 63.115 - Requirements for multiple barriers.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false Requirements for multiple barriers. 63.115 Section 63.115 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Technical Criteria Postclosure Performance Assessment §...

  4. 10 CFR 63.115 - Requirements for multiple barriers.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Requirements for multiple barriers. 63.115 Section 63.115 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Technical Criteria Postclosure Performance Assessment §...

  5. Sustaining multiple ecosystem functions in grassland communities requires higher biodiversity

    E-print Network

    Thomas, David D.

    Sustaining multiple ecosystem functions in grassland communities requires higher biodiversity Erika by ongoing biodiversity losses. Recent empirically based models using individual species' traits sug- gest.Weusedatafromthelongest-running biodiversity-functioning field experiment to date totest how species diversity affects the ability of grassland

  6. 10 CFR 63.115 - Requirements for multiple barriers.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false Requirements for multiple barriers. 63.115 Section 63.115 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Technical Criteria Postclosure Performance Assessment §...

  7. 10 CFR 63.115 - Requirements for multiple barriers.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Requirements for multiple barriers. 63.115 Section 63.115 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) DISPOSAL OF HIGH-LEVEL RADIOACTIVE WASTES IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Technical Criteria Postclosure Performance Assessment §...

  8. Revealing Pathway Dynamics in Heart Diseases by Analyzing Multiple Differential Networks

    PubMed Central

    Ma, Xiaoke; Gao, Long; Karamanlidis, Georgios; Gao, Peng; Lee, Chi Fung; Garcia-Menendez, Lorena; Tian, Rong; Tan, Kai

    2015-01-01

    Development of heart diseases is driven by dynamic changes in both the activity and connectivity of gene pathways. Understanding these dynamic events is critical for understanding pathogenic mechanisms and development of effective treatment. Currently, there is a lack of computational methods that enable analysis of multiple gene networks, each of which exhibits differential activity compared to the network of the baseline/healthy condition. We describe the iMDM algorithm to identify both unique and shared gene modules across multiple differential co-expression networks, termed M-DMs (multiple differential modules). We applied iMDM to a time-course RNA-Seq dataset generated using a murine heart failure model generated on two genotypes. We showed that iMDM achieves higher accuracy in inferring gene modules compared to using single or multiple co-expression networks. We found that condition-specific M-DMs exhibit differential activities, mediate different biological processes, and are enriched for genes with known cardiovascular phenotypes. By analyzing M-DMs that are present in multiple conditions, we revealed dynamic changes in pathway activity and connectivity across heart failure conditions. We further showed that module dynamics were correlated with the dynamics of disease phenotypes during the development of heart failure. Thus, pathway dynamics is a powerful measure for understanding pathogenesis. iMDM provides a principled way to dissect the dynamics of gene pathways and its relationship to the dynamics of disease phenotype. With the exponential growth of omics data, our method can aid in generating systems-level insights into disease progression. PMID:26083688

  9. CSF Proteomics Identifies Specific and Shared Pathways for Multiple Sclerosis Clinical Subtypes

    PubMed Central

    Avsar, Timucin; Duras?, ?lknur Melis; Uyguno?lu, U?ur; Tütüncü, Melih; Demirci, Nuri Onat; Saip, Sabahattin; Sezerman, O. U?ur; Siva, Aksel; Tahir Turanl?, Eda

    2015-01-01

    Multiple sclerosis (MS) is an immune-mediated, neuro-inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS) with a heterogeneous clinical presentation and course. There is a remarkable phenotypic heterogeneity in MS, and the molecular mechanisms underlying it remain unknown. We aimed to investigate further the etiopathogenesis related molecular pathways in subclinical types of MS using proteomic and bioinformatics approaches in cerebrospinal fluids of patients with clinically isolated syndrome, relapsing remitting MS and progressive MS (n=179). Comparison of disease groups with controls revealed a total of 151 proteins that are differentially expressed in clinically different MS subtypes. KEGG analysis using PANOGA tool revealed the disease related pathways including aldosterone-regulated sodium reabsorption (p=8.02x10-5) which is important in the immune cell migration, renin-angiotensin (p=6.88x10-5) system that induces Th17 dependent immunity, notch signaling (p=1.83x10-10) pathway indicating the activated remyelination and vitamin digestion and absorption pathways (p=1.73x10-5). An emerging theme from our studies is that whilst all MS clinical forms share common biological pathways, there are also clinical subtypes specific and pathophysiology related pathways which may have further therapeutic implications. PMID:25942430

  10. Multiple myeloma acquires resistance to EGFR inhibitor via induction of pentose phosphate pathway

    PubMed Central

    Chen, Yan; Huang, Ruibin; Ding, Jianghua; Ji, Dexiang; Song, Bing; Yuan, Liya; Chang, Hong; Chen, Guoan

    2015-01-01

    Multiple myeloma (MM) was characterized by frequent mutations in KRAS/NRAS/BRAF within the EGFR pathway that could induce resistance to EGFR inhibitors. We here report that EGFR inhibition solely exhibited moderate inhibition in KRAS/NRAS/BRAF wildtype (triple-WT) MM cells, whilst had no effect in myeloma cells with any of the mutated genes. The moderate inhibitory effect was conferred by induction of pentose phosphate pathway (PPP) when cells were treated with Gefitinib, the EGFR inhibitor. Combination of Gefitinib with PPP inhibitor 6AN effected synergistically in triple-WT cells. The inhibition could be restored by addition of NADPH. Dual EGFR/ERBB2 inhibitor Afatinib also exhibited similar effects. Further genetic silencing of EGFR, ERBB2 and mTOR indicated that major effect conferred by ERBB2 was via convergence to EGFR pathway in MM. Our results contributed to the individualized targeted therapy with EGFR inhibitors in MM. PMID:25894462

  11. Targeting the Fanconi Anemia/BRCA Pathway Circumvents Drug Resistance in Multiple Myeloma

    PubMed Central

    Yarde, Danielle N.; Oliveira, Vasco; Mathews, Linda; Wang, Xingyu; Villagra, Alejandro; Boulware, David; Shain, Kenneth H.; Hazlehurst, Lori A.; Alsina, Melissa; Chen, Dung-Tsa; Beg, Amer A.; Dalton, William S.

    2015-01-01

    The Fanconi Anemia (FA)/BRCA DNA damage repair pathway plays a pivotal role in the cellular response to replicative stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA genes are overexpressed and causative for drug resistance in human melphalan-resistant multiple myeloma (MM) cell lines. However, the transcriptional regulation of the FA/BRCA pathway is not understood. In this report, we describe for the first time a novel function of the NF-?B subunits, RelB/p50, as transcriptional activators of the FA/BRCA pathway. Specifically, our findings point to constitutive phosphorylation of I?B Kinase IKK? and subsequent alterations in FANCD2 expression and function as underlying events leading to melphalan resistance in repeatedly exposed MM cells. Inhibiting NF-?B by siRNA, blocking the IKK complex with BMS-345541, or using the proteasome inhibitor bortezomib drastically reduced FA/BRCA gene expression and FANCD2 protein expression in myeloma cells, resulting in diminished DNA damage repair and enhanced melphalan sensitivity. Importantly, we also found that bortezomib decreases FA/BRCA gene expression in multiple myeloma patients. These results show for the first time that NF-?B transcriptionally regulates the FA/BRCA pathway, and provide evidence for targeting FA-mediated DNA repair to enhance chemotherapeutic response and circumvent drug resistance in myeloma patients. PMID:19934314

  12. The Phosphoenolpyruvate Phosphotransferase System Regulates Vibrio cholerae Biofilm Formation through Multiple Independent Pathways?

    PubMed Central

    Houot, Laetitia; Chang, Sarah; Pickering, Bradley S.; Absalon, Cedric; Watnick, Paula I.

    2010-01-01

    The bacterial phosphoenolpyruvate phosphotransferase system (PTS) is a highly conserved phosphotransfer cascade that participates in the transport and phosphorylation of selected carbohydrates and modulates many cellular functions in response to carbohydrate availability. It plays a role in the virulence of many bacterial pathogens. Components of the carbohydrate-specific PTS include the general cytoplasmic components enzyme I (EI) and histidine protein (HPr), the sugar-specific cytoplasmic components enzymes IIA (EIIA) and IIB (EIIB), and the sugar-specific membrane-associated multisubunit components enzymes IIC (EIIC) and IID (EIID). Many bacterial genomes also encode a parallel PTS pathway that includes the EI homolog EINtr, the HPr homolog NPr, and the EIIA homolog EIIANtr. This pathway is thought to be nitrogen specific because of the proximity of the genes encoding this pathway to the genes encoding the nitrogen-specific ? factor ?54. We previously reported that phosphorylation of HPr and FPr by EI represses Vibrio cholerae biofilm formation in minimal medium supplemented with glucose or pyruvate. Here we report two additional PTS-based biofilm regulatory pathways that are active in LB broth but not in minimal medium. These pathways involve the glucose-specific enzyme EIIA (EIIAGlc) and two nitrogen-specific EIIA homologs, EIIANtr1 and EIIANtr2. The presence of multiple, independent biofilm regulatory circuits in the PTS supports the hypothesis that the PTS and PTS-dependent substrates have a central role in sensing environments suitable for a surface-associated existence. PMID:20400550

  13. Carbon dioxide fixation in 'Archaeoglobus lithotrophicus': are there multiple autotrophic pathways?

    PubMed

    Estelmann, Sebastian; Ramos-Vera, Walter Hugo; Gad'on, Nasser; Huber, Harald; Berg, Ivan A; Fuchs, Georg

    2011-06-01

    Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with 'Archaeoglobus lithotrophicus' being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin-Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in 'A. lithotrophicus', but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO(2)) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-D-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO(2) fixation pathway in 'A. lithotrophicus'. PMID:21410513

  14. Differential Requirements for Clathrin Endocytic Pathway Components in Cellular Entry by Ebola and Marburg Glycoprotein Pseudovirions

    PubMed Central

    Bhattacharyya, Suchita; Hope, Thomas J.; Young, John A. T.

    2011-01-01

    Clathrin-mediated endocytosis was previously implicated as one of the cellular pathways involved in filoviral glycoprotein mediated viral entry into target cells. Here we have further dissected the requirements for different components of this pathway in Ebola versus Marburg virus glycoprotein (GP) mediated viral infection. Although a number of these components were involved in both cases; Ebola GP-dependent viral entry specifically required the cargo recognition proteins Eps15 and DAB2 as well as the clathrin adaptor protein AP-2. In contrast, Marburg GP-mediated infection was independent of these three proteins and instead required beta-arrestin 1 (ARRB1). These findings have revealed an unexpected difference between the clathrin pathway requirements for Ebola GP- versus Marburg GP pseudovirion infection. Anthrax toxin entry also uses a clathrin-, and ARRB1-dependent pathway for cellular entry, indicating that the mechanism used by Marburg GP pseudovirions may be more generally important for pathogen entry. PMID:21855102

  15. The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome.

    PubMed

    Driscoll, James J; Pelluru, Dheeraj; Lefkimmiatis, Konstantinos; Fulciniti, Mariateresa; Prabhala, Rao H; Greipp, Philip R; Barlogie, Bart; Tai, Yu-Tzu; Anderson, Kenneth C; Shaughnessy, John D; Annunziata, Christina M; Munshi, Nikhil C

    2010-04-01

    Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies. PMID:19965618

  16. Efficient inhibition of infectious prions multiplication and release by targeting the exosomal pathway.

    PubMed

    Vilette, Didier; Laulagnier, Karine; Huor, Alvina; Alais, Sandrine; Simoes, Sabrina; Maryse, Romao; Provansal, Monique; Lehmann, Sylvain; Andreoletti, Olivier; Schaeffer, Laurent; Raposo, Graça; Leblanc, Pascal

    2015-11-01

    Exosomes are secreted membrane vesicles of endosomal origin present in biological fluids. Exosomes may serve as shuttles for amyloidogenic proteins, notably infectious prions, and may participate in their spreading in vivo. To explore the significance of the exosome pathway on prion infectivity and release, we investigated the role of the endosomal sorting complex required for transport (ESCRT) machinery and the need for ceramide, both involved in exosome biogenesis. Silencing of HRS-ESCRT-0 subunit drastically impairs the formation of cellular infectious prion due to an altered trafficking of cholesterol. Depletion of Tsg101-ESCRT-I subunit or impairment of the production of ceramide significantly strongly decreases infectious prion release. Together, our data reveal that ESCRT-dependent and -independent pathways can concomitantly regulate the exosomal secretion of infectious prion, showing that both pathways operate for the exosomal trafficking of a particular cargo. These data open up a new avenue to regulate prion release and propagation. PMID:26047659

  17. The Toll-Dorsal Pathway Is Required for Resistance to Viral Oral Infection in Drosophila

    PubMed Central

    Ferreira, Álvaro Gil; Naylor, Huw; Esteves, Sara Santana; Pais, Inês Silva; Martins, Nelson Eduardo; Teixeira, Luis

    2014-01-01

    Pathogen entry route can have a strong impact on the result of microbial infections in different hosts, including insects. Drosophila melanogaster has been a successful model system to study the immune response to systemic viral infection. Here we investigate the role of the Toll pathway in resistance to oral viral infection in D. melanogaster. We show that several Toll pathway components, including Spätzle, Toll, Pelle and the NF-kB-like transcription factor Dorsal, are required to resist oral infection with Drosophila C virus. Furthermore, in the fat body Dorsal is translocated from the cytoplasm to the nucleus and a Toll pathway target gene reporter is upregulated in response to Drosophila C Virus infection. This pathway also mediates resistance to several other RNA viruses (Cricket paralysis virus, Flock House virus, and Nora virus). Compared with control, viral titres are highly increased in Toll pathway mutants. The role of the Toll pathway in resistance to viruses in D. melanogaster is restricted to oral infection since we do not observe a phenotype associated with systemic infection. We also show that Wolbachia and other Drosophila-associated microbiota do not interact with the Toll pathway-mediated resistance to oral infection. We therefore identify the Toll pathway as a new general inducible pathway that mediates strong resistance to viruses with a route-specific role. These results contribute to a better understanding of viral oral infection resistance in insects, which is particularly relevant in the context of transmission of arboviruses by insect vectors. PMID:25473839

  18. Associations between Proprioceptive Neural Pathway Structural Connectivity and Balance in People with Multiple Sclerosis

    PubMed Central

    Fling, Brett W.; Dutta, Geetanjali Gera; Schlueter, Heather; Cameron, Michelle H.; Horak, Fay B.

    2014-01-01

    Mobility and balance impairments are a hallmark of multiple sclerosis (MS), affecting nearly half of patients at presentation and resulting in decreased activity and participation, falls, injuries, and reduced quality of life. A growing body of work suggests that balance impairments in people with mild MS are primarily the result of deficits in proprioception, the ability to determine body position in space in the absence of vision. A better understanding of the pathophysiology of balance disturbances in MS is needed to develop evidence-based rehabilitation approaches. The purpose of the current study was to (1) map the cortical proprioceptive pathway in vivo using diffusion-weighted imaging and (2) assess associations between proprioceptive pathway white matter microstructural integrity and performance on clinical and behavioral balance tasks. We hypothesized that people with MS (PwMS) would have reduced integrity of cerebral proprioceptive pathways, and that reduced white matter microstructure within these tracts would be strongly related to proprioceptive-based balance deficits. We found poorer balance control on proprioceptive-based tasks and reduced white matter microstructural integrity of the cortical proprioceptive tracts in PwMS compared with age-matched healthy controls (HC). Microstructural integrity of this pathway in the right hemisphere was also strongly associated with proprioceptive-based balance control in PwMS and controls. Conversely, while white matter integrity of the right hemisphere’s proprioceptive pathway was significantly correlated with overall balance performance in HC, there was no such relationship in PwMS. These results augment existing literature suggesting that balance control in PwMS may become more dependent upon (1) cerebellar-regulated proprioceptive control, (2) the vestibular system, and/or (3) the visual system. PMID:25368564

  19. Mechanically Untying a Protein Slipknot: Multiple Pathways Revealed by Force Spectroscopy and Steered Molecular Dynamics Simulations

    PubMed Central

    He, Chengzhi; Genchev, Georgi Z.; Lu, Hui; Li, Hongbin

    2013-01-01

    Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechancial unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie, and the polypeptide chain to completely extend. These distinct unfolding pathways proceed either via a two-state or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus provding a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechancial unfolding pathway of AFV3-109, and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding. PMID:22626004

  20. Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways.

    PubMed

    Contreras, Janette; Elnagar, Ahmed Y O; Hamm-Alvarez, Sarah F; Camarero, Julio A

    2011-10-30

    Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands. PMID:21906641

  1. Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways

    PubMed Central

    Contreras, Janette; Elnagar, Ahmed Y. O.; Hamm-Alvarez, Sarah F.; Camarero, Julio A.

    2011-01-01

    Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands. PMID:21906641

  2. Ventromedial hypothalamic lesions downregulate multiple immune signaling pathways in rat pancreatic islets.

    PubMed

    Kiba, Takayoshi

    2016-01-01

    It was recently reported that ventromedial hypothalamic lesions change the expression of cell proliferation-related genes and metabolism-related genes in rat pancreatic islets. This study has examined how gene families involved in immune responses are regulated in rat pancreatic islets after VMH lesions formation. Total pancreatic islets RNA was extracted, and differences in the gene expression profiles between rats at day 3 after VMH lesioning and sham-VMH-lesioned rats were investigated using DNA microarray and real-time polymerase chain reaction. VMH lesions downregulated multiple immune signaling pathways in rat pancreatic islets. Real-time polymerase chain reaction also confirmed that gene expressions of RT1 class II, locus Bb (RT1-Bb) was up-regulated and Spi-B transcription factor (Spib) was downregulated at day 3 after the VMH lesions. Ventromedial hypothalamic lesions may change the expression of multiple immune response genes in rat pancreatic islets. PMID:26555798

  3. Thermal comfort requirements: A study of people with multiple sclerosis

    SciTech Connect

    Webb, L.H.; Parsons, K.C.; Hodder, S.G.

    1999-07-01

    Existing specifications for thermal comfort in built environments are coming under increased criticism for failing to consider the requirements of specific populations. People with physical disabilities are an example of one such population. This paper presents the results of a study on the thermal comfort requirements of 32 people with multiple sclerosis. Subjects were exposed to three conditions: 18.5 C, PMV = {minus}1.5, slightly cool to cool; 23 C, PMV = 0, neutral; 29 C, PMV = +1.5, slightly warm to warm. Results indicate that people with multiple sclerosis have a wide range of responses to the three experimental conditions. The actual percentage dissatisfied was much higher than predicted by Fange's (1970) predicted percentage dissatisfied. Their preferred environment is slightly warmer than 23 C, PMV = 0, neutral. A subgroup of the population prefers an environment that is slightly cooler than 23 C. Further work is needed to qualify if their preferred environments match that of PMV+1 and PMV{minus}1 and to identify if any of the factors such as age, duration of disability, and medication affect the actual mean vote.

  4. Caspase 3 is not essential for the induction of anergy or multiple pathways of CD8+ T-cell death.

    PubMed

    Murakami, Kiichi; Liadis, Nicole; Sarmiento, Janice; Elford, Alisha R; Woo, Minna; Nguyen, Linh T; Mak, Tak W; Ohashi, Pamela S

    2010-12-01

    T-cell death is a fundamental process that is intricately regulated at multiple phases during T-cell differentiation, tolerance induction and the decline of the immune response. Caspase 3 is a crucial molecule regulating both mitochondrial and death receptor apoptotic pathways and therefore we were interested in examining the role of caspase 3 in T cells. Using P14 and H-Y CD8(+) TCR-transgenic models, our analysis has shown that caspase 3 is not required for thymic negative selection. In addition, caspase 3 does not play a prominent role in the contraction phase following acute viral infection, nor clonal deletion of CD8(+) T cells under tolerizing conditions. Surprisingly, our studies demonstrate that caspase 3 was not required for the induction of CD8(+) T-cell anergy in vivo, contrary to published reports using CD4(+) T cells. Therefore, these results demonstrate that caspase 3 is not essential in CD8(+) T cells for multiple forms of thymic or peripheral tolerance, nor the contraction phase after an acute anti-viral response. PMID:21110320

  5. Estimating trajectory correction requirements for multiple outer planet missions.

    NASA Technical Reports Server (NTRS)

    Friedman, L. D.; Hamilton, T. W.; Stanton, R. H.

    1972-01-01

    General approach to the problem of estimating trajectory correction requirements for multiple outer planet flyby missions when the navigation system uses onboard optical measurements made during approach to each target planet to complement the ground-based radio measurements. The accuracy and reliability of the onboard measurement system plays a critical role in sizing the trajectory correction capability required. An illustration of the combined use of radio and optical measurements is provided for the particular case of a Jupiter-Uranus-Neptune mission. Use of the statistical technique developed for computing the trajectory correction margin required to account for uncertainties in subsystem performance, permits trajectory correction savings of 100 to 20 m/sec over 'worst case' designs. This represents weight savings of about 50% of the science payload. For the example case trajectory correction requirements are estimated for two candidate optical systems and the radio alone case. The use of onboard measurements allows a trajectory correction savings of approximately 140 m/sec.

  6. ?-Tocotrienol suppresses prostate cancer cell proliferation and invasion through multiple-signalling pathways

    PubMed Central

    Yap, W N; Chang, P N; Han, H Y; Lee, D T W; Ling, M T; Wong, Y C; Yap, Y L

    2008-01-01

    Tocotrienol-rich fraction (TRF) has demonstrated antiproliferative effect on prostate cancer (PCa) cells. To elucidate this anticancer property in PCa cells, this study aimed, first, to identify the most potent isomer for eliminating PCa cells; and second, to decipher the molecular pathway responsible for its activity. Results showed that the inhibitory effect of ?-tocotrienol was most potent, which resulted in induction of apoptosis as evidenced by activation of pro-caspases and the presence of sub-G1 cell population. Examination of the pro-survival genes revealed that the ?-tocotrienol-induced cell death was associated with suppression of NF-?B, EGF-R and Id family proteins (Id1 and Id3). Meanwhile, ?-tocotrienol treatment also resulted in the induction of JNK-signalling pathway and inhibition of JNK activity by a specific inhibitor (SP600125) was able to partially block the effect of ?-tocotrienol. Interestingly, ?-tocotrienol treatment led to suppression of mesenchymal markers and the restoration of E-cadherin and ?-catenin expression, which was associated with suppression of cell invasion capability. Furthermore, a synergistic effect was observed when cells were co-treated with ?-tocotrienol and Docetaxel. Our results suggested that the antiproliferative effect of ?-tocotrienol act through multiple-signalling pathways, and demonstrated for the first time the anti-invasion and chemosensitisation effect of ?-tocotrienol against PCa cells. PMID:19002171

  7. Toward Multiple Conductance Pathways with Heterocycle-Based Oligo(phenyleneethynylene) Derivatives.

    PubMed

    Miguel, Delia; Álvarez de Cienfuegos, Luis; Martín-Lasanta, Ana; Morcillo, Sara P; Zotti, Linda A; Leary, Edmund; Bürkle, Marius; Asai, Yoshihiro; Jurado, Rocío; Cárdenas, Diego J; Rubio-Bollinger, Gabino; Agraït, Nicolás; Cuerva, Juan M; González, M Teresa

    2015-11-01

    In this paper, we have systematically studied how the replacement of a benzene ring by a heterocyclic compound in oligo(phenyleneethynylene) (OPE) derivatives affects the conductance of a molecular wire using the scanning tunneling microscope-based break junction technique. We describe for the first time how OPE derivatives with a central pyrimidine ring can efficiently link to the gold electrode by two pathways presenting two different conductance G values. We have demonstrated that this effect is associated with the presence of two efficient conductive pathways of different length: the conventional end-to-end configuration, and another with one of the electrodes linked directly to the central ring. This represents one of the few examples in which two defined conductive states can be set up in a single molecule without the aid of an external stimulus. Moreover, we have observed that the conductance through the full length of the heterocycle-based OPEs is basically unaffected by the presence of the heterocycle. All these results and the simplicity of the proposed molecules push forward the development of compounds with multiple conductance pathways, which would be a breakthrough in the field of molecular electronics. PMID:26452050

  8. A gene pathway analysis highlights the role of cellular adhesion molecules in multiple sclerosis susceptibility.

    PubMed

    Damotte, V; Guillot-Noel, L; Patsopoulos, N A; Madireddy, L; El Behi, M; De Jager, P L; Baranzini, S E; Cournu-Rebeix, I; Fontaine, B

    2014-03-01

    Genome-wide association studies (GWASs) perform per-SNP association tests to identify variants involved in disease or trait susceptibility. However, such an approach is not powerful enough to unravel genes that are not individually contributing to the disease/trait, but that may have a role in interaction with other genes as a group. Pathway analysis is an alternative way to highlight such group of genes. Using SNP association P-values from eight multiple sclerosis (MS) GWAS data sets, we performed a candidate pathway analysis for MS susceptibility by considering genes interacting in the cell adhesion molecule (CAMs) biological pathway using Cytoscape software. This network is a strong candidate, as it is involved in the crossing of the blood-brain barrier by the T cells, an early event in MS pathophysiology, and is used as an efficient therapeutic target. We drew up a list of 76 genes belonging to the CAM network. We highlighted 64 networks enriched with CAM genes with low P-values. Filtering by a percentage of CAM genes up to 50% and rejecting enriched signals mainly driven by transcription factors, we highlighted five networks associated with MS susceptibility. One of them, constituted of ITGAL, ICAM1 and ICAM3 genes, could be of interest to develop novel therapeutic targets. PMID:24430173

  9. Genistein inhibits tumor invasion by suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells

    PubMed Central

    2014-01-01

    Background Genistein (Gen) exhibits anti-mutagenic and anti-metastatic activities in hepatoma cell lines. Gen has suppressive effects on tumor growth and angiogenesis in nude mice. Gen suppresses the enzymatic activity of matrix metalloproteinase (MMP)-9; however, the mechanism underlying its anti-invasive activity on hepatocellular carcinoma (HCC) cells is unclear. Methods In this study, the possible mechanisms underlying Gen-mediated reduction of 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced cell invasion and inhibition of secreted and cytosolic MMP-9 production in human hepatoma cells (HepG2, Huh-7, and HA22T) and murine embryonic liver cells (BNL CL2) were investigated. Results Gen suppressed MMP-9 transcription by inhibiting activator protein (AP)-1 and nuclear factor-? B (NF-?B) activity. Gen suppressed TPA-induced AP-1 activity through inhibitory phosphorylation of extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and TPA-stimulated inhibition of NF-?B nuclear translocation through I?B inhibitory signaling pathways. Moreover, Gen suppressed TPA-induced activation of ERK/phosphatidylinositol 3-kinase/Akt upstream of NF-?B and AP-1. Conclusions Gen and its inhibition of multiple signal transduction pathways can control the invasiveness and metastatic potential of HCC. PMID:24433534

  10. Partitioning the effects of an ecosystem engineer: kangaroo rats control community structure via multiple pathways.

    PubMed

    Prugh, Laura R; Brashares, Justin S

    2012-05-01

    1.?Ecosystem engineers impact communities by altering habitat conditions, but they can also have strong effects through consumptive, competitive and other non-engineering pathways. 2.?Engineering effects can lead to fundamentally different community dynamics than non-engineering effects, but the relative strengths of these interactions are seldom quantified. 3.?We combined structural equation modelling and exclosure experiments to partition the effects of a keystone engineer, the giant kangaroo rat (Dipodomys ingens), on plants, invertebrates and vertebrates in a semi-arid California grassland. 4.?We separated the effects of burrow creation from kangaroo rat density and found that kangaroo rats increased the diversity and abundance of other species via both engineering and non-engineering pathways. 5.?Engineering was the primary factor structuring plant and small mammal communities, whereas non-engineering effects structured invertebrate communities and increased lizard abundance. 6.?These results highlight the importance of the non-engineering effects of ecosystem engineers and shed new light on the multiple pathways by which strong-interactors shape communities. PMID:22098534

  11. Mycobacterial Esx-3 Requires Multiple Components for Iron Acquisition

    PubMed Central

    Siegrist, M. Sloan; Steigedal, Magnus; Ahmad, Rushdy; Mehra, Alka; Dragset, Marte S.; Schuster, Brian M.; Philips, Jennifer A.; Carr, Steven A.

    2014-01-01

    ABSTRACT The type VII secretion systems are conserved across mycobacterial species and in many Gram-positive bacteria. While the well-characterized Esx-1 pathway is required for the virulence of pathogenic mycobacteria and conjugation in the model organism Mycobacterium smegmatis, Esx-3 contributes to mycobactin-mediated iron acquisition in these bacteria. Here we show that several Esx-3 components are individually required for function under low-iron conditions but that at least one, the membrane-bound protease MycP3 of M. smegmatis, is partially expendable. All of the esx-3 mutants tested, including the ?mycP3ms mutant, failed to export the native Esx-3 substrates EsxHms and EsxGms to quantifiable levels, as determined by targeted mass spectrometry. Although we were able to restore low-iron growth to the esx-3 mutants by genetic complementation, we found a wide range of complementation levels for protein export. Indeed, minute quantities of extracellular EsxHms and EsxGms were sufficient for iron acquisition under our experimental conditions. The apparent separation of Esx-3 function in iron acquisition from robust EsxGms and EsxHms secretion in the ?mycP3ms mutant and in some of the complemented esx-3 mutants compels reexamination of the structure-function relationships for type VII secretion systems. PMID:24803520

  12. Targeting TopBP1 at a convergent point of multiple oncogenic pathways for cancer therapy

    PubMed Central

    Chowdhury, Pinki; Lin, Gregory E.; Liu, Kang; Song, Yongcheng; Lin, Fang-Tsyr; Lin, Weei-Chin

    2014-01-01

    The progression of many solid tumors is driven by de-regulation of multiple common pathways, particularly Rb, PI (3) K/Akt and p53. Prior studies identified TopBP1as a key mediator for the oncogenic gain-of-function activities of mutant p53 (mutp53) in cancer. In Akt-hyperactive cancer, TopBP1 forms oligomers and represses E2F1-dependent apoptosis. Here we perform a molecular docking screening and identify a lead compound, calcein, capable of blocking TopBP1 oligomerization and p53 binding, resulting in re-activation of E2F1-dependent apoptosis and blockade of mutp53 gain-of-function. Calcein AM, the cell permeable derivative of calcein, shows significant anti-tumor activity in a wide-spectrum of cultured cancer cells harboring high TopBP1 levels. These biochemical findings are recapitulated in breast cancer xenograft models. Thus, our study provides proof-of-concept evidence for targeting TopBP1, a convergent point of multiple pathways, as a cancer therapy. PMID:25400145

  13. Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells.

    PubMed

    Deng, Xiaobei; Zhang, Fang; Wang, Lijuan; Rui, Wei; Long, Fang; Zhao, Yong; Chen, Deliang; Ding, Wenjun

    2014-07-01

    Our group was the first one reporting that autophagy could be triggered by airborne fine particulate matter (PM) with a mean diameter of less than 2.5 ?m (PM2.5) in human lung epithelial A549 cells, which could potentially lead to cell death. In the present study, we further explored the potential interactions between autophagy and apoptosis because it was well documented that PM2.5 could induce apoptosis in A549 cells. Much to our surprise, we found that PM2.5-exposure caused oxidative stress, resulting in activation of multiple cell death pathways in A549 cells, that is, the tumor necrosis factor-alpha (TNF-?)-induced pathway as evidenced by TNF-? secretion and activation of caspase-8 and -3, the intrinsic apoptosis pathway as evidenced by increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic protein Bcl-2, disruption of mitochondrial membrane potential, and activation of caspase-9 and -3, and autophagy as evidenced by an increased number of double-membrane vesicles, accompanied by increases of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) and expression of Beclin 1. It appears that reactive oxygen species (ROS) function as signaling molecules for all the three pathways because pretreatment with N-acetylcysteine, a scavenger of ROS, almost completely abolished TNF-? secretion and significantly reduced the number of apoptotic and autophagic cells. In another aspect, inhibiting autophagy with 3-methyladenine, a specific autophagy inhibitor, enhanced PM2.5-induced apoptosis and cytotoxicity. Intriguingly, neutralization of TNF-? with an anti-TNF-? special antibody not only abolished activation of caspase-8, but also drastically reduced LC3-II conversion. Thus, the present study has provided novel insights into the mechanism of cytotoxicity and even pathogenesis of diseases associated with PM2.5 exposure. PMID:24722831

  14. Multiple genetic pathways regulate replicative senescence in telomerase-deficient yeast.

    PubMed

    Ballew, Bari J; Lundblad, Victoria

    2013-08-01

    Most human tissues express low levels of telomerase and undergo telomere shortening and eventual senescence; the resulting limitation on tissue renewal can lead to a wide range of age-dependent pathophysiologies. Increasing evidence indicates that the decline in cell division capacity in cells that lack telomerase can be influenced by numerous genetic factors. Here, we use telomerase-defective strains of budding yeast to probe whether replicative senescence can be attenuated or accelerated by defects in factors previously implicated in handling of DNA termini. We show that the MRX (Mre11-Rad50-Xrs2) complex, as well as negative (Rif2) and positive (Tel1) regulators of this complex, comprise a single pathway that promotes replicative senescence, in a manner that recapitulates how these proteins modulate resection of DNA ends. In contrast, the Rad51 recombinase, which acts downstream of the MRX complex in double-strand break (DSB) repair, regulates replicative senescence through a separate pathway operating in opposition to the MRX-Tel1-Rif2 pathway. Moreover, defects in several additional proteins implicated in DSB repair (Rif1 and Sae2) confer only transient effects during early or late stages of replicative senescence, respectively, further suggesting that a simple analogy between DSBs and eroding telomeres is incomplete. These results indicate that the replicative capacity of telomerase-defective yeast is controlled by a network comprised of multiple pathways. It is likely that telomere shortening in telomerase-depleted human cells is similarly under a complex pattern of genetic control; mechanistic understanding of this process should provide crucial information regarding how human tissues age in response to telomere erosion. PMID:23672410

  15. Multiple Requirements of PLK1 during Mouse Oocyte Maturation

    PubMed Central

    Solc, Petr; Kitajima, Tomoya S.; Yoshida, Shuhei; Brzakova, Adela; Kaido, Masako; Baran, Vladimir; Mayer, Alexandra; Samalova, Pavlina; Motlik, Jan; Ellenberg, Jan

    2015-01-01

    Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals. PMID:25658810

  16. Multiple signaling pathways promote B lymphocyte stimulator–dependent B-cell growth and survival

    PubMed Central

    Fox, Casey J.; Schmidt, Madelyn R.; Hammerman, Peter S.; Opferman, Joseph T.; Korsmeyer, Stanley J.; Hilbert, David M.; Thompson, Craig B.

    2008-01-01

    We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2–deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells. PMID:17942753

  17. Multiple folding pathways of proteins with shallow knots and co-translational folding

    E-print Network

    Chwastyk, Mateusz

    2015-01-01

    We study the folding process in the shallowly knotted protein MJ0366 within two variants of a structure-based model. We observe that the resulting topological pathways are much richer than identified in previous studies. In addition to the single knot-loop events, we find novel, and dominant, two-loop mechanisms. We demonstrate that folding takes place in a range of temperatures and the conditions of most successful folding are at temperatures which are higher than those required for the fastest folding. We also demonstrate that nascent conditions are more favorable to knotting than off-ribosome folding.

  18. Multiple folding pathways of proteins with shallow knots and co-translational folding

    NASA Astrophysics Data System (ADS)

    Chwastyk, Mateusz; Cieplak, Marek

    2015-07-01

    We study the folding process in the shallowly knotted protein MJ0366 within two variants of a structure-based model. We observe that the resulting topological pathways are much richer than identified in previous studies. In addition to the single knot-loop events, we find novel, and dominant, two-loop mechanisms. We demonstrate that folding takes place in a range of temperatures and the conditions of most successful folding are at temperatures which are higher than those required for the fastest folding. We also demonstrate that nascent conditions are more favorable to knotting than off-ribosome folding.

  19. PARADIGM-SHIFT predicts the function of mutations in multiple cancers using pathway impact analysis

    PubMed Central

    Ng, Sam; Collisson, Eric A.; Sokolov, Artem; Goldstein, Theodore; Gonzalez-Perez, Abel; Lopez-Bigas, Nuria; Benz, Christopher; Haussler, David; Stuart, Joshua M.

    2012-01-01

    Motivation: A current challenge in understanding cancer processes is to pinpoint which mutations influence the onset and progression of disease. Toward this goal, we describe a method called PARADIGM-SHIFT that can predict whether a mutational event is neutral, gain-or loss-of-function in a tumor sample. The method uses a belief-propagation algorithm to infer gene activity from gene expression and copy number data in the context of a set of pathway interactions. Results: The method was found to be both sensitive and specific on a set of positive and negative controls for multiple cancers for which pathway information was available. Application to the Cancer Genome Atlas glioblastoma, ovarian and lung squamous cancer datasets revealed several novel mutations with predicted high impact including several genes mutated at low frequency suggesting the approach will be complementary to current approaches that rely on the prevalence of events to reach statistical significance. Availability: All source code is available at the github repository http:github.org/paradigmshift. Contact: jstuart@soe.ucsc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22962493

  20. Angiogenic activity of sesamin through the activation of multiple signal pathways

    SciTech Connect

    Chung, Byung-Hee; Division of Food Biotechnology, School of Biotechnology, Kangwon National University, Chuncheon ; Lee, Jung Joon; Kim, Jong-Dai; Jeoung, Dooil; Lee, Hansoo; Choe, Jongseon; Ha, Kwon-Soo; Kwon, Young-Geun; Kim, Young-Myeong

    2010-01-01

    The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125{sup FAK}-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.

  1. Integrity of the Anterior Visual Pathway and Its Association with Ambulatory Performance in Multiple Sclerosis

    PubMed Central

    Sandroff, Brian M.; Pula, John H.; Motl, Robert W.

    2013-01-01

    Background. Retinal nerve fiber layer thickness (RNFLT) and total macular volume (TMV) represent markers of neuroaxonal degeneration within the anterior visual pathway that might correlate with ambulation in persons with multiple sclerosis (MS). Objective. This study examined the associations between RNFLT and TMV with ambulatory parameters in MS. Methods. Fifty-eight MS patients underwent a neurological examination for generation of an expanded disability status scale (EDSS) score and measurement of RNFLT and TMV using optical coherence tomography (OCT). Participants completed the 6-minute walk (6MW) and the timed 25-foot walk (T25FW). The associations were examined using generalized estimating equation models that accounted for within-patient, inter-eye correlations, and controlled for disease duration, EDSS score, and age. Results. RNFLT was not significantly associated with 6MW (P = 0.99) or T25FW (P = 0.57). TMV was significantly associated with 6MW (P = 0.023) and T25FW (P = 0.005). The coefficients indicated that unit differences in 6MW (100 feet) and T25FW (1 second) were associated with 0.040 and ?0.048 unit differences in TMV (mm3), respectively. Conclusion. Integrity of the anterior visual pathway, particularly TMV, might represent a noninvasive measure of neuroaxonal degeneration that is correlated with ambulatory function in MS. PMID:23864950

  2. Resistance to a Bacterial Toxin Is Mediated by Removal of a Conserved Glycosylation Pathway Required for

    E-print Network

    Aroian, Raffi V.

    Resistance to a Bacterial Toxin Is Mediated by Removal of a Conserved Glycosylation Pathway Required for Toxin-Host Interactions* Received for publication, July 25, 2003, and in revised form, August, California 92093 Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins

  3. Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants

    PubMed Central

    Tao, Yi; Ferrer, Jean-Luc; Ljung, Karin; Pojer, Florence; Hong, Fangxin; Long, Jeff A.; Li, Lin; Moreno, Javier E.; Bowman, Marianne E.; Ivans, Lauren J.; Cheng, Youfa; Lim, Jason; Zhao, Yunde; Ballaré, Carlos L.; Sandberg, Göran; Noel, Joseph P.; Chory, Joanne

    2008-01-01

    SUMMARY Plants grown at high densities perceive a decrease in the red to far-red (R:FR) ratio of incoming light, resulting from absorption of red light by canopy leaves and reflection of far-red light from neighboring plants. These changes in light quality trigger a series of responses known collectively as the shade avoidance syndrome. During shade avoidance, stems elongate at the expense of leaf and storage organ expansion, branching is inhibited, and flowering is accelerated. We identified several loci in Arabidopsis, mutations in which lead to plants defective in multiple shade avoidance outputs. Here we describe SAV3, an aminotransferase, and show that SAV3 catalyzes the formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a previously proposed, but uncharacterized, auxin biosynthetic pathway. This pathway is rapidly deployed to biosynthesize auxin at the high levels required to initiate the multiple changes in body plan associated with shade avoidance. PMID:18394996

  4. Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment.

    PubMed

    Ichijo, Yuta; Mochimaru, Yuta; Azuma, Morio; Satou, Kazuhiro; Negishi, Jun; Nakakura, Takashi; Oshima, Natsuki; Mogi, Chihiro; Sato, Koichi; Matsuda, Kouhei; Okajima, Fumikazu; Tomura, Hideaki

    2016-01-01

    Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A. PMID:26614909

  5. The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

    NASA Technical Reports Server (NTRS)

    Halfon, M. S.; Keshishian, H.

    1998-01-01

    The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

  6. Pathways targeted by antidiabetes drugs are enriched for multiple genes associated with type 2 diabetes risk.

    PubMed

    Segrè, Ayellet V; Wei, Nancy; Altshuler, David; Florez, Jose C

    2015-04-01

    Genome-wide association studies (GWAS) have uncovered >65 common variants associated with type 2 diabetes (T2D); however, their relevance for drug development is not yet clear. Of note, the first two T2D-associated loci (PPARG and KCNJ11/ABCC8) encode known targets of antidiabetes medications. We therefore tested whether other genes/pathways targeted by antidiabetes drugs are associated with T2D. We compiled a list of 102 genes in pathways targeted by marketed antidiabetic medications and applied Gene Set Enrichment Analysis (MAGENTA [Meta-Analysis Gene-set Enrichment of variaNT Associations]) to this gene set, using available GWAS meta-analyses for T2D and seven quantitative glycemic traits. We detected a strong enrichment of drug target genes associated with T2D (P = 2 × 10(-5); 14 potential new associations), primarily driven by insulin and thiazolidinedione (TZD) targets, which was replicated in an independent meta-analysis (Metabochip). The glycemic traits yielded no enrichment. The T2D enrichment signal was largely due to multiple genes of modest effects (P = 4 × 10(-4), after removing known loci), highlighting new associations for follow-up (ACSL1, NFKB1, SLC2A2, incretin targets). Furthermore, we found that TZD targets were enriched for LDL cholesterol associations, illustrating the utility of this approach in identifying potential side effects. These results highlight the potential biomedical relevance of genes revealed by GWAS and may provide new avenues for tailored therapy and T2D treatment design. PMID:25368101

  7. Multiple pathways to the same end: Mechanisms of myonuclear apoptosis in sarcopenia of aging

    PubMed Central

    Marzetti, Emanuele; Privitera, Giuseppe; Simili, Vincenzo; Wohlgemuth, Stephanie E.; Aulisa, Lorenzo; Pahor, Marco; Leeuwenburgh, Christiaan

    2015-01-01

    Sarcopenia, the age-related decline in muscle mass and function, represents a significant health issue due to the high prevalence of frailty and disability associated with this condition. Nevertheless, the cellular mechanisms responsible for the loss of muscle mass in old age are still largely unknown. An altered regulation of myocyte apoptosis has recently emerged as a possible contributor to the pathogenesis of sarcopenia. Studies in animal models have shown that the severity of skeletal muscle apoptosis increases over the course of aging and correlates with the degree of muscle mass and strength decline. Several apoptotic pathways are operative in aged muscles, with the mitochondria- and TNF-?-mediated pathways likely being the most relevant to sarcopenia. However, despite the growing number of studies on the subject, a definite mechanistic link between myocyte apoptosis and age-related muscle atrophy has not yet been established. Furthermore, the evidence on the role played by apoptosis in human sarcopenia is still sparse. Clearly, further research is required to better define the involvement of myocyte apoptosis in the pathogenesis of muscle loss at advanced age. This knowledge will likely help in the design of more effective therapeutic strategies to preserve muscle mass into old age, thus fostering independence of the elderly population and reducing the socioeconomic burden associated with sarcopenia. PMID:20191247

  8. Defects in the Secretory Pathway and High Ca2+ Induce Multiple P-bodies

    PubMed Central

    Kilchert, Cornelia; Weidner, Julie; Prescianotto-Baschong, Cristina

    2010-01-01

    mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellular stresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathway mutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress. Exposure of wild-type cells to osmotic stress or high extracellular Ca2+ mimicked this increase in PB number. Conversely, intracellular Ca2+-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction through starvation or osmotic stress, PB formation in secretory mutants and by Ca2+ required the PB components Pat1 and Scd6, and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, when stresses were combined, PB number did not correlate with the strength of the translational block, but rather with the type of stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of ?40–100 nm connected to the endoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatially coordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends on intracellular signals that regulate PB number. PMID:20519435

  9. Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

    PubMed Central

    Wold, Mitchell S.; Gong, Shiaoching; Phillips, Greg R.; Dou, Zhixun; Zhao, Yanxiang; Heintz, Nathaniel; Zong, Wei-Xing; Yue, Zhenyu

    2014-01-01

    Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis. PMID:25275521

  10. Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum

    PubMed Central

    2012-01-01

    Background Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30?Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination. Results The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases). Conclusions The results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation. PMID:22672912

  11. Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

    PubMed Central

    Bernard, Delphine G.; Netz, Daili J. A.; Lagny, Thibaut J.; Pierik, Antonio J.; Balk, Janneke

    2013-01-01

    The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids. PMID:23754812

  12. Hedgehog signaling is required at multiple stages of zebrafish tooth development

    PubMed Central

    2010-01-01

    Background The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. Results We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. Conclusion We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution. PMID:21118524

  13. DEPTOR is linked to a TORC1-p21 survival proliferation pathway in multiple myeloma cells

    PubMed Central

    Yang, Yonghui; Bardeleben, Carolyne; Frost, Patrick; Hoang, Bao; Shi, Yijiang; Finn, Richard; Gera, Joseph; Lichtenstein, Alan

    2014-01-01

    We investigated the mechanism by which gene silencing of the mTOR inhibitor, DEPTOR, induces cytoreductive effects on multiple myeloma (MM) cells. DEPTOR knockdown resulted in anti-MM effects in several MM cell lines. Using an inducible shRNA to silence DEPTOR, 8226 MM cells underwent TORC1 activation, downregulation of AKT/SGK activity, apoptosis, cell cycle arrest and senescence. These latter cytotoxic effects were prevented by TORC1 paralysis (Raptor knockdown) but not by over-expression of AKT activity. In addition, DEPTOR knockdown-induced MM death was not associated with activation of the unfolded protein response, suggesting that enhanced ER stress did not play a role. In contrast, DEPTOR knockdown in 8226 cells induced p21 expression, independent of p53, and p21 knockdown prevented all of the cytotoxic effects following DEPTOR silencing. DEPTOR silencing resulted in p21 upregulation in additional MM cell lines. Furthermore, DEPTOR silencing in a murine xenograft model resulted in anti-MM effects associated with p21 upregulation. DEPTOR knockdown also resulted in a decreased expression of p21-targeting miRNAs and transfection of miRNA mimics prevented p21 upregulation and apoptosis following DEPTOR silencing. Use of a shRNA-resistant DEPTOR construct ruled out off-target effects of the shRNA. These results indicate that DEPTOR regulates growth and survival of MM cells via a TORC1/p21 pathway and suggest an involvement of p21-targeted miRNAs. PMID:25568666

  14. Multiplicity of n-heptane oxidation pathways catalyzed by cytochrome P450.

    PubMed

    Szutowski, Miros?aw M; Rakoto, Joseph S

    2009-01-01

    Modeling, mutagenesis, and kinetic studies have demonstrated that the substrate-binding site of cytochrome P450 is composed of multiple interactive regions that are capable of simultaneously binding two or more xenobiotics. Substrate molecules can interact with each other after docking. Thus, substrates can compete for the activated oxygen-ferrous complex or alter the spatial orientation of other molecules. Cytochrome P450 is a unique enzyme that produces n-heptane metabolites of different oxidation states. Metabolism of n-heptane was investigated with rat liver microsomes and a reconstituted rat liver system. Ethanol, n-propanol, and n-butanol molecules interacted with the n-heptane molecule and resulted in cytochrome P450 spectral changes as well as alterations in the n-heptane metabolic profile. The observed modifications in the biotransformation of n-heptane indicated that there are three distinct pathways for oxidation of n-heptane to heptanols, heptanones, and one-side oxygen-oriented heptanediones. PMID:19705363

  15. The G alpha i homologue gna-1 controls multiple differentiation pathways in Neurospora crassa.

    PubMed Central

    Ivey, F D; Hodge, P N; Turner, G E; Borkovich, K A

    1996-01-01

    Heterotrimeric G proteins are components of principal signaling pathways in eukaryotes. In higher organisms, alpha subunits of G proteins have been divided into four families, Gi, Gs, Gq, and G12. We previously identified a G alpha i homologue gna-1 in the filamentous fungus Neurospora crassa. Now we report that deletion of gna-1 leads to multiple phenotypes during the vegetative and sexual cycles in N. crassa. On solid medium, delta gna-1 strains have a slower rate of hyphal apical extension than wild type, a rate that is more pronounced under hyperosmotic conditions or in the presence of a cellophane overlay. delta gna-1 mutants accumulate less mass than wild-type strains, and their mass accumulation is not affected in the same way by exposure to light. delta gna-1 strains are defective in macroconidiation, possessing aerial hyphae that are shorter, contain abnormal swellings, and differentiate adherent macroconidia. During the sexual cycle, delta gna-1 strains are fertile as males. However, the mutants are female-sterile, producing small, aberrant female reproductive structures. After fertilization, delta gna-1 female structures do not enlarge and develop normally, and no sexual spores are produced. Thus, mutation of gna-1 results in sex-specific loss of fertility. Images PMID:8856670

  16. Curcumin induces apoptosis of upper aerodigestive tract cancer cells by targeting multiple pathways.

    PubMed

    Amin, A R M Ruhul; Haque, Abedul; Rahman, Mohammad Aminur; Chen, Zhuo Georgia; Khuri, Fadlo Raja; Shin, Dong Moon

    2015-01-01

    Curcumin, a natural compound isolated from the Indian spice "Haldi" or "curry powder", has been used for centuries as a traditional remedy for many ailments. Recently, the potential use of curcumin in cancer prevention and therapy urges studies to uncover the molecular mechanisms associated with its anti-tumor effects. In the current manuscript, we investigated the mechanism of curcumin-induced apoptosis in upper aerodigestive tract cancer cell lines and showed that curcumin-induced apoptosis is mediated by the modulation of multiple pathways such as induction of p73, and inhibition of p-AKT and Bcl-2. Treatment of cells with curcumin induced both p53 and the related protein p73 in head and neck and lung cancer cell lines. Inactivation of p73 by dominant negative p73 significantly protected cells from curcumin-induced apoptosis, whereas ablation of p53 by shRNA had no effect. Curcumin treatment also strongly inhibited p-AKT and Bcl-2 and overexpression of constitutively active AKT or Bcl-2 significantly inhibited curcumin-induced apoptosis. Taken together, our findings suggest that curcumin-induced apoptosis is mediated via activating tumor suppressor p73 and inhibiting p-AKT and Bcl-2. PMID:25910231

  17. Multiple modes of proepicardial cell migration require heartbeat

    PubMed Central

    2014-01-01

    Background The outermost layer of the vertebrate heart, the epicardium, forms from a cluster of progenitor cells termed the proepicardium (PE). PE cells migrate onto the myocardium to give rise to the epicardium. Impaired epicardial development has been associated with defects in valve development, cardiomyocyte proliferation and alignment, cardiac conduction system maturation and adult heart regeneration. Zebrafish are an excellent model for studying cardiac development and regeneration; however, little is known about how the zebrafish epicardium forms. Results We report that PE migration occurs through multiple mechanisms and that the zebrafish epicardium is composed of a heterogeneous population of cells. Heterogeneity is first observed within the PE and persists through epicardium formation. Using in vivo imaging, histology and confocal microscopy, we show that PE cells migrate through a cellular bridge that forms between the pericardial mesothelium and the heart. We also observed the formation of PE aggregates on the pericardial surface, which were released into the pericardial cavity. It was previously reported that heartbeat-induced pericardiac fluid advections are necessary for PE cluster formation and subsequent epicardium development. We manipulated heartbeat genetically and pharmacologically and found that PE clusters clearly form in the absence of heartbeat. However, when heartbeat was inhibited the PE failed to migrate to the myocardium and the epicardium did not form. We isolated and cultured hearts with only a few epicardial progenitor cells and found a complete epicardial layer formed. However, pharmacologically inhibiting contraction in culture prevented epicardium formation. Furthermore, we isolated control and silent heart (sih) morpholino (MO) injected hearts prior to epicardium formation (60 hpf) and co-cultured these hearts with “donor” hearts that had an epicardium forming (108 hpf). Epicardial cells from donor hearts migrated on to control but not sih MO injected hearts. Conclusions Epicardial cells stem from a heterogeneous population of progenitors, suggesting that the progenitors in the PE have distinct identities. PE cells attach to the heart via a cellular bridge and free-floating cell clusters. Pericardiac fluid advections are not necessary for the development of the PE cluster, however heartbeat is required for epicardium formation. Epicardium formation can occur in culture without normal hydrodynamic and hemodynamic forces, but not without contraction. PMID:24885804

  18. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    SciTech Connect

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-08-15

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  19. Integrated QSAR study for inhibitors of hedgehog signal pathway against multiple cell lines:a collaborative filtering method

    PubMed Central

    2012-01-01

    Background The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. Results In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Conclusions Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1. PMID:22849868

  20. Flux analysis of cholesterol biosynthesis in vivo reveals multiple tissue and cell-type specific pathways

    PubMed Central

    Mitsche, Matthew A; McDonald, Jeffrey G; Hobbs, Helen H; Cohen, Jonathan C

    2015-01-01

    Two parallel pathways produce cholesterol: the Bloch and Kandutsch-Russell pathways. Here we used stable isotope labeling and isotopomer analysis to trace sterol flux through the two pathways in mice. Surprisingly, no tissue used the canonical K–R pathway. Rather, a hybrid pathway was identified that we call the modified K–R (MK–R) pathway. Proportional flux through the Bloch pathway varied from 8% in preputial gland to 97% in testes, and the tissue-specificity observed in vivo was retained in cultured cells. The distribution of sterol isotopomers in plasma mirrored that of liver. Sterol depletion in cultured cells increased flux through the Bloch pathway, whereas overexpression of 24-dehydrocholesterol reductase (DHCR24) enhanced usage of the MK–R pathway. Thus, relative use of the Bloch and MK–R pathways is highly variable, tissue-specific, flux dependent, and epigenetically fixed. Maintenance of two interdigitated pathways permits production of diverse bioactive sterols that can be regulated independently of cholesterol. DOI: http://dx.doi.org/10.7554/eLife.07999.001 PMID:26114596

  1. Ancient and Novel Small RNA Pathways Compensate for the Loss of piRNAs in Multiple Independent Nematode Lineages

    PubMed Central

    Sarkies, Peter; Selkirk, Murray E.; Jones, John T.; Blok, Vivian; Boothby, Thomas; Goldstein, Bob; Hanelt, Ben; Ardila-Garcia, Alex; Fast, Naomi M.; Schiffer, Phillip M.; Kraus, Christopher; Taylor, Mark J.; Koutsovoulos, Georgios; Blaxter, Mark L.; Miska, Eric A.

    2015-01-01

    Small RNA pathways act at the front line of defence against transposable elements across the Eukaryota. In animals, Piwi interacting small RNAs (piRNAs) are a crucial arm of this defence. However, the evolutionary relationships among piRNAs and other small RNA pathways targeting transposable elements are poorly resolved. To address this question we sequenced small RNAs from multiple, diverse nematode species, producing the first phylum-wide analysis of how small RNA pathways evolve. Surprisingly, despite their prominence in Caenorhabditis elegans and closely related nematodes, piRNAs are absent in all other nematode lineages. We found that there are at least two evolutionarily distinct mechanisms that compensate for the absence of piRNAs, both involving RNA-dependent RNA polymerases (RdRPs). Whilst one pathway is unique to nematodes, the second involves Dicer-dependent RNA-directed DNA methylation, hitherto unknown in animals, and bears striking similarity to transposon-control mechanisms in fungi and plants. Our results highlight the rapid, context-dependent evolution of small RNA pathways and suggest piRNAs in animals may have replaced an ancient eukaryotic RNA-dependent RNA polymerase pathway to control transposable elements. PMID:25668728

  2. Applied electro-optics educational and training program with multiple entrance and exit pathways

    NASA Astrophysics Data System (ADS)

    Scott, Patricia; Zhou, Feng; Zilic, Dorothy

    2007-06-01

    This paper presents an innovative hands-on training program designed to create a pipeline of highly-skilled technical workers for today's workforce economy. The 2+2+2 Pennsylvania Integrated Workforce Leadership Program in Electro-Optics prepares students for a career in this new high-tech field. With seamless transition from high school into college, the program offers the versatility of multiple entrance and exit pathways. After completion of each educational level, students can exit the program with various skill levels, including certificates, an associate's degree, or a bachelor's degree. Launched by Indiana University of Pennsylvania (IUP) in partnership with Lenape Vocational School (Lenape), the 2+2+2 educational pathway program was implemented to promote early training of high-school students. During the first level, students in their junior and/or senior year enroll in four Electro-Optics courses at Lenape. Upon completion of these courses and an Advanced Placement Equivalency course with an appropriate exam score, students can earn a certificate from Lenape for the 15+ credits, which also can be articulated into IUP's associate degree program in Electro-Optics. During the second level, students can earn an associate's degree in Electro-Optics, offered only at the IUP Northpointe Campus. After completion of the Associate in Applied Science (A.A.S.), students are prepared to enter the workforce as senior technicians. During the third level, students who have completed the Associate of Science (A.S.) in Electro-Optics have the opportunity to matriculate at IUP's Indiana Campus to earn a Bachelor of Science (B.S.) degree in Applied Physics with a track in Electro-Optics. Hence, the name 2+2+2 refers to getting started in high school, continuing the educational experience with an associate's degree program, and optionally moving on to a bachelor's degree. Consequently, students move from one educational level to the next with advanced credits toward the next degree. This program was made possible by two grants from the Pennsylvania Department of Community and Economic Development (PA DCED). The intent of the grant is to foster partnerships that will develop programs in high-tech fields, such as biotechnology/life sciences, information technology, opto-electronics, and advanced manufacturing and materials. Topics of discussion will include program development, curriculum development, course descriptions, course sequencing, outreach and recruitment efforts, and program challenges.

  3. The major cellular sterol regulatory pathway is required for Andes virus infection.

    PubMed

    Petersen, Josiah; Drake, Mary Jane; Bruce, Emily A; Riblett, Amber M; Didigu, Chukwuka A; Wilen, Craig B; Malani, Nirav; Male, Frances; Lee, Fang-Hua; Bushman, Frederic D; Cherry, Sara; Doms, Robert W; Bates, Paul; Briley, Kenneth

    2014-02-01

    The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV). Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P) of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection. PMID:24516383

  4. Morbillivirus V Proteins Exhibit Multiple Mechanisms to Block Type 1 and Type 2 Interferon Signalling Pathways

    PubMed Central

    Chinnakannan, Senthil K.; Nanda, Sambit K.; Baron, Michael D.

    2013-01-01

    Morbilliviruses form a closely related group of pathogenic viruses which encode three non-structural proteins V, W and C in their P gene. Previous studies with rinderpest virus (RPV) and measles virus (MeV) have demonstrated that these non-structural proteins play a crucial role in blocking type I (IFN?/?) and type II (IFN?) interferon action, and various mechanisms have been proposed for these effects. We have directly compared four important morbilliviruses, rinderpest (RPV), measles virus (MeV), peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). These viruses and their V proteins could all block type I IFN action. However, the viruses and their V proteins had varying abilities to block type II IFN action. The ability to block type II IFN-induced gene transcription correlated with co-precipitation of STAT1 with the respective V protein, but there was no correlation between co-precipitation of either STAT1 or STAT2 and the abilities of the V proteins to block type I IFN-induced gene transcription or the creation of the antiviral state. Further study revealed that the V proteins of RPV, MeV, PPRV and CDV could all interfere with phosphorylation of the interferon-receptor-associated kinase Tyk2, and the V protein of highly virulent RPV could also block the phosphorylation of another such kinase, Jak1. Co-precipitation studies showed that morbillivirus V proteins all form a complex containing Tyk2 and Jak1. This study highlights the ability of morbillivirus V proteins to target multiple components of the IFN signalling pathways to control both type I and type II IFN action. PMID:23431397

  5. Pseudomonas aeruginosa Uses Multiple Pathways To Acquire Iron during Chronic Infection in Cystic Fibrosis Lungs

    PubMed Central

    Konings, Anna F.; Martin, Lois W.; Sharples, Katrina J.; Roddam, Louise F.; Latham, Roger; Reid, David W.

    2013-01-01

    Pseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription–quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF. PMID:23690396

  6. Precise lamination of retinal axons generates multiple parallel input pathways in the tectum

    PubMed Central

    Robles, Estuardo; Filosa, Alessandro; Baier, Herwig

    2013-01-01

    The axons of retinal ganglion cells (RGCs) form topographic connections in the optic tectum, recreating a two-dimensional map of the visual field in the midbrain. RGC axons are also targeted to specific positions along the laminar axis of the tectum. Understanding the sensory transformations performed by the tectum requires identification of the rules that control the formation of synaptic laminae by RGC axons. However, there is little information regarding the spatial relationships between multiple axons as they establish laminar and retinotopic arborization fields within the same region of neuropil. Moreover, the contribution of RGC axon lamination to the processing of visual information is unknown. We have utilized Brainbow genetic labeling to visualize groups of individually identifiable axons during the assembly of a precise laminar map in the tectum. Live imaging of multiple RGCs revealed that axons target specific sublaminar positions during initial innervation and maintain their relative laminar positions throughout early larval development, ruling out a model for lamina selection based on iterative refinements. During this period of laminar stability, RGC arbors undergo structural rearrangements that shift their relative retinotopic positions. Analysis of cell type-specific lamination patterns revealed that distinct combinations of RGCs converge to form each sublamina, and this input heterogeneity correlates with different functional responses to visual stimuli. These findings suggest that lamina-specific sorting of retinal inputs provides an anatomical blueprint for the integration of visual features in the tectum. PMID:23486973

  7. Multiple Pathways Control the Reactivation of Telomerase in HTLV-I-Associated Leukemia

    PubMed Central

    Bellon, Marcia; Nicot, Christophe

    2015-01-01

    While telomerase (hTERT) activity is absent from normal somatic cells, reactivation of hTERT expression is a hallmark of cancer cells. Telomerase activity is required for avoiding replicative senescence and supports immortalization of cellular proliferation. Only a minority of cancer cells rely on a telomerase-independent process known as alternative lengthening of telomeres, ALT, to sustain cancer cell proliferation. Multiple genetic, epigenetic, and viral mechanisms have been found to de-regulate telomerase gene expression, thereby increasing the risk of cellular transformation. Here, we review the different strategies used by the Human T-cell leukemia virus type 1, HTLV-I, to activate hTERT expression and stimulate its enzymatic activity in virally infected CD4 T cells. The implications of hTERT reactivation in HTLV-I pathogenesis and disease treatment are discussed. PMID:26430700

  8. Simultaneous Identification of Multiple Driver Pathways Mark D. M. Leiserson1

    E-print Network

    Sharan, Roded

    -Dendrix identifies sets of mutations in genes that overlap with known pathways ­ including Rb, p53, PI(3)K, and cell the somatic mutations responsible for cancer (driver mutations) from random, passenger mutations is a key challenge in cancer genomics. Driver mutations generally target cellular signaling and regulatory pathways

  9. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity.

    PubMed

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-01-01

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the ?-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of ?-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish. PMID:26672484

  10. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity

    PubMed Central

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-01-01

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the ?-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of ?-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish. PMID:26672484

  11. Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

    PubMed Central

    May, Victor; Lutz, Eve; MacKenzie, Christopher; Schutz, Kristin C.; Dozark, Kate; Braas, Karen M.

    2010-01-01

    MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on G?q/phosphatidylinositol 3-kinase ? activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase ?-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways. PMID:20093365

  12. Dentate Gyrus Development Requires ERK Activity to Maintain Progenitor Population and MAPK Pathway Feedback Regulation

    PubMed Central

    Vithayathil, Joseph; Pucilowska, Joanna; Goodnough, L. Henry; Atit, Radhika P.

    2015-01-01

    The ERK/MAPK pathway is an important developmental signaling pathway. Mutations in upstream elements of this pathway result in neuro-cardio-facial cutaneous (NCFC) syndromes, which are typified by impaired neurocognitive abilities that are reliant upon hippocampal function. The role of ERK signaling during hippocampal development has not been examined and may provide critical insight into the cause of hippocampal dysfunction in NCFC syndromes. In this study, we have generated ERK1 and conditional ERK2 compound knock-out mice to determine the role of ERK signaling during development of the hippocampal dentate gyrus. We found that loss of both ERK1 and ERK2 resulted in 60% fewer granule cells and near complete absence of neural progenitor pools in the postnatal dentate gyrus. Loss of ERK1/2 impaired maintenance of neural progenitors as they migrate from the dentate ventricular zone to the dentate gyrus proper, resulting in premature depletion of neural progenitor cells beginning at E16.5, which prevented generation of granule cells later in development. Finally, loss of ERK2 alone does not impair development of the dentate gyrus as animals expressing only ERK1 developed a normal hippocampus. These findings establish that ERK signaling regulates maintenance of progenitor cells required for development of the dentate gyrus. PMID:25926459

  13. Control of the Wnt pathways by nephrocystin-4 is required for morphogenesis of the zebrafish pronephros.

    PubMed

    Burcklé, Céline; Gaudé, Helori-Mael; Vesque, Christine; Silbermann, Flora; Salomon, Rémi; Jeanpierre, Cécile; Antignac, Corinne; Saunier, Sophie; Schneider-Maunoury, Sylvie

    2011-07-01

    Nephronophthisis is a hereditary nephropathy characterized by interstitial fibrosis and cyst formation. It is caused by mutations in NPHP genes encoding the ciliary proteins, nephrocystins. In this paper, we investigate the function of nephrocystin-4, the product of the nphp4 gene, in vivo by morpholino-mediated knockdown in zebrafish and in vitro in mammalian kidney cells. Depletion of nephrocystin-4 results in convergence and extension defects, impaired laterality, retinal anomalies and pronephric cysts associated with alterations in early cloacal morphogenesis. These defects are accompanied by abnormal ciliogenesis in the cloaca and in the laterality organ. We show that nephrocystin-4 is required for the elongation of the caudal pronephric primordium and for the regulation of cell rearrangements during cloaca morphogenesis. Moreover, depletion of either inversin, the product of the nphp2 gene, or of the Wnt-planar cell polarity (PCP) pathway component prickle2 increases the proportion of cyst formation in nphp4-depleted embryos. Nephrocystin-4 represses the Wnt-?-catenin pathway in the zebrafish cloaca and in mammalian kidney cells in culture. In these cells, nephrocystin-4 interacts with inversin and dishevelled, and regulates dishevelled stability and subcellular localization. Our data point to a function of nephrocystin-4 in a tight regulation of the Wnt-?-catenin and Wnt-PCP pathways, in particular during morphogenesis of the zebrafish pronephros. Moreover, they highlight common signalling functions for inversin and nephrocystin-4, suggesting that these two nephrocystins are involved in common physiopathological mechanisms. PMID:21498478

  14. The Root Hair Assay Facilitates the Use of Genetic and Pharmacological Tools in Order to Dissect Multiple Signalling Pathways That Lead to Programmed Cell Death

    PubMed Central

    Kacprzyk, Joanna; Devine, Aoife; McCabe, Paul F.

    2014-01-01

    The activation of programmed cell death (PCD) is often a result of complex signalling pathways whose relationship and intersection are not well understood. We recently described a PCD root hair assay and proposed that it could be used to rapidly screen genetic or pharmacological modulators of PCD. To further assess the applicability of the root hair assay for studying multiple signalling pathways leading to PCD activation we have investigated the crosstalk between salicylic acid, autophagy and apoptosis-like PCD (AL-PCD) in Arabidopsis thaliana. The root hair assay was used to determine rates of AL-PCD induced by a panel of cell death inducing treatments in wild type plants treated with chemical modulators of salicylic acid synthesis or autophagy, and in genetic lines defective in autophagy or salicylic acid signalling. The assay demonstrated that PCD induced by exogenous salicylic acid or fumonisin B1 displayed a requirement for salicylic acid signalling and was partially dependent on the salicylic acid signal transducer NPR1. Autophagy deficiency resulted in an increase in the rates of AL-PCD induced by salicylic acid and fumonisin B1, but not by gibberellic acid or abiotic stress. The phenylalanine ammonia lyase-dependent salicylic acid synthesis pathway contributed only to death induced by salicylic acid and fumonisin B1. 3-Methyladenine, which is commonly used as an inhibitor of autophagy, appeared to influence PCD induction in all treatments suggesting a possible secondary, non-autophagic, effect on a core component of the plant PCD pathway. The results suggest that salicylic acid signalling is negatively regulated by autophagy during salicylic acid and mycotoxin-induced AL-PCD. However, this crosstalk does not appear to be directly involved in PCD induced by gibberellic acid or abiotic stress. This study demonstrates that the root hair assay is an effective tool for relatively rapid investigation of complex signalling pathways leading to the activation of PCD. PMID:24755572

  15. Gene expression profiles from discordant monozygotic twins suggest that molecular pathways are shared among multiple systemic autoimmune diseases

    PubMed Central

    2011-01-01

    Introduction The objective of this study is to determine if multiple systemic autoimmune diseases (SAID) share gene expression pathways that could provide insights into pathogenic mechanisms common to these disorders. Methods RNA microarray analyses (Agilent Human 1A(V2) 20K oligo arrays) were used to quantify gene expression in peripheral blood cells from 20 monozygotic (MZ) twin pairs discordant for SAID. Six affected probands with systemic lupus erythematosus (SLE), six with rheumatoid arthritis (RA), eight with idiopathic inflammatory myopathies (IIM), and their same-gendered unaffected twins, were enrolled. Comparisons were made between discordant twin pairs and these were also each compared to 40 unrelated control subjects (matched 2:1 to each twin by age, gender and ethnicity) using statistical and molecular pathway analyses. Relative quantitative PCR was used to verify independently measures of differential gene expression assessed by microarray analysis. Results Probands and unrelated, matched controls differed significantly in gene expression for 104 probes corresponding to 92 identifiable genes (multiple-comparison adjusted P values < 0.1). Differentially expressed genes involved several overlapping pathways including immune responses (16%), signaling pathways (24%), transcription/translation regulators (26%), and metabolic functions (15%). Interferon (IFN)-response genes (IFI27, OASF, PLSCR1, EIF2AK2, TNFAIP6, and TNFSF10) were up-regulated in probands compared to unrelated controls. Many of the abnormally expressed genes played regulatory roles in multiple cellular pathways. We did not detect any probes expressed differentially in comparisons among the three SAID phenotypes. Similarly, we found no significant differences in gene expression when comparing probands to unaffected twins or unaffected twins to unrelated controls. Gene expression levels for unaffected twins appeared intermediate between that of probands and unrelated controls for 6535 probes (32% of the total probes) as would be expected by chance. By contrast, in unaffected twins intermediate ordering was observed for 84 of the 104 probes (81%) whose expression differed significantly between probands and unrelated controls. Conclusions Alterations in expression of a limited number of genes may influence the dysregulation of numerous, integrated immune response, cell signaling and regulatory pathways that are common to a number of SAID. Gene expression profiles in peripheral blood suggest that for genes in these critical pathways, unaffected twins may be in a transitional or intermediate state of immune dysregulation between twins with SAID and unrelated controls, perhaps predisposing them to the development of SAID given the necessary and sufficient environmental exposures. PMID:21521520

  16. mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells

    SciTech Connect

    Yang, Xiaojun; Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000 ; Zhong, Xiaomin; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011 ; Tanyi, Janos L.; Shen, Jianfeng; Xu, Congjian; Gao, Peng; Zheng, Tim M.; DeMichele, Angela; Zhang, Lin

    2013-02-15

    Highlights: ? Gene set enrichment analysis indicated mir-30d might regulate the autophagy pathway. ? mir-30d represses the expression of BECN1, BNIP3L, ATG12, ATG5 and ATG2. ? BECN1, BNIP3L, ATG12, ATG5 and ATG2 are direct targets of mir-30d. ? mir-30d inhibits autophagosome formation and LC3B-I conversion to LC3B-II. ? mir-30d regulates the autophagy process. -- Abstract: In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasing evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis, and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, and ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy.

  17. User Interface Requirements for Web-Based Integrated Care Pathways: Evidence from the Evaluation of an Online Care Pathway Investigation Tool.

    PubMed

    Balatsoukas, Panos; Williams, Richard; Davies, Colin; Ainsworth, John; Buchan, Iain

    2015-11-01

    Integrated care pathways (ICPs) define a chronological sequence of steps, most commonly diagnostic or treatment, to be followed in providing care for patients. Care pathways help to ensure quality standards are met and to reduce variation in practice. Although research on the computerisation of ICP progresses, there is still little knowledge on what are the requirements for designing user-friendly and usable electronic care pathways, or how users (normally health care professionals) interact with interfaces that support design, analysis and visualisation of ICPs. The purpose of the study reported in this paper was to address this gap by evaluating the usability of a novel web-based tool called COCPIT (Collaborative Online Care Pathway Investigation Tool). COCPIT supports the design, analysis and visualisation of ICPs at the population level. In order to address the aim of this study, an evaluation methodology was designed based on heuristic evaluations and a mixed method usability test. The results showed that modular visualisation and direct manipulation of information related to the design and analysis of ICPs is useful for engaging and stimulating users. However, designers should pay attention to issues related to the visibility of the system status and the match between the system and the real world, especially in relation to the display of statistical information about care pathways and the editing of clinical information within a care pathway. The paper concludes with recommendations for interface design. PMID:26446014

  18. Wolfberry Water Soluble Phytochemicals Down-Regulate ER Stress Biomarkers and Modulate Multiple Signaling Pathways Leading To Inhibition of Proliferation and Induction of Apoptosis in Jurkat Cells

    PubMed Central

    Jiang, Yu; Zhang, Yunong; Wark, Logan; Ortiz, Edlin; Lim, Soyoung; He, Hui; Wang, Weiqun; Medeiros, Denis; Lin, Dingbo

    2012-01-01

    Phytochemicals have received much recent attention in cancer prevention through simultaneous targeting multiple pathways in the disease progression. Here we determined that wolfberry phytochemicals was chemopreventive on the leukemic Jurkat cell. The water soluble wolfberry fractions (i.e., wolfberry phytochemicals) were enriched in carbohydrates (73.4 ± 4.5 % (w/w)), polyphenolics (1555 ± 112 mg quercetin equivalent/100 g freeze dry powder, including 213 mg rutin/100 g freeze dry powder), and had enhanced antioxidant activity (7771 ± 207 ?M Trolox equivalent/100 g freeze dry powder). Wolfberry phytochemicals, but not purified wolfberry polysaccharide fractions, inhibited Jurkat cell proliferation, induced cycle arrest at the G2/M phase in a dose dependent manner starting at 1 mg/ml for 48 h. Wolfberry phytochemicals eliminated cellular reactive oxygen species, declined expression of endoplasmic reticulum (ER) stress biomarkers, including glucose regulated protein 78, inositol-requiring protein 1(IRE1), activating transcription factor 6 (ATF6), protein kinase RNA-like ER kinase (PERK), and c/EBP-homologous protein, and induced activation of AMP activated protein kinase, stabilization of ?-catenin, and inhibition of NF?B, and AKT activity. Simultaneous siRNA knockdown of ATF6, IRE1 and PERK caused inhibition of cell proliferation and induction of apoptosis. Data suggested that ER stress and multiple survival/apoptosis signaling pathways were modulated by wolfberry phytochemicals during the apoptotic progression. Consumption of wolfberry could be an efficacious dietary strategy for preventing leukemia. PMID:22685690

  19. Wolfberry Water Soluble Phytochemicals Down-Regulate ER Stress Biomarkers and Modulate Multiple Signaling Pathways Leading To Inhibition of Proliferation and Induction of Apoptosis in Jurkat Cells.

    PubMed

    Jiang, Yu; Zhang, Yunong; Wark, Logan; Ortiz, Edlin; Lim, Soyoung; He, Hui; Wang, Weiqun; Medeiros, Denis; Lin, Dingbo

    2011-11-24

    Phytochemicals have received much recent attention in cancer prevention through simultaneous targeting multiple pathways in the disease progression. Here we determined that wolfberry phytochemicals was chemopreventive on the leukemic Jurkat cell. The water soluble wolfberry fractions (i.e., wolfberry phytochemicals) were enriched in carbohydrates (73.4 ± 4.5 % (w/w)), polyphenolics (1555 ± 112 mg quercetin equivalent/100 g freeze dry powder, including 213 mg rutin/100 g freeze dry powder), and had enhanced antioxidant activity (7771 ± 207 ?M Trolox equivalent/100 g freeze dry powder). Wolfberry phytochemicals, but not purified wolfberry polysaccharide fractions, inhibited Jurkat cell proliferation, induced cycle arrest at the G2/M phase in a dose dependent manner starting at 1 mg/ml for 48 h. Wolfberry phytochemicals eliminated cellular reactive oxygen species, declined expression of endoplasmic reticulum (ER) stress biomarkers, including glucose regulated protein 78, inositol-requiring protein 1(IRE1), activating transcription factor 6 (ATF6), protein kinase RNA-like ER kinase (PERK), and c/EBP-homologous protein, and induced activation of AMP activated protein kinase, stabilization of ?-catenin, and inhibition of NF?B, and AKT activity. Simultaneous siRNA knockdown of ATF6, IRE1 and PERK caused inhibition of cell proliferation and induction of apoptosis. Data suggested that ER stress and multiple survival/apoptosis signaling pathways were modulated by wolfberry phytochemicals during the apoptotic progression. Consumption of wolfberry could be an efficacious dietary strategy for preventing leukemia. PMID:22685690

  20. Anode Biofilms of Geoalkalibacter ferrihydriticus Exhibit Electrochemical Signatures of Multiple Electron Transport Pathways.

    PubMed

    Yoho, Rachel A; Popat, Sudeep C; Rago, Laura; Guisasola, Albert; Torres, César I

    2015-11-17

    Thriving under alkaliphilic conditions, Geoalkalibacter ferrihydriticus (Glk. ferrihydriticus) provides new applications in treating alkaline waste streams as well as a possible new model organism for microbial electrochemistry. We investigated the electrochemical response of biofilms of the alkaliphilic anode-respiring bacterium (ARB) Glk. ferrihydriticus voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry. We observed there to be at least four dominant electron transfer pathways, with their contribution to the overall current produced dependent on the set anode potential. These pathways appear to be manifested at midpoint potentials of approximately -0.14 V, -0.2 V, -0.24 V, and -0.27 V vs standard hydrogen electrode. The individual contributions of the pathways change upon equilibration from a set anode potential to another anode potential. Additionally, the contribution of each pathway to the overall current produced is reversible when the anode potential is changed back to the original set potential. The pathways involved in anode respiration in Glk. ferrihydriticus biofilms follow a similar, but more complicated, pattern as compared to those in the model ARB, Geobacter sulfurreducens. This greater diversity of electron transport pathways in Glk. ferrihydriticus could be related to its wider metabolic capability (e.g., higher pH and larger set of possible substrates, among others). PMID:26488071

  1. Integration of multiple signaling pathway activities resolves K-RAS/N-RAS mutation paradox in colon epithelial cell response to inflammatory cytokine stimulation

    E-print Network

    Kreeger, Pamela K.

    Colon tumors frequently harbor mutation in K-RAS and/or N-RAS, members of a GTPase family operating as a central hub for multiple key signaling pathways. While these proteins are strongly homologous, they exhibit diverse ...

  2. Multiple pathways for steel regulation suggested by genomic and sequence analysis of the murine Steel gene

    SciTech Connect

    Bedell, M.A.; Copeland, N.G.; Jenkins, N.A.

    1996-03-01

    The Steel (Sl) locus encodes mast cell growth factor (Mgf) that is required for the development of germ cells, hematopoietic cells and melanocytes. Although the expression patterns of the Mgf gene are well characterized, little is known of the factors which regulate its expression. Here, we describe the cloning and sequence of the full-length transcription unit and the 5{prime} flanking region of the murine Mgf gene. The full-length Mgf mRNA consists of a short 5{prime} untranslated region (UTR), a 0.8-kb ORF and a long 3{prime} UTR. A single transcription initiation site is used in a number of mouse tissues and is located just downstream of binding sites for several known transcription factors. In the 5{prime} UTR, two ATGs were found upstream of the initiator methionine and are conserved among different species, suggesting that Mgf may be translationally regulated. At least two Mgf mRNAs are produced by alternative use of polyadenylation sites, but numerous other potential polyadenylation sites were found in the 3{prime} UTR. In addition, the 3{prime} UTR contains numerous sequence motifs that may regulate Mgf mRNA stability. These studies suggest multiple ways in which expression of Mgf may be regulated. 39 refs., 4 figs.

  3. An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

    PubMed Central

    Huard, Jérémy; Mueller, Stephanie; Gilles, Ernst D; Klingmüller, Ursula; Klamt, Steffen

    2012-01-01

    During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor ?, interleukin-6, insulin and transforming growth factor ? orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels. PMID:22443451

  4. Dissection of Biological Property of Chinese Acupuncture Point Zusanli Based on Long-Term Treatment via Modulating Multiple Metabolic Pathways.

    PubMed

    Yan, Guangli; Zhang, Aihua; Sun, Hui; Cheng, Weiping; Meng, Xiangcai; Liu, Li; Zhang, Yingzhi; Xie, Ning; Wang, Xijun

    2013-01-01

    Acupuncture has a history of over 3000 years and is a traditional Chinese medical therapy that uses hair-thin metal needles to puncture the skin at specific points on the body to promote wellbeing, while its molecular mechanism and ideal biological pathways are still not clear. High-throughput metabolomics is the global assessment of endogenous metabolites within a biologic system and can potentially provide a more accurate snap shot of the actual physiological state. We hypothesize that acupuncture-treated human would produce unique characterization of metabolic phenotypes. In this study, UPLC/ESI-HDMS coupled with pattern recognition methods and system analysis were carried out to investigate the mechanism and metabolite biomarkers for acupuncture treatment at "Zusanli" acupoint (ST-36) as a case study. The top 5 canonical pathways including alpha-linolenic acid metabolism, d-glutamine and d-glutamate metabolism, citrate cycle, alanine, aspartate, and glutamate metabolism, and vitamin B6 metabolism pathways were acutely perturbed, and 53 differential metabolites were identified by chemical profiling and may be useful to clarify the physiological basis and mechanism of ST-36. More importantly, network construction has led to the integration of metabolites associated with the multiple perturbation pathways. Urine metabolic profiling might be a promising method to investigate the molecular mechanism of acupuncture. PMID:24073005

  5. Matrix-Vector Multiplication in Sub-Quadratic Time (Some Preprocessing Required)

    E-print Network

    Boneh, Dan

    Matrix-Vector Multiplication in Sub-Quadratic Time (Some Preprocessing Required) Ryan Williams Abstract We show that any n × n matrix A over any finite semiring can be preprocessed in O(n2+ ) time applications are described. 1 Introduction Matrix-vector multiplication is an absolutely fundamental operation

  6. OAS/PKR Pathways and ?/? TCR+ T Cells are Required for Ad: IFN-? Inhibition of HSV-1 in Cornea1

    PubMed Central

    Austin, Bobbie Ann; Halford, William P.; Williams, Bryan R. G.; Carr, Daniel J. J.

    2007-01-01

    An adenoviral vector containing the muIFN-? transgene (Ad:IFN-?) was evaluated for its capacity to inhibit HSV-1. To measure effectiveness, viral titers were analyzed in cornea and trigeminal ganglia (TG) during acute ocular HSV-1 infection. Ad: IFN-? potently suppressed HSV-1 replication in a dose-dependent fashion, requiring IFN-? R. Moreover, Ad:IFN-? was effective when delivered -72 and -24 h prior to infection as well as 24 h post infection. Associated with anti-viral opposition, TG from Ad: IFN-? transduced mice harbored fewer T cells. Also related to T cell involvement, Ad:IFN-? was effective but attenuated in TG from ?/? TCR deficient mice. In corneas, ?/? TCR+ T cells were obligatory for protection against viral multiplication. Type I IFN involvement amid anti-viral efficacy of Ad: IFN-? was further investigated because type I and II IFN pathways have synergistic anti-HSV-1 activity. Ad:IFN-? inhibited viral reproduction in corneas and TG from IFN-?/? R deficient (CD118 ?/?) mice, although viral titers were 2–3 fold higher in cornea and TG, compared to wild type. The absence of IFN-stimulated anti-viral proteins, 2’-5’ oligoadenylate synthetase/RNase L and ds RNA dependent protein kinase R, completely eliminated the anti-viral effectiveness of Ad:IFN-?. Collectively, the results demonstrate: (1) nonexistence of type I IFN R does not abolish defense of Ad:IFN-? against HSV-1; (2) anti-viral pathways, OAS/RNase L and PKR are mandatory; and (3) ?/? TCR+ T cells are compulsory for Ad: IFN-? effectiveness against HSV-1 in cornea but not in TG. PMID:17404299

  7. 40 CFR 63.1356 - Sources with multiple emissions limit or monitoring requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...AIR POLLUTANTS FOR SOURCE CATEGORIES (CONTINUED) National Emission Standards for Hazardous Air Pollutants From the Portland Cement Manufacturing Industry Other § 63.1356 Sources with multiple emissions limit or monitoring requirements....

  8. 40 CFR 63.1356 - Sources with multiple emissions limit or monitoring requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...AIR POLLUTANTS FOR SOURCE CATEGORIES (CONTINUED) National Emission Standards for Hazardous Air Pollutants From the Portland Cement Manufacturing Industry Other § 63.1356 Sources with multiple emissions limit or monitoring requirements....

  9. 40 CFR 63.1356 - Sources with multiple emission limits or monitoring requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES National Emission Standards for Hazardous Air Pollutants From the Portland Cement Manufacturing Industry Other § 63.1356 Sources with multiple emission limits or monitoring requirements....

  10. 40 CFR 63.1356 - Sources with multiple emission limits or monitoring requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES National Emission Standards for Hazardous Air Pollutants From the Portland Cement Manufacturing Industry Other § 63.1356 Sources with multiple emission limits or monitoring requirements....

  11. Transcriptional Activation by NF-?B Requires Multiple Coactivators

    PubMed Central

    Sheppard, Kelly-Ann; Rose, David W.; Haque, Zaffar K.; Kurokawa, Riki; McInerney, Eileen; Westin, Stefan; Thanos, Dimitris; Rosenfeld, Michael G.; Glass, Christopher K.; Collins, Tucker

    1999-01-01

    Nuclear factor-?B (NF-?B) plays a role in the transcriptional regulation of genes involved in inflammation and cell survival. In this report we demonstrate that NF-?B recruits a coactivator complex that has striking similarities to that recruited by nuclear receptors. Inactivation of either cyclic AMP response element binding protein (CREB)-binding protein (CBP), members of the p160 family of coactivators, or the CBP-associated factor (p/CAF) by nuclear antibody microinjection prevents NF-?B-dependent transactivation. Like nuclear receptor-dependent gene expression, NF-?B-dependent gene expression requires specific LXXLL motifs in one of the p160 family members, and enhancement of NF-?B activity requires the histone acetyltransferase (HAT) activity of p/CAF but not that of CBP. This coactivator complex is differentially recruited by members of the Rel family. The p50 homodimer fails to recruit coactivators, although the p50-p65 heterodimeric form of the transcription factor assembles the integrator complex. These findings provide new mechanistic insights into how this family of dimeric transcription factors has a differential effect on gene expression. PMID:10454583

  12. TGF-? induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways

    PubMed Central

    Strand, Douglas W; Liang, Yao-Yun; Yang, Feng; Barron, David A; Ressler, Steven J; Schauer, Isaiah G; Feng, Xin-Hua; Rowley, David R

    2014-01-01

    Transforming Growth Factor-? (TGF-?) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-? is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-? regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-?1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-?1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-?1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-?1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-? action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention. PMID:25374926

  13. Genetic Dissection of Differential Signaling Threshold Requirements for the Wnt/?-Catenin Pathway In Vivo

    PubMed Central

    Burke, Zoe D.; Faux, Maree C.; Samuel, Michael S.; Jarnicki, Andrew G.; Winbanks, Catherine E.; Newton, Ian P.; Meniel, Valerie S.; Suzuki, Hiromu; Stacker, Steven A.; Näthke, Inke S.; Tosh, David; Huelsken, Joerg; Clarke, Alan R.; Heath, Joan K.; Sansom, Owen J.; Ernst, Matthias

    2010-01-01

    Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/?-catenin pathway, we challenged the allele combinations by genetically restricting intracellular ?-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/?-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/?-catenin signaling capacity similar to that in the germline of the Apcmin mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apcmin mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/?-catenin pathway that regulate both physiological and pathophysiological conditions. PMID:20084116

  14. Multiple phytohormone signalling pathways modulate susceptibility of tomato plants to Alternaria alternata f. sp. lycopersici

    PubMed Central

    Jia, Chengguo; Zhang, Liping; Wang, Qiaomei

    2013-01-01

    Three phytohormone molecules – ethylene (ET), jasmonic acid (JA) and salicylic acid (SA) – play key roles in mediating disease response to necrotrophic fungal pathogens. This study investigated the roles of the ET, JA, and SA pathways as well as their crosstalk during the interaction between tomato (Solanum lycopersicum) plants and a necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). Both the ET and JASMONIC ACID INSENSITIVE1 (JAI1) receptor-dependent JA signalling pathways are necessary for susceptibility, while SA response promotes resistance to AAL infection. In addition, the role of JA in susceptibility to AAL is partly dependent on ET biosynthesis and perception, while the SA pathway enhances resistance to AAL and antagonizes the ET response. Based on these results, it is proposed that ET, JA, and SA each on their own can influence the susceptibility of tomato to AAL. Furthermore, the functions of JA and SA in susceptibility to the pathogen are correlated with the enhanced or decreased action of ET, respectively. This study has revealed the functional relationship among the three key hormone pathways in tomato defence against AAL. PMID:23264518

  15. Hepatocyte growth factor pathway upregulation in the bone marrow microenvironment in multiple myeloma is associated with lytic bone disease.

    PubMed

    Kristensen, Ida B; Christensen, Jacob H; Lyng, Maria B; Møller, Michael B; Pedersen, Lise; Rasmussen, Lars M; Ditzel, Henrik J; Abildgaard, Niels

    2013-05-01

    Lytic bone disease (LBD) in multiple myeloma (MM) is caused by osteoclast hyperactivation and osteoblast inhibition. Based on in vitro studies, the hepatocyte growth factor (HGF) pathway is thought to be central in osteoblast inhibition. We evaluated the gene expression of the HGF pathway in vivo using bone marrow biopsies (BMBs) of patients with MM and monoclonal gammopathy of undetermined significance (MGUS), and healthy volunteers (HV). BMBs (N = 110) obtained at diagnosis were snap-frozen and used to evaluate gene expression by quantitative reverse transcription polymerase chain reaction. LBD was evaluated using standard radiographs. Enzyme-linked immunosorbent assay (ELISA) was performed on matched bone marrow plasma and immunohistochemistry on matched formalin-fixed paraffin-embedded biopsies. Gene expression of HGF, SDC1, and MET in BMBs were significantly altered in MM versus HV and MGUS, and HGF and MET correlated with the extent of LBD. A significant correlation between gene and protein expression levels was observed for SDC1 (Syndecan-1) and HGF. The HGF bone marrow plasma level was significantly lower in MM patients with no/limited versus advanced LBD. Our novel approach using snap-frozen BMBs seems generally applicable because it allows evaluation of gene expression independent of the extent of MM plasma-cell infiltration. Our study highlights the importance of the HGF pathway in MM LBD. PMID:23431957

  16. Multiple Propionyl Coenzyme A-Supplying Pathways for Production of the Bioplastic Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) in Haloferax mediterranei

    PubMed Central

    Han, Jing; Hou, Jing; Zhang, Fan; Ai, Guomin; Li, Ming; Cai, Shuangfeng; Liu, Hailong; Wang, Lei; Wang, Zejian; Zhang, Siliang; Cai, Lei; Zhao, Dahe; Zhou, Jian

    2013-01-01

    Haloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO2 assimilation with PHBV biosynthesis was further confirmed by analysis of 13C positional enrichment in 3HV. Notably, 13C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ?phaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction. PMID:23435886

  17. Multiple propionyl coenzyme A-supplying pathways for production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Haloferax mediterranei.

    PubMed

    Han, Jing; Hou, Jing; Zhang, Fan; Ai, Guomin; Li, Ming; Cai, Shuangfeng; Liu, Hailong; Wang, Lei; Wang, Zejian; Zhang, Siliang; Cai, Lei; Zhao, Dahe; Zhou, Jian; Xiang, Hua

    2013-05-01

    Haloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV) from unrelated carbon sources. However, the pathways that produce propionyl coenzyme A (propionyl-CoA), an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, the aspartate/2-oxobutyrate pathway, the methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Cofeeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO2 assimilation with PHBV biosynthesis was further confirmed by analysis of (13)C positional enrichment in 3HV. Notably, (13)C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. In addition, genetic perturbation on the transcriptome of the ?phaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly downregulated. We determined for the first time four propionyl-CoA-supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction. PMID:23435886

  18. Aberrant over-expression of COX-1 intersects multiple pro-tumorigenic pathways in high-grade serous ovarian cancer

    PubMed Central

    Wilson, Andrew J.; Fadare, Oluwole; Beeghly-Fadiel, Alicia; Son, Deok-Soo; Liu, Qi; Zhao, Shilin; Saskowski, Jeanette; Uddin, Md. Jashim; Daniel, Cristina; Crews, Brenda; Lehmann, Brian D.; Pietenpol, Jennifer A.; Crispens, Marta A.; Marnett, Lawrence J.; Khabele, Dineo

    2015-01-01

    Cyclooxygenase-1 (COX-1) is implicated in ovarian cancer. However, patterns of COX expression and function have been unclear and controversial. In this report, patterns of COX-1 and COX-2 gene expression were obtained from RNA-seq data through The Cancer Genome Atlas. Our analysis revealed markedly higher COX-1 mRNA expression than COX-2 in high-grade serous ovarian cancers (HGSOC) and higher COX-1 expression in HGSOC tumors than 10 other tumor types. High expression of COX-1 in HGSOC tumors was confirmed in an independent tissue microarray. In contrast, lower or similar expression of COX-1 compared to COX-2 was observed in endometrioid, mucinous and clear cell tumors. Stable COX-1 knockdown in HGSOC-representative OVCAR-3 ovarian cancer cells reduced gene expression in multiple pro-tumorigenic pathways. Functional cell viability, clonogenicity, and migration/invasion assays were consistent with transcriptomic changes. These effects were reversed by stable over-expression of COX-1 in SKOV-3 cells. Our results demonstrate a distinct pattern of COX-1 over-expression in HGSOC tumors and strong association of COX-1 with multiple pro-tumorigenic pathways in ovarian cancer cells. These findings provide additional insight into the role of COX-1 in human ovarian cancer and support further development of methods to selectively target COX-1 in the management of HGSOC tumors. PMID:25972361

  19. Oyster Shell Proteins Originate from Multiple Organs and Their Probable Transport Pathway to the Shell Formation Front

    PubMed Central

    Zhu, Yabing; Du, Yishuai; Song, Xiaorui; Chen, Yuanxin; Huang, Ronglian; Que, Huayong; Fang, Xiaodong; Zhang, Guofan

    2013-01-01

    Mollusk shell is one kind of potential biomaterial, but its vague mineralization mechanism hinders its further application. Mollusk shell matrix proteins are important functional components that are embedded in the shell, which play important roles in shell formation. The proteome of the oyster shell had been determined based on the oyster genome sequence by our group and gives the chance for further deep study in this area. The classical model of shell formation posits that the shell proteins are mantle-secreted. But, in this study, we further analyzed the shell proteome data in combination with organ transcriptome data and we found that the shell proteins may be produced by multiple organs though the mantle is still the most important organ for shell formation. To identify the transport pathways of these shell proteins not in classical model of shell formation, we conducted a shell damage experiment and we determined the shell-related gene set to identify the possible transport pathways from multiple organs to the shell formation front. We also found that there may exist a remodeling mechanism in the process of shell formation. Based on these results along with some published results, we proposed a new immature model, which will help us think about the mechanism of shell formation in a different way. PMID:23840499

  20. Microbial effectors target multiple steps in the salicylic acid production and signaling pathway

    PubMed Central

    Tanaka, Shigeyuki; Han, Xiaowei; Kahmann, Regine

    2015-01-01

    Microbes attempting to colonize plants are recognized through the plant immune surveillance system. This leads to a complex array of global as well as specific defense responses, which are often associated with plant cell death and subsequent arrest of the invader. The responses also entail complex changes in phytohormone signaling pathways. Among these, salicylic acid (SA) signaling is an important pathway because of its ability to trigger plant cell death. As biotrophic and hemibiotrophic pathogens need to invade living plant tissue to cause disease, they have evolved efficient strategies to downregulate SA signaling by virulence effectors, which can be proteins or secondary metabolites. Here we review the strategies prokaryotic pathogens have developed to target SA biosynthesis and signaling, and contrast this with recent insights into how plant pathogenic eukaryotic fungi and oomycetes accomplish the same goal. PMID:26042138

  1. Noise-induced quantum coherence in photosynthetic complexes with multiple energy transfer pathways

    E-print Network

    Voronine, Dmitri V; Cao, Bin; Joshi, Amitabh

    2013-01-01

    We theoretically investigate exciton relaxation dynamics in molecular aggregates based on model photosynthetic complexes under various conditions of incoherent excitation. We show that noise-induced quantum coherence is generated between spatially-separated exciton states which belong to the same or different energy transfer pathways, coupled via real and virtual transfer processes. Such quantum coherence effects may be used to improve light-harvesting efficiency and to reveal quantum phenomena in biology.

  2. Noise-induced quantum coherence in photosynthetic complexes with multiple energy transfer pathways

    E-print Network

    Dmitri V. Voronine; Konstantin E. Dorfman; Bin Cao; Amitabh Joshi

    2014-08-12

    We theoretically investigate exciton relaxation dynamics in molecular aggregates based on model photosynthetic complexes under various conditions of incoherent excitation. We show that noise-induced quantum coherence is generated between spatially-separated exciton states which belong to the same or different energy transfer pathways, coupled via real and virtual transfer processes. Such quantum coherence effects may be used to improve light-harvesting efficiency and to reveal quantum phenomena in biology.

  3. Multispecific Drug Transporter Slc22a8 (Oat3) Regulates Multiple Metabolic and Signaling Pathways

    PubMed Central

    Wu, Wei; Jamshidi, Neema; Eraly, Satish A.; Liu, Henry C.; Bush, Kevin T.; Palsson, Bernhard O.

    2013-01-01

    Multispecific drug transporters of the solute carrier and ATP-binding cassette families are highly conserved through evolution, but their true physiologic role remains unclear. Analyses of the organic anion transporter 3 (OAT3; encoded by Slc22a8/Oat3, originally Roct) knockout mouse have confirmed its critical role in the renal handling of common drugs (e.g., antibiotics, antivirals, diuretics) and toxins. Previous targeted metabolomics of the knockout of the closely related Oat1 have demonstrated a central metabolic role, but the same approach with Oat3 failed to reveal a similar set of endogenous substrates. Nevertheless, the Oat3 knockout is the only Oat described so far with a physiologically significant phenotype, suggesting the disturbance of metabolic or signaling pathways. Here we analyzed global gene expression in Oat3 knockout tissue, which implicated OAT3 in phase I and phase II metabolism (drug metabolizing enzymes or DMEs), as well as signaling pathways. Metabolic reconstruction with the recently developed “mouse Recon1” supported the involvement of Oat3 in the aforementioned pathways. Untargeted metabolomics were used to determine whether the predicted metabolic alterations could be confirmed. Many significant changes were observed; several metabolites were tested for direct interaction with mOAT3, whereas others were supported by published data. Oat3 thus appears critical for the handling of phase I (hydroxylation) and phase II (glucuronidation) metabolites. Oat3 also plays a role in bioenergetic pathways (e.g., the tricarboxylic acid cycle), as well as those involving vitamins (e.g., folate), steroids, prostaglandins, gut microbiome products, uremic toxins, cyclic nucleotides, amino acids, glycans, and possibly hyaluronic acid. The data seemingly consistent with the Remote Sensing and Signaling Hypothesis (Ahn and Nigam, 2009; Wu et al., 2011), also suggests that Oat3 is essential for the handling of dietary flavonoids and antioxidants. PMID:23920220

  4. Evaluations of the trans-sulfuration pathway in multiple liver toxicity studies

    SciTech Connect

    Schnackenberg, Laura K. Chen Minjun; Sun, Jinchun; Holland, Ricky D.; Dragan, Yvonne; Tong Weida; Welsh, William; Beger, Richard D.

    2009-02-15

    Drug-induced liver injury has been associated with the generation of reactive metabolites, which are primarily detoxified via glutathione conjugation. In this study, it was hypothesized that molecules involved in the synthesis of glutathione would be diminished to replenish the glutathione depleted through conjugation reactions. Since S-adenosylmethionine (SAMe) is the primary source of the sulfur atom in glutathione, UPLC/MS and NMR were used to evaluate metabolites involved with the transulfuration pathway in urine samples collected during studies of eight liver toxic compounds in Sprague-Dawley rats. Urinary levels of creatine were increased on day 1 or day 2 in 8 high dose liver toxicity studies. Taurine concentration in urine was increased in only 3 of 8 liver toxicity studies while SAMe was found to be reduced in 4 of 5 liver toxicity studies. To further validate the results from the metabonomic studies, microarray data from rat liver samples following treatment with acetaminophen was obtained from the Gene Expression Omnibus (GEO) database. Some genes involved in the trans-sulfuration pathway, including guanidinoacetate N-methyltransferase, glycine N-methyltransferase, betaine-homocysteine methyltransferase and cysteine dioxygenase were found to be significantly decreased while methionine adenosyl transferase II, alpha increased at 24 h post-dosing, which is consistent with the SAMe and creatine findings. The metabolic and transcriptomic results show that the trans-sulfuration pathway from SAMe to glutathione was disturbed due to the administration of heptatotoxicants.

  5. Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells

    PubMed Central

    Wong, Kimberly A.; Trembley, Michael; Abd Wahab, Syafiq; Viczian, Andrea S.

    2015-01-01

    Retina formation requires the correct spatiotemporal patterning of key regulatory factors. While it is known that repression of several signaling pathways lead to specification of retinal fates, addition of only Noggin, a known BMP antagonist, can convert pluripotent Xenopus laevis animal cap cells to functional retinal cells. The aim of this study is to determine the intracellular molecular events that occur during this conversion. Surprisingly, blocking BMP signaling alone failed to mimic Noggin treatment. Overexpressing Noggin in pluripotent cells resulted in a concentration-dependent suppression of both Smad1 and Smad2 phosphorylation, which act downstream of BMP and Activin signaling, respectively. This caused a decrease in downstream targets: endothelial marker, xk81, and mesodermal marker, xbra. We treated pluripotent cells with dominant-negative receptors or the chemical inhibitors, dorsomorphin and SB431542, which each target either the BMP or Activin signaling pathway. We determined the effect of these treatments on retina formation using the Animal Cap Transplant (ACT) assay; in which treated pluripotent cells were transplanted into the eye field of host embryos. We found that inhibition of Activin signaling, in the presence of BMP signaling inhibition, promotes efficient retinal specification in Xenopus tissue, mimicking the affect of adding Noggin alone. In whole embryos, we found that the eye field marker, rax, expanded when adding both dominant-negative Smad1 and Smad2, as did treating the cells with both dorsomorphin and SB431542. Future studies could translate these findings to a mammalian culture assay, in order to more efficiently produce retinal cells in culture. PMID:25750435

  6. Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway

    PubMed Central

    Cinq-Frais, Christel; Coatrieux, Christelle; Savary, Aude; D’Angelo, Romina; Bernis, Corinne; Salvayre, Robert; Nègre-Salvayre, Anne; Augé, Nathalie

    2014-01-01

    Actin remodeling is a dynamic process associated with cell shape modification occurring during cell cycle and proliferation. Oxidative stress plays a role in actin reorganization via various systems including p38MAPK. Beside, the mitogenic response evoked by hydrogen peroxide (H2O2) in fibroblasts and smooth muscle cells (SMC) involves the metalloproteinase (MMPs)/sphingomyelinase 2 (nSMase2) signaling pathway. The aim of this work was to investigate whether this system plays a role in actin remodeling induced by H2O2. Low H2O2 dose (5 µM) rapidly triggered a signaling cascade leading to nSMase2 activation, src and annexin 2 (AnxA2) phosphorylation, and actin remodeling, in fibroblasts and SMC. These events were blocked by pharmacological inhibitors of MMPs (Ro28-2653) and p38MAPK (SB203580), and were lacking in MMP2?/? and in nSMase2-mutant (fro) fibroblasts. Likewise, H2O2 was unable to induce actin remodeling in fro and MMP2?/? fibroblasts or in cells pretreated with p38MAPK, or MMP inhibitors. Finally we show that nSMase2 activation by H2O2, depends on MMP2 and p38MAPK, and is required for the src-dependent phosphorylation of AnxA2, and actin remodeling. Taken together, these findings indicate for the first time that AnxA2 phosphorylation and actin remodeling evoked by oxidative stress depend on the sphingolipid pathway, via MMP2 and p38MAPK. PMID:25574848

  7. Ten-m3 Is Required for the Development of Topography in the Ipsilateral Retinocollicular Pathway

    PubMed Central

    Dharmaratne, Nuwan; Glendining, Kelly A.; Young, Timothy R.; Tran, Heidi; Sawatari, Atomu; Leamey, Catherine A.

    2012-01-01

    Background The alignment of ipsilaterally and contralaterally projecting retinal axons that view the same part of visual space is fundamental to binocular vision. While much progress has been made regarding the mechanisms which regulate contralateral topography, very little is known of the mechanisms which regulate the mapping of ipsilateral axons such that they align with their contralateral counterparts. Results Using the advantageous model provided by the mouse retinocollicular pathway, we have performed anterograde tracing experiments which demonstrate that ipsilateral retinal axons begin to form terminal zones (TZs) in the superior colliculus (SC), within the first few postnatal days. These appear mature by postnatal day 11. Importantly, TZs formed by ipsilaterally-projecting retinal axons are spatially offset from those of contralaterally-projecting axons arising from the same retinotopic location from the outset. This pattern is consistent with that required for adult visuotopy. We further demonstrate that a member of the Ten-m/Odz/Teneurin family of homophilic transmembrane glycoproteins, Ten-m3, is an essential regulator of ipsilateral retinocollicular topography. Ten-m3 mRNA is expressed in a high-medial to low-lateral gradient in the developing SC. This corresponds topographically with its high-ventral to low-dorsal retinal gradient. In Ten-m3 knockout mice, contralateral ventrotemporal axons appropriately target rostromedial SC, whereas ipsilateral axons exhibit dramatic targeting errors along both the mediolateral and rostrocaudal axes of the SC, with a caudal shift of the primary TZ, as well as the formation of secondary, caudolaterally displaced TZs. In addition to these dramatic ipsilateral-specific mapping errors, both contralateral and ipsilateral retinocollicular TZs exhibit more subtle changes in morphology. Conclusions We conclude that important aspects of adult visuotopy are established via the differential sensitivity of ipsilateral and contralateral axons to intrinsic guidance cues. Further, we show that Ten-m3 plays a critical role in this process and is particularly important for the mapping of the ipsilateral retinocollicular pathway. PMID:23028443

  8. Bmi1 Is Required for Hedgehog Pathway-Driven Medulloblastoma Expansion12

    PubMed Central

    Michael, Lowell Evan; Westerman, Bart A; Ermilov, Alexandre N; Wang, Aiqin; Ferris, Jennifer; Liu, Jianhong; Blom, Marleen; Ellison, David W; van Lohuizen, Maarten; Dlugosz, Andrzej A

    2008-01-01

    Inappropriate Hedgehog (Hh) signaling underlies development of a subset of medulloblastomas, and tumors with elevated HH signaling activity express the stem cell self-renewal gene BMI1. To test whether Bmi1 is required for Hh-driven medulloblastoma development, we varied Bmi1 gene dosage in transgenic mice expressing an oncogenic Hh effector, SmoA1, driven by a glial fibrillary acidic protein (GFAP) promoter. Whereas 100% of SmoA1; Bmi1+/+ or SmoA1;Bmi1+/- mice examined between postnatal (P) days 14 and 26 had typical medulloblastomas (N = 29), tumors were not detected in any of the SmoA1;Bmi1-/- animals examined (N = 6). Instead, small ectopic collections of cells were present in the region of greatest tumor load in SmoA1 animals, suggesting that medulloblastomas were initiated but failed to undergo expansion into frank tumors. Cells within these Bmi1-/- lesions expressed SmoA1 but were largely nonproliferative, in contrast to cells in Bmi1+/+ tumors (6.2% vs 81.9% PCNA-positive, respectively). Ectopic cells were negative for the progenitor marker nestin, strongly GFAP-positive, and highly apoptotic, relative to Bmi1+/+ tumor cells (29.6% vs 6.3% TUNEL-positive). The alterations in proliferation and apoptosis in SmoA1;Bmi1-/- ectopic cells are associated with reduced levels of Cyclin D1 and elevated expression of cyclin-dependent kinase inhibitor p19Arf, two inversely regulated downstream targets of Bmi1. These data provide the first demonstration that Bmi1 is required for spontaneous de novo development of a solid tumor arising in the brain, suggest a crucial role for Bmi1-dependent, nestin-expressing progenitor cells in medulloblastoma expansion, and implicate Bmi1 as a key factor required for Hh pathway-driven tumorigenesis. PMID:19048113

  9. Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory Pathways

    E-print Network

    Gillette, Martha U.

    Immortalized Suprachiasmatic Nucleus Cells Express Components of Multiple Circadian Regulatory fibroblasts, Rat-1 fibroblasts and spontaneously immortalized embryonic mouse fibroblasts after syn is a pluripotent and immortal- ized line of SCN progenitor cells from fetal rat hypo- thalamic tissue that exhibits

  10. Modulation of Cell Metabolic Pathways and Oxidative Stress Signaling Contribute to Acquired Melphalan Resistance in Multiple Myeloma Cells

    PubMed Central

    Zub, Kamila Anna; de Sousa, Mirta Mittelstedt Leal; Sarno, Antonio; Sharma, Animesh; Demirovic, Aida; Rao, Shalini; Young, Clifford; Aas, Per Arne; Ericsson, Ida; Sundan, Anders; Jensen, Ole Nørregaard; Slupphaug, Geir

    2015-01-01

    Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70–80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance. PMID:25769101

  11. Modulation of cell metabolic pathways and oxidative stress signaling contribute to acquired melphalan resistance in multiple myeloma cells.

    PubMed

    Zub, Kamila Anna; Sousa, Mirta Mittelstedt Leal de; Sarno, Antonio; Sharma, Animesh; Demirovic, Aida; Rao, Shalini; Young, Clifford; Aas, Per Arne; Ericsson, Ida; Sundan, Anders; Jensen, Ole Nørregaard; Slupphaug, Geir

    2015-01-01

    Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. Melphalan is the most commonly used chemotherapeutic agent against multiple myeloma. However, despite a 70-80% initial response rate, virtually all patients eventually relapse due to the emergence of drug-resistant tumour cells. By using global proteomic and transcriptomic profiling on melphalan sensitive and resistant RPMI8226 cell lines followed by functional assays, we discovered changes in cellular processes and pathways not previously associated with melphalan resistance in multiple myeloma cells, including a metabolic switch conforming to the Warburg effect (aerobic glycolysis), and an elevated oxidative stress response mediated by VEGF/IL8-signaling. In addition, up-regulated aldo-keto reductase levels of the AKR1C family involved in prostaglandin synthesis contribute to the resistant phenotype. Finally, selected metabolic and oxidative stress response enzymes were targeted by inhibitors, several of which displayed a selective cytotoxicity against the melphalan-resistant cells and should be further explored to elucidate their potential to overcome melphalan resistance. PMID:25769101

  12. Multiple effects of digoxin on subsets of cancer-associated genes through the alternative splicing pathway.

    PubMed

    Lu, Guan-Yu; Liu, Shu-Ting; Huang, Shih-Ming; Chang, Yung-Lung; Lin, Wei-Shiang

    2014-11-01

    The signaling characteristics of Na(+)/K(+)-ATPase are distinct from its ion pumping activity. Cardiac glycosides modulate the Na(+)/K(+)-ATPase protein complex upon binding, activate downstream signaling pathways and increase [Ca(2+)]i. Recent studies demonstrate that the depletion of p53 and hypoxia-induced factor 1? proteins is caused by cardiac glycosides. However, the detailed mechanisms governing this process are not well known. In this study, we showed that the depletion of p53 proteins by digoxin involved not only inhibition of protein synthesis but also inhibition at the post-transcriptional level. Post-transcriptional regulation occurs via down-regulation of SRSF3, the primary splicing factor responsible for the switch from p53? to the p53? isoform. Digoxin also modulated G2/M arrest, DNA damage and apoptosis through the p53-dependent pathway in HeLa cells. In addition, digoxin was involved in epithelial-mesenchymal-transition progression via E-cadherin reduction and snail induction. Digoxin had similar effects to caffeine, another SRSF3-reduced agent, on the cell cycle profile and DNA damage of cells. Interestingly, combined digoxin and caffeine treatment blocked cell cycle progression and conferred resistance to cell death via snail induction. These findings demonstrate that down-regulation of splicing factor, such as SRSF3, to alter cell cycle progression, cell death and invasion is a potential target for the drug repositioning of cardiac glycosides. PMID:25193633

  13. The proinflammatory cytokine interleukin-18 alters multiple signaling pathways to inhibit natural killer cell death

    USGS Publications Warehouse

    Hodge, D.L.; Subleski, J.J.; Reynolds, D.A.; Buschman, M.D.; Schill, W.B.; Burkett, M.W.; Malyguine, A.M.; Young, H.A.

    2006-01-01

    The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-?? (IFN-??) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors. ?? Mary Ann Liebert, Inc.

  14. Multiple signaling pathways involved in the effect of endothelin type B receptor in rat median eminence.

    PubMed

    Mathison, Yaira; del Garrido, María Rosario; Israel, Anita

    2007-06-01

    We assessed the possible link between endothelin receptor mediated phosphoinositide breakdown and NO/cGMP signaling pathways in rat arcuate nucleus-median eminence fragments (AN-ME), brain structures known to contain a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers, together with densely arranged endothelin ETB-receptors-like immunoreactive fibres. Our data show that ET-1, ET-3 and the ETB-receptors agonist, IRL 1620, increased inositol monophosphate (InsP1) accumulation, NOS activity and cGMP formation, in a similar degree. The stimulatory effect of ETs on InsP1 accumulation and cGMP formation was inhibited by the phospholipase C (PLC) inhibitor, neomycin, and the absence of extracellular calcium, suggesting that calcium is involved in endothelin receptor-induced PLC activation. The L-arginine analog, L-NAME, inhibited ET-1 or IRL1620-stimulated cGMP formation. The ETA receptor antagonists BQ 123, did not alter, while the ETB receptor antagonists BQ788 inhibited ETs-induced increase in the PI metabolism, NOS activity and cGMP generation. Our data indicate that in AN-ME, ETB receptor signals through receptor-mediated calcium dependent-stimulation of phosphoinositide breakdown and activation of NOS/cGMP signaling pathway. PMID:17585504

  15. Peptidoglycan Up-Regulates CXCL8 Expression via Multiple Pathways in Monocytes/Macrophages

    PubMed Central

    Lee, Chung Won; Chung, Sung Woon; Bae, Mi Ju; Song, Seunghwan; Kim, Sang-pil; Kim, Koanhoi

    2015-01-01

    Peptidoglycan (PG), the gram positive bacterial pathogen-associated molecular patterns (PAMP), is detected in a high proportion in macrophage-rich atheromatous regions, and expression of chemokine CXCL8, which triggers monocyte arrest on early atherosclerotic endothelium, is elevated in monocytes/macrophages in human atherosclerotic lesion. The aim of this study was to investigate whether PG induced CXCL8 expression in the cell type and to determine cellular signaling pathways involved in that process. Exposure of THP-1 cell, human monocyte/macrophage cell line, to PG not only enhanced CXCL8 release but also profoundly induced il8 gene transcription. PG-induced release of CXCL8 and induction of il8 gene transcription were blocked by OxPAPC, an inhibitor of TLR-2/4 and TLR4, but not by polymyxin B, an inhibitor of LPS. PG-mediated CXCL8 release was significantly attenuated by inhibitors of PI3K-Akt-mTOR pathways. PKC inhibitors, MAPK inhibitors, and ROS quenchers also significantly attenuated expression of CXCL8. The present study proposes that PG contributes to inflammatory reaction and progression of atherosclerosis by inducing CXCL8 expression in monocytes/macrophages, and that TLR-2, PI3K-Akt-mTOR, PKC, ROS, and MAPK are actively involved in the process. PMID:26535082

  16. Propofol attenuates hydrogenperoxide-induced apoptosis in human umbilical vein endothelial cells via multiple signaling pathways

    PubMed Central

    Xie, Cheng Lan; Pan, Yin Bing; Hu, Liu Qing

    2015-01-01

    Background Propofol has been reported to protect vascular endothelial cells against oxidative stress. In this study we investigated its effect on hydrogen peroxide (H2O2)-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and examined the possible signaling pathways. Methods HUVECs were pretreated with propofol (1, 5, 25, and 50 µM) for 30 min and then co-incubated with 0.4 mM H2O2 for 4 h. Cell viability was assessed using a Cell Counting Kit-8. Cell apoptosis was analyzed using flow cytometry with annexin V/propidium iodide staining, and evaluated by quantifying caspase-3, Bax, and Bcl-2 expression levels. The expression levels of p38 mitogen activated protein kinase (MAPK), phosphorylated (p)-p38 MAPK, cJun-N-terminal kinases (JNK), phosphorylated (p)-JNK, Akt and phosphorylated Akt [(p)-Akt] (Ser473) were measured by western blotting. Results H2O2 treatment induced the activation of caspase-3, downregulated Bcl-2 expression, and up-regulated Bax expression, all of which were dose-dependently attenuated by propofol pretreatment. Furthermore, propofol significantly ameliorated H2O2-induced phosphorylation of p38 MAPK, JNK, and Akt in HUVECs. Conclusions Propofol can protect HUVECs against H2O2-induced apoptosis via a mechanism that may involve p38 MAPK, JNK, and Akt signaling pathways. PMID:26495060

  17. Multiple Independent Fusions of Glucose-6-Phosphate Dehydrogenase with Enzymes in the Pentose Phosphate Pathway

    PubMed Central

    Stover, Nicholas A.; Dixon, Thomas A.; Cavalcanti, Andre R. O.

    2011-01-01

    Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconolactonase (6PGL), have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD) in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms. PMID:21829610

  18. Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway.

    PubMed

    Hassan, Ihab H; Zhang, Michael S; Powers, Linda S; Shao, Jian Q; Baltrusaitis, Jonas; Rutkowski, D Thomas; Legge, Kevin; Monick, Martha M

    2012-02-10

    Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications. PMID:22194594

  19. Vitamin D receptor pathway is required for probiotic protection in colitis.

    PubMed

    Wu, Shaoping; Yoon, Sonia; Zhang, Yong-Guo; Lu, Rong; Xia, Yinglin; Wan, Jiandi; Petrof, Elaine O; Claud, Erika C; Chen, Di; Sun, Jun

    2015-09-01

    Low expression of vitamin D receptor (VDR) and dysfunction of vitamin D/VDR signaling are reported in patients with inflammatory bowel disease (IBD); therefore, restoration of VDR function to control inflammation in IBD is desirable. Probiotics have been used in the treatment of IBD. However, the role of probiotics in the modulation of VDR signaling to effectively reduce inflammation is unknown. We identified a novel role of probiotics in activating VDR activity, thus inhibiting inflammation, using cell models and VDR knockout mice. We found that the probiotics Lactobacillus rhamnosus strain GG (LGG) and Lactobacillus plantarum (LP) increased VDR protein expression in both mouse and human intestinal epithelial cells. Using the VDR luciferase reporter vector, we detected increased transcriptional activity of VDR after probiotic treatment. Probiotics increased the expression of the VDR target genes, such as antimicrobial peptide cathelicidin, at the transcriptional level. Furthermore, the role of probiotics in regulating VDR signaling was tested in vivo using a Salmonella-colitis model in VDR knockout mice. Probiotic treatment conferred physiological and histologic protection from Salmonella-induced colitis in VDR(+/+) mice, whereas probiotics had no effects in the VDR(-/-) mice. Probiotic treatment also enhanced numbers of Paneth cells, which secrete AMPs for host defense. These data indicate that the VDR pathway is required for probiotic protection in colitis. Understanding how probiotics enhance VDR signaling and inhibit inflammation will allow probiotics to be used effectively, resulting in innovative approaches to the prevention and treatment of chronic inflammation. PMID:26159695

  20. Multiple stalk formation as a pathway of defect-induced membrane fusion

    E-print Network

    D. B. Lukatsky; Daan Frenkel

    2004-03-29

    We propose that the first stage of membrane fusion need not be the formation of a single stalk. Instead, we consider a scenario for defect-induced membrane fusion that proceeds cooperatively via multiple stalk formation. The defects (stalks or pores) attract each other via membrane-mediated capillary interactions that result in a condensation transition of the defects. The resulting dense phase of stalks corresponds to the so-called fusion intermediate.

  1. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis

    PubMed Central

    Jeanblanc, Jerome; Logrip, Marian L.; Janak, Patricia H.; Ron, Dorit

    2013-01-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494–13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-? (PLC-?) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-? or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. PMID:23189980

  2. BDNF-mediated regulation of ethanol consumption requires the activation of the MAP kinase pathway and protein synthesis.

    PubMed

    Jeanblanc, Jerome; Logrip, Marian L; Janak, Patricia H; Ron, Dorit

    2013-02-01

    We previously found that the brain-derived neurotrophic factor (BDNF) in the dorsolateral striatum (DLS) is part of a homeostatic pathway that gates ethanol self-administration [Jeanblanc et al. (2009). J Neurosci, 29, 13494-13502)]. Specifically, we showed that moderate levels (10%) of ethanol consumption increase BDNF expression within the DLS, and that direct infusion of BDNF into the DLS decreases operant self-administration of a 10% ethanol solution. BDNF binding to its receptor, TrkB, activates the mitogen-activated protein kinase (MAPK), phospholipase C-? (PLC-?) and phosphatidylinositol 3-kinase (PI3K) pathways. Thus, here, we set out to identify which of these intracellular pathway(s) plays a role in the regulation of ethanol consumption by BDNF. We found that inhibition of the MAPK, but not PLC-? or PI3K, activity blocks the BDNF-mediated reduction of ethanol consumption. As activation of the MAPK pathway leads to the initiation of transcription and/or translation events, we tested whether the BDNF-mediated reduction of ethanol self-administration requires de novo protein synthesis. We found that the inhibitory effect of BDNF on ethanol intake is blocked by the protein synthesis inhibitor cycloheximide. Together, our results show that BDNF attenuates ethanol drinking via activation of the MAPK pathway in a protein synthesis-dependent manner within the DLS. PMID:23189980

  3. Targeting multiple angiogenic pathways simultaneously: experience with nintedanib in non-small-cell lung cancer.

    PubMed

    Durm, Greg; Hanna, Nasser

    2014-05-01

    Angiogenesis plays a major role in the growth and progression of non-small-cell lung cancer (NSCLC), and there is increasing interest in the development of therapies that block this particular aspect of tumorigenesis. Bevacizumab was the first US FDA-approved inhibitor of angiogenesis after demonstrating improved progression-free survival and overall survival in combination with chemotherapy in treating NSCLC. However, the benefit of bevacizumab is only modest and transient as the tumors inevitably develop resistance to this particular treatment. Therefore, new therapies are being developed that attempt to inhibit angiogenesis through several different pathways. One promising new drug, nintedanib, is an oral triple angiokinase inhibitor that acts by blocking not only VEGFR, but also FGFR and PDGFR, which are involved in the development of resistance to bevacizumab. This article discusses the rationale for this approach and summarizes the clinical trial data on nintedanib, including the two most recent Phase III trials. PMID:24947258

  4. Capping protein integrates multiple MAMP signalling pathways to modulate actin dynamics during plant innate immunity.

    PubMed

    Li, Jiejie; Henty-Ridilla, Jessica L; Staiger, Benjamin H; Day, Brad; Staiger, Christopher J

    2015-01-01

    Plants and animals perceive diverse microbe-associated molecular patterns (MAMPs) via pattern recognition receptors and activate innate immune signalling. The actin cytoskeleton has been suggested as a target for innate immune signalling and a key transducer of cellular responses. However, the molecular mechanisms underlying actin remodelling and the precise functions of these rearrangements during innate immunity remain largely unknown. Here we demonstrate rapid actin remodelling in response to several distinct MAMP signalling pathways in plant epidermal cells. The regulation of actin dynamics is a convergence point for basal defence machinery, such as cell wall fortification and transcriptional reprogramming. Our quantitative analyses of actin dynamics and genetic studies reveal that MAMP-stimulated actin remodelling is due to the inhibition of capping protein (CP) by the signalling lipid, phosphatidic acid. In addition, CP promotes resistance against bacterial and fungal phytopathogens. These findings demonstrate that CP is a central target for the plant innate immune response. PMID:26018794

  5. Capping protein integrates multiple MAMP signalling pathways to modulate actin dynamics during plant innate immunity

    PubMed Central

    Li, Jiejie; Henty-Ridilla, Jessica L.; Staiger, Benjamin H.; Day, Brad; Staiger, Christopher J.

    2015-01-01

    Plants and animals perceive diverse microbe-associated molecular patterns (MAMPs) via pattern recognition receptors and activate innate immune signalling. The actin cytoskeleton has been suggested as a target for innate immune signalling and a key transducer of cellular responses. However, the molecular mechanisms underlying actin remodelling and the precise functions of these rearrangements during innate immunity remain largely unknown. Here we demonstrate rapid actin remodelling in response to several distinct MAMP signalling pathways in plant epidermal cells. The regulation of actin dynamics is a convergence point for basal defence machinery, such as cell wall fortification and transcriptional reprogramming. Our quantitative analyses of actin dynamics and genetic studies reveal that MAMP-stimulated actin remodelling is due to the inhibition of capping protein (CP) by the signalling lipid, phosphatidic acid. In addition, CP promotes resistance against bacterial and fungal phytopathogens. These findings demonstrate that CP is a central target for the plant innate immune response. PMID:26018794

  6. Multiple signaling pathways are responsible for prostaglandin E2-induced murine keratinocyte proliferation

    PubMed Central

    Ansari, Kausar M.; Rundhaug, Joyce E.; Fischer, Susan M.

    2008-01-01

    Although prostaglandin E2 (PGE2) has been shown by pharmacological and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor growth is not well understood. In this study we investigated the mechanisms by which PGE2 stimulates murine keratinocyte proliferation using in vitro and in vivo models. In primary mouse keratinocyte (PMK) cultures, PGE2 activated the epidermal growth factor receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP2), nor by a protein kinase A inhibitor (H-89). However, PGE2-stimulated extracellularly-regulated kinase1/2 (ERK1/2) activation was completely blocked by EGFR, ERK1/2 and phosphatidylinositol 3-kinase (PI3-K) pathway inhibitors. In addition, these inhibitors attenuated the PGE2-induced proliferation, nuclear factor-?B (NF-?B), activator protein-1 (AP-1) and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF in PMKs. Similarly, in vivo, we found that wild type (WT) mice treated with PGE2 and untreated COX-2 overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, NF-?B, AP-1 and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between COX-2 overexpression and EGFR-, ERK-, PI3-K-, cAMP-mediated cell proliferation, and the tumor promoting activity of PGE2 in mouse skin. PMID:18567804

  7. Ubiquitin-proteasome pathway function is required for lens cell proliferation and differentiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin proteasome pathway is involved in the regulation of many cellular processes, such as cell cycle control, signal transduction, transcription, and removal of obsolete proteins. The objective of this work was to investigate roles for this proteolytic pathway in controlling the differentia...

  8. Mild depletion of dietary folate combined with other B vitamins alters multiple components of the Wnt pathway in mouse colon.

    PubMed

    Liu, Zhenhua; Choi, Sang-Woon; Crott, Jimmy W; Keyes, Mary K; Jang, Hyeran; Smith, Donald E; Kim, Myungjin; Laird, Peter W; Bronson, Roderick; Mason, Joel B

    2007-12-01

    Preclinical and clinical studies suggest that diminished folate status increases the risk of colorectal carcinogenesis. However, many biochemical functions of folate are dependent on the adequate availability of other 1-carbon nutrients, including riboflavin, vitamin B-6, and vitamin B-12. Aberrations in the Wnt pathway are thought to play an important role in human colorectal cancers. This study therefore investigated if mild depletion of folate combined with depletion of riboflavin, vitamin B-6, and vitamin B-12 could induce alterations in the Wnt pathway in the colonic mucosa. Ninety-six mice were pair-fed diets with different combinations of B vitamin depletion for 10 wk. Genomic DNA methylation and uracil misincorporation were measured by LC/MS and GC/MS. Gene-specific methylation, strand breaks, and expressions were measured by real-time PCR and immunoblotting. Proliferation and apoptosis were determined by immunohistochemistry. DNA strand breaks within the Apc mutation cluster region were induced by folate depletion combined with inadequacies of riboflavin, vitamin B-6, and vitamin B-12 (P < 0.05), but such effects were not induced by folate depletion alone. Similarly, minor changes in the expression of Apc, beta-catenin, and cyclin D1 produced by mild folate depletion were significantly magnified by multiple vitamin depletion. Apoptosis, which can be suppressed by increased Wnt-signaling, was attenuated by the combined deficiency state (P < 0.05) but not by singlet or doublet deficiencies. These findings indicate that a mild depletion of folate that is of insufficient magnitude by itself to induce alterations in components of the Wnt pathway may produce such effects when present in conjunction with mild inadequacies of other 1-carbon nutrients. PMID:18029487

  9. Thalidomide decreases gelatinase production by malignant B lymphoid cell lines through disruption of multiple integrin-mediated signaling pathways

    PubMed Central

    Segarra, Marta; Lozano, Ester; Corbera-Bellalta, Marc; Vilardell, Carme; Cibeira, Maria-Teresa; Esparza, Jordi; Izco, Nora; Bladé, Joan; Cid, Maria C.

    2010-01-01

    Background Thalidomide and its analogs are effective agents in the treatment of multiple myeloma. Since gelatinases (matrix metalloproteinases-2 and -9) play a crucial role in tumor progression, we explored the effect of thalidomide on gelatinase production by malignant B lymphoid cell lines. Design and Methods We investigated the effect of therapeutic doses of thalidomide on integrin-mediated production of gelatinases by malignant B lymphoid cell lines by gelatin zymography, western-blot, reverse transcriptase polymerase chain reaction and invasive capacity through Matrigel-coated Boyden chambers. We also explored the effect of thalidomide on the activation status of the main signaling pathways involved in this process. Results Thalidomide strongly inhibited gelatinase production by B-cell lines and primary myeloma cells in response to fibronectin, the most efficient gelatinase inducer identified in lymphoid cells. Thalidomide disrupted integrin-mediated signaling pathways involved in gelatinase induction and release, such as Src and MAP-kinase ERK activation, resulting in decreased cell motility and invasiveness. Unexpectedly, treatment with thalidomide elicited an increase in fibronectin-induced Akt phosphorylation through phosphoinositide 3-kinase-independent pathways since thalidomide decreased fibronectin-induced phosphoinositide 3-kinase phosphorylation and reversed the inhibition of Akt phosphorylation achieved by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Conclusions Disruption of integrin-mediated signaling may be an important mechanism through which thalidomide and its analogs impair tumor cell interactions with the microenvironment. The unexpected effects of thalidomide on Akt activation indicate the need for further studies to elucidate whether the interference with Akt downstream effects would synergize with the anti-tumor activity of thalidomide. PMID:19815837

  10. The 3-Hydroxy-2-Butanone Pathway Is Required for Pectobacterium carotovorum Pathogenesis

    PubMed Central

    Marquez-Villavicencio, Maria del Pilar; Weber, Brooke; Witherell, R. Andrews; Willis, David K.; Charkowski, Amy O.

    2011-01-01

    Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the ?-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH. PMID:21876734

  11. Number of repetitions required to retain single-digit multiplication math facts for elementary students.

    PubMed

    Burns, Matthew K; Ysseldyke, Jim; Nelson, Peter M; Kanive, Rebecca

    2015-09-01

    Computational fluency is an important aspect of math proficiency. Despite widely held beliefs about the differential difficulty of single-digit multiplication math facts, little empirical work has examined this issue. The current study analyzed the number of repetitions needed to master multiplication math facts. Data from 15,402 3rd, 4th, and 5th graders were analyzed using a national database. Results suggested that (a) students with lower math skills required significantly (p < .001) more repetitions than more skilled students; (b) across all students, single-digit multiplication facts with 4s, 5s, 6s, and 7s required significantly (p < .001) more repetition than did 2s and 3s; and (c) the number of practice sessions needed to attain mastery significantly (p < .001) decreased with increase in grade level. Implications for instructional planning and implementation are discussed. PMID:25420042

  12. 40 CFR 63.1356 - Sources with multiple emissions limit or monitoring requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 12 2014-07-01 2014-07-01 false Sources with multiple emissions limit or monitoring requirements. 63.1356 Section 63.1356 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES (CONTINUED)...

  13. 40 CFR 63.1356 - Sources with multiple emissions limit or monitoring requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 12 2013-07-01 2013-07-01 false Sources with multiple emissions limit or monitoring requirements. 63.1356 Section 63.1356 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES (CONTINUED)...

  14. 40 CFR 63.1356 - Sources with multiple emission limits or monitoring requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 11 2011-07-01 2011-07-01 false Sources with multiple emission limits or monitoring requirements. 63.1356 Section 63.1356 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES National...

  15. Multiple Smaller Missions as a Direct Pathway to Mars Sample Return

    NASA Technical Reports Server (NTRS)

    Niles, P. B.; Draper, D. S.; Evans, C. A.; Gibson, E. K.; Graham, L. D.; Jones, J. H.; Lederer, S. M.; Ming, D.; Seaman, C. H.; Archer, P. D.; Andrews-Hanna, J.; Baldridge, A. M.; Bourke, M. C.; Crown, D. A.; Fries, M.; Knudson, A. T.; Michalski, J.; Dobrea, E. Noe; Vaniman, D.; Weitz, C. M.; Williams, R. M. E.; Bell, J. F., III; Knauth, L. P.

    2012-01-01

    Recent discoveries by the Mars Exploration Rovers, Mars Express, Mars Odyssey, and Mars Reconnaissance Orbiter spacecraft include multiple, tantalizing astrobiological targets representing both past and present environments on Mars. The most desirable path to Mars Sample Return (MSR) would be to collect and return samples from that site which provides the clearest examples of the variety of rock types considered a high priority for sample return (pristine igneous, sedimentary, and hydrothermal). Here we propose an MSR architecture in which the next steps (potentially launched in 2018) would entail a series of smaller missions, including caching, to multiple landing sites to verify the presence of high priority sample return targets through in situ analyses. This alternative architecture to one flagship-class sample caching mission to a single site would preserve a direct path to MSR as stipulated by the Planetary Decadal Survey, while permitting investigation of diverse deposit types and providing comparison of the site of returned samples to other aqueous environments on early Mars

  16. Free Energy Landscape and Multiple Folding Pathways of an H-Type RNA Pseudoknot

    PubMed Central

    Bian, Yunqiang; Zhang, Jian; Wang, Jun; Wang, Jihua; Wang, Wei

    2015-01-01

    How RNA sequences fold to specific tertiary structures is one of the key problems for understanding their dynamics and functions. Here, we study the folding process of an H-type RNA pseudoknot by performing a large-scale all-atom MD simulation and bias-exchange metadynamics. The folding free energy landscapes are obtained and several folding intermediates are identified. It is suggested that the folding occurs via multiple mechanisms, including a step-wise mechanism starting either from the first helix or the second, and a cooperative mechanism with both helices forming simultaneously. Despite of the multiple mechanism nature, the ensemble folding kinetics estimated from a Markov state model is single-exponential. It is also found that the correlation between folding and binding of metal ions is significant, and the bound ions mediate long-range interactions in the intermediate structures. Non-native interactions are found to be dominant in the unfolded state and also present in some intermediates, possibly hinder the folding process of the RNA. PMID:26030098

  17. NDR1 interaction with RIN4 mediates the differential activation of multiple disease resistance pathways in Arabidopsis.

    PubMed

    Day, Brad; Dahlbeck, Douglas; Staskawicz, Brian J

    2006-10-01

    Recognition of pathogens by plants involves the coordinated efforts of molecular chaperones, disease resistance (R) proteins, and components of disease resistance signaling pathways. Characterization of events associated with pathogen perception in Arabidopsis thaliana has advanced understanding of molecular genetic mechanisms associated with disease resistance and protein interactions critical for the activation of resistance signaling. Regulation of R protein-mediated signaling in response to the bacterial pathogen Pseudomonas syringae in Arabidopsis involves the physical association of at least two R proteins with the negative regulator RPM1 INTERACTING PROTEIN4 (RIN4). While the RIN4-RPS2 (for RESISTANCE TO P. SYRINGAE2) and RIN4-RPM1 (for RESISTANCE TO P. SYRINGAE PV MACULICOLA1) signaling pathways exhibit differential mechanisms of activation in terms of effector action, the requirement for NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) is shared. Using a yeast two-hybrid screen, followed by a series of coimmunoprecipitation experiments, we demonstrate that the RIN4-NDR1 interaction occurs on the cytoplasmically localized N-terminal portion of NDR1 and that this interaction is required for the activation of resistance signaling following infection by P. syringae expressing the Cys protease Type III effector protein AvrRpt2. We demonstrate that like RPS2 and RPM1, NDR1 also associates with RIN4 in planta. We suggest that this interaction serves to further regulate activation of disease resistance signaling following recognition of P. syringae DC3000-AvrRpt2 by Arabidopsis. PMID:17012600

  18. Evidence for multiple pathways of sup 125 I-insulin internalization in isolated rat hepatocytes

    SciTech Connect

    Moss, A.L.

    1988-01-01

    Insulin internalization has been characterized frequently as occurring by the coated pit pathway of receptor-mediated endocytosis. The present study in rat hepatocytes demonstrates that insulin internalization is, in part, receptor-mediated, but also occurs by nonreceptor-mediated or fluid-phase endocytosis. Endocytosis was probed with four perturbations: depletion of metabolic energy with anoxia, inhibition of endocytosis with phenylarsine oxide, disruption of coated pits with hyperosmolar sucrose, and inhibition of receptor recycling or ligand-receptor dissociation with monensin. Internalization of {sup 125}I-epidermal growth factor and {sup 125}I-asialofetuin was compared to {sup 125}I-insulin internalization. Pretreatment of cells with anoxia or hyperosmolarity inhibited {sup 125}I-insulin internalization by 40%; pretreatment with phenylarsine oxide resulted in inhibition by 54%. Monensin has no effect on uptake or degradation of a high insulin concentration, but inhibited degradation of a low insulin concentration resulting in intracellular accumulation of insulin. In contract, all four perturbations inhibited {sup 125}I-asialofetuin internalization by greater than 90%. Phenylarsine oxide almost completely abolished {sup 125}I-epidermal growth factor uptake; the other perturbations caused partial inhibition. Competition studies demonstrated that insulin internalization was receptor-mediated over a wide concentration range.

  19. Multiple Pathways Suppress Telomere Addition to DNA Breaks in the Drosophila Germline

    PubMed Central

    Beaucher, Michelle; Zheng, Xiao-Feng; Amariei, Flavia; Rong, Yikang S.

    2012-01-01

    Telomeres protect chromosome ends from being repaired as double-strand breaks (DSBs). Just as DSB repair is suppressed at telomeres, de novo telomere addition is suppressed at the site of DSBs. To identify factors responsible for this suppression, we developed an assay to monitor de novo telomere formation in Drosophila, an organism in which telomeres can be established on chromosome ends with essentially any sequence. Germline expression of the I-SceI endonuclease resulted in precise telomere formation at its cut site with high efficiency. Using this assay, we quantified the frequency of telomere formation in different genetic backgrounds with known or possible defects in DNA damage repair. We showed that disruption of DSB repair factors (Rad51 or DNA ligase IV) or DSB sensing factors (ATRIP or MDC1) resulted in more efficient telomere formation. Interestingly, partial disruption of factors that normally regulate telomere protection (ATM or NBS) also led to higher frequencies of telomere formation, suggesting that these proteins have opposing roles in telomere maintenance vs. establishment. In the ku70 mutant background, telomere establishment was preceded by excessive degradation of DSB ends, which were stabilized upon telomere formation. Most strikingly, the removal of ATRIP caused a dramatic increase in telomeric retrotransposon attachment to broken ends. Our study identifies several pathways thatsuppress telomere addition at DSBs, paving the way for future mechanistic studies. PMID:22446318

  20. Multiple independent regulatory pathways control UBI4 expression after heat shock in Saccharomyces cerevisiae.

    PubMed

    Simon, J R; Treger, J M; McEntee, K

    1999-02-01

    Transcription of the polyubiquitin gene UBI4 of Saccharomyces cerevisiae is strongly induced by a variety of environmental stresses, such as heat shock, nutrient depletion and exposure to DNA-damaging agents. This transcriptional response of UBI4 is likely to be the primary mechanism for increasing the pool of ubiquitin for degradation of stress-damaged proteins. Deletion and promoter fusion studies of the 5' regulatory sequences indicated that two different elements, heat shock elements (HSEs) and stress response element (STREs), contributed independently to heat shock regulation of the UBI4 gene. In the absence of HSEs, STRE sequences localized to the intervals -264 to -238 and -215 to -183 were needed for stress control of transcription after heat shock. Site-directed mutagenesis of the STRE (AG4) at -252 to -248 abolished heat shock induction of UBI4 transcription. Northern analysis demonstrated that cells containing either a temperature-sensitive HSF or non-functional Msn2p/Msn4p transcription factors induced high levels of UBI4 transcripts after heat shock. In cells deficient in both heat stress pathways, heat-induced UBI4 transcript levels were considerably lower but not abolished, suggesting a role for another factor(s) in stress control of its expression. PMID:10048026

  1. Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways

    PubMed Central

    Kitchen, Philip; Öberg, Fredrik; Sjöhamn, Jennie; Hedfalk, Kristina; Bill, Roslyn M.; Conner, Alex C.; Conner, Matthew T.; Törnroth-Horsefield, Susanna

    2015-01-01

    Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow. PMID:26569106

  2. NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation

    SciTech Connect

    Adachi, Atsuo; Takahashi, Tomosaburo; Ogata, Takehiro; Imoto-Tsubakimoto, Hiroko; Nakanishi, Naohiko; Ueyama, Tomomi; Matsubara, Hiroaki

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer NFAT5 protein expression is downregulated during cardiomyogenesis. Black-Right-Pointing-Pointer Inhibition of NFAT5 function suppresses canonical Wnt signaling. Black-Right-Pointing-Pointer Inhibition of NFAT5 function attenuates mesodermal induction. Black-Right-Pointing-Pointer NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2 Prime Z, 3 Prime E]-6-bromoindirubin-3 Prime -oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

  3. PPAR? inhibition modulates multiple reprogrammed metabolic pathways in kidney cancer and attenuates tumor growth.

    PubMed

    Abu Aboud, Omran; Donohoe, Dallas; Bultman, Scott; Fitch, Mark; Riiff, Tim; Hellerstein, Marc; Weiss, Robert H

    2015-06-01

    Kidney cancer [renal cell carcinoma (RCC)] is the sixth-most-common cancer in the United States, and its incidence is increasing. The current progression-free survival for patients with advanced RCC rarely extends beyond 1-2 yr due to the development of therapeutic resistance. We previously identified peroxisome proliferator-activating receptor-? (PPAR?) as a potential therapeutic target for this disease and showed that a specific PPAR? antagonist, GW6471, induced apoptosis and cell cycle arrest at G0/G1 in RCC cell lines associated with attenuation of cell cycle regulatory proteins. We now extend that work and show that PPAR? inhibition attenuates components of RCC metabolic reprogramming, capitalizing on the Warburg effect. The specific PPAR? inhibitor GW6471, as well as a siRNA specific to PPAR?, attenuates the enhanced fatty acid oxidation and oxidative phosphorylation associated with glycolysis inhibition, and PPAR? antagonism also blocks the enhanced glycolysis that has been observed in RCC cells; this effect did not occur in normal human kidney epithelial cells. Such cell type-specific inhibition of glycolysis corresponds with changes in protein levels of the oncogene c-Myc and has promising clinical implications. Furthermore, we show that treatment with GW6471 results in RCC tumor growth attenuation in a xenograft mouse model, with minimal obvious toxicity, a finding associated with the expected on-target effects on c-Myc. These studies demonstrate that several pivotal cancer-relevant metabolic pathways are inhibited by PPAR? antagonism. Our data support the concept that targeting PPAR?, with or without concurrent inhibition of glycolysis, is a potential novel and effective therapeutic approach for RCC that targets metabolic reprogramming in this tumor. PMID:25810260

  4. Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways.

    PubMed

    Willett, Julia L E; Gucinski, Grant C; Fatherree, Jackson P; Low, David A; Hayes, Christopher S

    2015-09-01

    Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria. PMID:26305955

  5. Ablation of Vacuole Protein Sorting 18 (Vps18) Gene Leads to Neurodegeneration and Impaired Neuronal Migration by Disrupting Multiple Vesicle Transport Pathways to Lysosomes*?

    PubMed Central

    Peng, Chao; Ye, Jian; Yan, Shunfei; Kong, Shanshan; Shen, Ye; Li, Chenyu; Li, Qinyu; Zheng, Yufang; Deng, Kejing; Xu, Tian; Tao, Wufan

    2012-01-01

    Intracellular vesicle transport pathways are critical for neuronal survival and central nervous system development. The Vps-C complex regulates multiple vesicle transport pathways to the lysosome in lower organisms. However, little is known regarding its physiological function in mammals. We deleted Vps18, a central member of Vps-C core complex, in neural cells by generating Vps18F/F; Nestin-Cre mice (Vps18 conditional knock-out mice). These mice displayed severe neurodegeneration and neuronal migration defects. Mechanistic studies revealed that Vps18 deficiency caused neurodegeneration by blocking multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis, and biosynthetic pathways. Our study also showed that ablation of Vps18 resulted in up-regulation of ?1 integrin in mouse brain probably due to lysosome dysfunction but had no effects on the reelin pathway, expression of N-cadherin, or activation of JNK, which are implicated in the regulation of neuronal migration. Finally, we demonstrated that knocking down ?1 integrin partially rescued the migration defects, suggesting that Vps18 deficiency-mediated up-regulation of ?1 integrin may contribute to the defect of neuronal migration in the Vps18-deficient brain. Our results demonstrate important roles of Vps18 in neuron survival and migration, which are disrupted in multiple neural disorders. PMID:22854957

  6. Endocannabinoid pathways and their role in multiple sclerosis-related muscular dysfunction.

    PubMed

    Di Marzo, Vincenzo

    2011-04-01

    Endocannabinoids are endogenous agonists of the mammalian cannabinoid receptors CB(1) and CB(2), and they appear to be produced in tissues as an adaptive reaction to re-establish normal homeostasis when this is acutely altered. However, the production of endocannabinoids can be altered pathologically. The two most widely studied endocannabinoids are anandamide and 2-arachidonoyl glycerol. The levels of these endogenous modulators are regulated in different and sometimes opposing ways, and alterations in cerebrospinal fluid and/or spinal cord levels have been documented in animal models of neurodegenerative diseases and in samples from patients with multiple sclerosis (MS). Modulation of the endocannabinoid system has been shown to have therapeutic potential in a number of disease states. Sativex(®) (nabiximols, USAN name) contains the two main phytocannabinoids from Cannabis sativa, tetrahydrocannabinol and cannabidiol in a 1:1 ratio, and it acts as an endocannabinoid system modulator. In an experimental mouse model of MS-related spasticity, Sativex dose-dependently improved hind limb flexion/stiffness and a dosage of 10 mg/kg was shown to be as effective as the most widely established anti-spasticity treatment baclofen (5 mg/kg). These findings with Sativex are very promising and offer encouragement for MS patients, the majority of whom will develop spasticity-related disabling and recalcitrant symptoms. Furthermore, research into the endocannabinoid system may offer potential in other neurodegenerative, inflammatory and pain disorders. PMID:21449854

  7. T cell-specific inhibition of multiple apoptotic pathways blocks negative selection and causes autoimmunity.

    PubMed

    Burger, Megan L; Leung, Kenneth K; Bennett, Margaux J; Winoto, Astar

    2014-01-01

    T cell self-tolerance is thought to involve peripheral tolerance and negative selection, involving apoptosis of autoreactive thymocytes. However, evidence supporting an essential role for negative selection is limited. Loss of Bim, a Bcl-2 BH3-only protein essential for thymocyte apoptosis, rarely results in autoimmunity on the C57BL/6 background. Mice with T cell-specific over-expression of Bcl-2, that blocks multiple BH3-only proteins, are also largely normal. The nuclear receptor Nur77, also implicated in negative selection, might function redundantly to promote apoptosis by associating with Bcl-2 and exposing its potentially pro-apoptotic BH3 domain. Here, we report that T cell-specific expression of a Bcl2 BH3 mutant transgene results in enhanced rescue of thymocytes from negative selection. Concomitantly, Treg development is increased. However, aged BH3 mutant mice progressively accumulate activated, autoreactive T cells, culminating in development of multi-organ autoimmunity and lethality. These data provide strong evidence that negative selection is crucial for establishing T cell tolerance. PMID:25182415

  8. Multiple pathways for uptake of paraquat, methylglyoxal bis(guanylhydrazone), and polyamines

    SciTech Connect

    Byers, T.L.; Kameji, R.; Rannels, D.E.; Pegg, A.E.

    1987-06-01

    The uptake of polyamines, methylglyoxal bis(guanylhydrazone) (MGBG), and paraquat (N,N-dimethyl-4,4'-bipyridylium) into control Chinese hamster ovary (CHO) cells and a mutant CHO cell line selected for resistance to the toxicity of MGBG was examined. In contrast to control CHO cells, the mutant cells had no detectable uptake of (/sup 14/C)-MGBG or any of the polyamines. There was no difference between the two cell lines in the uptake of ..cap alpha..-aminoisobutyric (/sup 3/H-AIB), which indicates that there was no general change in membrane transport processes. The mutant cells were also found to be resistant to the toxicity of paraquat and to have a reduced capability to take up the herbicide. This finding confirms that the uptake of paraquat is necessary for the toxicity of this compound and that the paraquat is taken up by a transport system that also transports MGBG. Competition experiments showed that an excess of unlabeled paraquat inhibited uptake of MGBG and, to a lesser extent, uptake of putrescine and spermidine, but no inhibitory action on spermine uptake could be detected. Studies with type II cells isolated from rat lung also demonstrated uptake of paraquat and spermidine, but paraquat was only a weak inhibitor of spermidine uptake in this system. These results suggest that there may be multiple systems for the uptake of MGBG and polyamines and that paraquat is taken up by at least one but not by all of these systems.

  9. Understanding the role of adjunctive nonpharmacological therapies in management of the multiple pathways to depression.

    PubMed

    Velehorschi, Corina; Bleau, Pierre; Vermani, Monica; Furtado, Melissa; Klassen, Larry J

    2014-12-01

    Major depressive disorder (MDD) is a common disorder with a lifetime prevalence of 16.2% and the fourth highest cause of disability globally. It is hypothesized to be a syndromatic manifestation of multiple pathological processes leading to similar clinical manifestation. MDD is associated with at least three categories of peripheral hormone-type factors including neurotrophic factors, proinflammatory cytokines, and processes that impair regulation of the hypothalamic-pituitary-adrenocortical axis. Neuroimaging studies have identified functional abnormalities including subcortical systems associated with reward and emotion processing, medial prefrontal and anterior cingulate cortical regions and the lateral prefrontal cortical systems involved in cognitive control and voluntary emotion regulation. Studies investigating the effects of psychotherapy and pharmacotherapy on functional brain measures show normalization of brain function with return to euthymia. Nevertheless, approximately 50% of patients with MDD will not respond sufficiently and 60 to 70% will not achieve full remission with first-line pharmacotherapy, therefore clinicians strive to improve patient responses through the use of adjunct therapies. This review discusses recent research in the various biological processes associated with MDD as well as recent data in support of the use of adjunctive non-pharmacological therapies including psychotherapy, bibliotherapy, Internet therapy, "natural" or herbal approaches, exercise therapy, and somatic therapies. PMID:25539873

  10. Mitochondrial thioredoxin reductase regulates major cytotoxicity pathways of proteasome inhibitors in multiple myeloma cells.

    PubMed

    Fink, E E; Mannava, S; Bagati, A; Bianchi-Smiraglia, A; Nair, J R; Moparthy, K; Lipchick, B C; Drokov, M; Utley, A; Ross, J; Mendeleeva, L P; Savchenko, V G; Lee, K P; Nikiforov, M A

    2016-01-01

    It is generally accepted that intracellular oxidative stress induced by proteasome inhibitors is a byproduct of endoplasmic reticulum (ER) stress. Here we report a mechanism underlying the ability of proteasome inhibitors bortezomib (BTZ) and carfilzomib (CFZ) to directly induce oxidative and ER stresses in multiple myeloma (MM) cells via transcriptional repression of a gene encoding mitochondrial thioredoxin reductase (TXNRD2). TXNRD2 is critical for maintenance of intracellular red-ox status and detoxification of reactive oxygen species. Depletion of TXNRD2 to the levels detected in BTZ- or CFZ-treated cells causes oxidative stress, ER stress and death similar to those induced by proteasome inhibitors. Reciprocally, restoration of near-wildtype TXNRD2 amounts in MM cells treated with proteasome inhibitors reduces oxidative stress, ER stress and cell death by ~46%, ~35% and ~50%, respectively, compared with cells with unrestored TXNRD2 levels. Moreover, cells from three MM cell lines selected for resistance to BTZ demonstrate elevated levels of TXNRD2, indirectly confirming its functional role in BTZ resistance. Accordingly, ectopic expression of TXNRD2 in MM cell xenografts in immunocompromised mice blunts therapeutic effects of BTZ. Our data identify TXNRD2 as a potentially clinically relevant target, inhibition of which is critical for proteasome inhibitor-dependent cytotoxicity, oxidative stress and ER stress. PMID:26205085

  11. NAViGaTing the Micronome – Using Multiple MicroRNA Prediction Databases to Identify Signalling Pathway-Associated MicroRNAs

    PubMed Central

    Shirdel, Elize A.; Xie, Wing; Mak, Tak W.; Jurisica, Igor

    2011-01-01

    Background MicroRNAs are a class of small RNAs known to regulate gene expression at the transcript level, the protein level, or both. Since microRNA binding is sequence-based but possibly structure-specific, work in this area has resulted in multiple databases storing predicted microRNA:target relationships computed using diverse algorithms. We integrate prediction databases, compare predictions to in vitro data, and use cross-database predictions to model the microRNA:transcript interactome – referred to as the micronome – to study microRNA involvement in well-known signalling pathways as well as associations with disease. We make this data freely available with a flexible user interface as our microRNA Data Integration Portal — mirDIP (http://ophid.utoronto.ca/mirDIP). Results mirDIP integrates prediction databases to elucidate accurate microRNA:target relationships. Using NAViGaTOR to produce interaction networks implicating microRNAs in literature-based, KEGG-based and Reactome-based pathways, we find these signalling pathway networks have significantly more microRNA involvement compared to chance (p<0.05), suggesting microRNAs co-target many genes in a given pathway. Further examination of the micronome shows two distinct classes of microRNAs; universe microRNAs, which are involved in many signalling pathways; and intra-pathway microRNAs, which target multiple genes within one signalling pathway. We find universe microRNAs to have more targets (p<0.0001), to be more studied (p<0.0002), and to have higher degree in the KEGG cancer pathway (p<0.0001), compared to intra-pathway microRNAs. Conclusions Our pathway-based analysis of mirDIP data suggests microRNAs are involved in intra-pathway signalling. We identify two distinct classes of microRNAs, suggesting a hierarchical organization of microRNAs co-targeting genes both within and between pathways, and implying differential involvement of universe and intra-pathway microRNAs at the disease level. PMID:21364759

  12. Analysis of Signal Transducer and Activator of Transcription 3 (Stat 3) Pathway in Multiple Myeloma

    PubMed Central

    Quintanilla-Martinez, Leticia; Kremer, Marcus; Specht, Katja; Calzada-Wack, Julia; Nathrath, Michaela; Schaich, Robert; Höfler, Heinz; Fend, Falko

    2003-01-01

    The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three groups in terms of proliferation rate or expression of anti-apoptotic proteins. However, cyclin D1+ cases were always well differentiated and were more likely to show a lymphoplasmocytoid differentiation (chi-square = 9.55). Overall, constitutive activation of Stat 3 was found in almost half (48%) of the investigated MM cases. However, this does not seem to have a major impact on the expression of anti-apoptotic proteins and proliferation. We showed that cyclin D1 overexpression and Stat 3 activation are, mutually exclusive events in MM (P = 0.0066). The universal expression of Mcl-1, independent of activated Stat 3, suggests that its expression is constitutive and that it might play an important role in the pathogenesis of MM. PMID:12707028

  13. Gambogic acid inhibits multiple myeloma mediated osteoclastogenesis through suppression of chemokine receptor CXCR4 signaling pathways.

    PubMed

    Pandey, Manoj K; Kale, Vijay P; Song, Chunhua; Sung, Shen-shu; Sharma, Arun K; Talamo, Giampaolo; Dovat, Sinisa; Amin, Shantu G

    2014-10-01

    Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1? (SDF-1?) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1?/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation, but rather GA suppresses CXCR4 mRNA expression by inhibiting nuclear factor-kappa B (NF-?B) DNA binding. This was further confirmed by quantitative chromatin immunoprecipitation assay, as GA inhibits p65 binding at the CXCR4 promoter. GA suppressed SDF-1?-induced chemotaxis of MM cells and downstream signaling of CXCR4 by inhibiting phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA abrogated the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. In addition, we found that MM cells induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Importantly, suppression of osteoclastogenesis by GA was mediated through IL-6 inhibition. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and has a strong potential to suppress osteoclastogenesis mediated by MM cells. PMID:25034231

  14. Interleukin 1? and tumour necrosis factor ? inhibit acid secretion in cultured rabbit parietal cells by multiple pathways

    PubMed Central

    Beales, I; Calam, J

    1998-01-01

    Background—The cytokines interleukin 1? (IL-1?) and tumour necrosis factor ? (TNF-?) are inhibitors of gastric acid secretion when administered systemically. ?Aims—To investigate the inhibitory effect of IL-1? and TNF-? on cultured, acid secreting parietal cells in order to determine the mechanism of this inhibition. ?Methods—Rabbit parietal cells were prepared by collagenase-EDTA digestion and counter flow elutriation. Acid secretory activity was assessed by aminopyrine accumulation. ?Results—IL-1? and TNF-? inhibited basal and stimulated acid secretion in a dose dependent manner; near maximal effects were seen with both at 10 ng/ml. Inhibition was maximal with 15 minutes pretreatment but seen with up to 18 hours of preincubation. Both cytokines inhibited histamine, carbachol, gastrin, forskolin, and A23187 stimulated acid secretion but had no effect on stimulation by dibutyryl-cAMP. Inhibition of acid secretion was not accompanied by a change in radioligand binding to histamine H2 or gastrin/CCKB receptors. Pertussis toxin abolished the inhibitory effects on histamine and forskolin stimulation. The tyrosine kinase inhibitor herbimycin reduced the inhibitory effects of TNF-? against all stimuli but only reduced the effects of IL-1? against histamine and forskolin stimulation. ?Conclusions—IL-1? and TNF-? seem to inhibit parietal cell acid secretion by multiple pathways; the inhibition occurs at postreceptor level and involves pertussis toxin and tyrosine kinase dependent and independent pathways. Mucosal production of cytokines may be important in the regulation of gastric acid secretion. ?? Keywords: acid secretion; aminopyrine; cytokines; interleukin 1?; tumour necrosis factor ?; parietal cell PMID:9536948

  15. Multiple Redox-Active Chlorophylls in the Secondary Electron-Transfer Pathways of Oxygen-Evolving Photosystem II†

    PubMed Central

    Tracewell, Cara A.; Brudvig, Gary W.

    2009-01-01

    Photosystem II (PS II) is unique among photosynthetic reaction centers in having secondary electron donors that compete with the primary electron donors for reduction of P680+. We have characterized the photooxidation and dark decay of the redox-active accessory chlorophylls (Chl) and ?-carotenes (Car) in oxygen-evolving PS II core complexes by near-IR absorbance and EPR spectroscopies at cryogenic temperatures. In contrast to previous results for Mn-depleted PS II, multiple near-IR absorption bands are resolved in the light-minus-dark difference spectra of oxygen-evolving PS II core complexes including two fast-decaying bands at 793 nm and 814 nm and three slow-decaying bands at 810 nm, 825 nm, and 840 nm. We assign these bands to chlorophyll cation radicals (Chl+). The fast-decaying bands observed after illumination at 20 K could be generated again by re-illuminating the sample. Quantization by EPR gives a yield of 0.85 radicals per PS II, and the yield of oxidized cytochrome b559 by optical difference spectroscopy is 0.15 per PS II. Potential locations of Chl+ and Car+ species, and the pathways of secondary electron transfer based on the rates of their formation and decay, are discussed. This is the first evidence that Chls in the light-harvesting proteins CP43 and CP47 are oxidized by P680+ and may have a role in Chl fluorescence quenching. We also suggest that a possible role for negatively charged lipids (phosphatidyldiacylglycerol and sulphoquinovosyldiacylglycerol identified in the PS II structure) could be to decrease the redox potential of specific Chl and Car cofactors. These results provide new insight into the alternate electron-donation pathways to P680+. PMID:18850718

  16. Twin Promotes the Maintenance and Differentiation of Germline Stem Cell Lineage through Modulation of Multiple Pathways.

    PubMed

    Fu, Ziwen; Geng, Cuiyun; Wang, Hui; Yang, Zhihao; Weng, Changjiang; Li, Hua; Deng, Lamei; Liu, Luping; Liu, Nan; Ni, Jianquan; Xie, Ting

    2015-11-17

    The central question in stem cell regulation is how the balance between self-renewal and differentiation is controlled at the molecular level. This study uses germline stem cells (GSCs) in the Drosophila ovary to demonstrate that the Drosophila CCR4 homolog Twin is required intrinsically to promote both GSC self-renewal and progeny differentiation. Twin/CCR4 is one of the two catalytic subunits in the highly conserved CCR4-NOT mRNA deadenylase complex. Twin works within the CCR4-NOT complex to intrinsically maintain GSC self-renewal, at least partly by sustaining E-cadherin-mediated GSC-niche interaction and preventing transposable element-induced DNA damage. It promotes GSC progeny differentiation by forming protein complexes with differentiation factors Bam and Bgcn independently of other CCR4-NOT components. Interestingly, Bam can competitively inhibit the association of Twin with Pop2 in the CCR4-NOT complex. Therefore, this study demonstrates that Twin has important intrinsic roles in promoting GSC self-renewal and progeny differentiation by functioning in different protein complexes. PMID:26549449

  17. Multiple regulation pathways and pivotal biological functions of STAT3 in cancer

    PubMed Central

    Yuan, Jie; Zhang, Fei; Niu, Ruifang

    2015-01-01

    STAT3 is both a transcription activator and an oncogene that is tightly regulated under normal physiological conditions. However, abundant evidence indicates that STAT3 is persistently activated in several cancers, with a crucial position in tumor onset and progression. In addition to its traditional role in cancer cell proliferation, invasion, and migration, STAT3 also promotes cancer through altering gene expression via epigenetic modification, inducing epithelial–mesenchymal transition (EMT) phenotypes in cancer cells, regulating the tumor microenvironment, and promoting cancer stem cells (CSCs) self-renewal and differentiation. STAT3 is regulated not only by the canonical cytokines and growth factors, but also by the G-protein-coupled receptors, cadherin engagement, Toll-like receptors (TLRs), and microRNA (miRNA). Despite the presence of diverse regulators and pivotal biological functions in cancer, no effective therapeutic inventions are available for inhibiting STAT3 and acquiring potent antitumor effects in the clinic. An improved understanding of the complex roles of STAT3 in cancer is required to achieve optimal therapeutic effects. PMID:26631279

  18. Multiple regulation pathways and pivotal biological functions of STAT3 in cancer.

    PubMed

    Yuan, Jie; Zhang, Fei; Niu, Ruifang

    2015-01-01

    STAT3 is both a transcription activator and an oncogene that is tightly regulated under normal physiological conditions. However, abundant evidence indicates that STAT3 is persistently activated in several cancers, with a crucial position in tumor onset and progression. In addition to its traditional role in cancer cell proliferation, invasion, and migration, STAT3 also promotes cancer through altering gene expression via epigenetic modification, inducing epithelial-mesenchymal transition (EMT) phenotypes in cancer cells, regulating the tumor microenvironment, and promoting cancer stem cells (CSCs) self-renewal and differentiation. STAT3 is regulated not only by the canonical cytokines and growth factors, but also by the G-protein-coupled receptors, cadherin engagement, Toll-like receptors (TLRs), and microRNA (miRNA). Despite the presence of diverse regulators and pivotal biological functions in cancer, no effective therapeutic inventions are available for inhibiting STAT3 and acquiring potent antitumor effects in the clinic. An improved understanding of the complex roles of STAT3 in cancer is required to achieve optimal therapeutic effects. PMID:26631279

  19. MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways.

    PubMed

    Patel, A; Burton, D G A; Halvorsen, K; Balkan, W; Reiner, T; Perez-Stable, C; Cohen, A; Munoz, A; Giribaldi, M G; Singh, S; Robbins, D J; Nguyen, D M; Rai, P

    2015-05-14

    Oncogenic RAS promotes production of reactive oxygen species (ROS), which mediate pro-malignant signaling but can also trigger DNA damage-induced tumor suppression. Thus RAS-driven tumor cells require redox-protective mechanisms to mitigate the damaging aspects of ROS. Here, we show that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase that sanitizes oxidative damage in the nucleotide pool, is important for maintaining several KRAS-driven pro-malignant traits in a nonsmall cell lung carcinoma (NSCLC) model. MTH1 suppression in KRAS-mutant NSCLC cells impairs proliferation and xenograft tumor formation. Furthermore, MTH1 levels modulate KRAS-induced transformation of immortalized lung epithelial cells. MTH1 expression is upregulated by oncogenic KRAS and correlates positively with high KRAS levels in NSCLC human tumors. At a molecular level, in p53-competent KRAS-mutant cells, MTH1 loss provokes DNA damage and induction of oncogene-induced senescence. In p53-nonfunctional KRAS-mutant cells, MTH1 suppression does not produce DNA damage but reduces proliferation and leads to an adaptive decrease in KRAS expression levels. Thus, MTH1 not only enables evasion of oxidative DNA damage and its consequences, but can also function as a molecular rheostat for maintaining oncogene expression at optimal levels. Accordingly, our results indicate MTH1 is a novel and critical component of oncogenic KRAS-associated malignancy and its inhibition is likely to yield significant tumor-suppressive outcomes in KRAS-driven tumors. PMID:25023700

  20. Pathways of Methylmercury Transfer to the Water Column Across Multiple Estuaries

    NASA Astrophysics Data System (ADS)

    Schartup, A. T.; Balcom, P. H.; Mason, R. P.; Chen, C.

    2014-12-01

    Estuarine water column methylmercury (MeHg) is an important driver of bioaccumulation in pelagic organisms so it is important to understand the sources and cycling of MeHg. As MeHg biomagnifies in food webs, increased water column concentrations can be transferred to fish consumed by humans. Few studies have taken a multi-estuary approach to look at MeHg cycling in the water column of these important MeHg producing areas. We examined the distributions and partitioning of sediment and water column MeHg across a geographic range of estuaries. In 2008 we sampled 10 shallow-water estuarine sites from Maine to New Jersey, sampled 11 sites in 4 estuaries in 2009, and sampled at 3 estuarine turbidity maximum (ETM) sites in 1 estuary in 2012. Sediment measurements included both solid phase and pore water MeHg and total mercury (HgT). Water column parameters included dissolved and particulate MeHg and HgT, total suspended solids, nutrients, and dissolved organic carbon. Average suspended particle MeHg was highest at Wells (ME; 6 to 11.5 pmol/g; 4.5 to 7% of HgT) and lowest at Portsmouth (NH) and in Long Island Sound (CT-NY; 0.2 to 5.5 pmol/g; 0.25 to 3.75% of HgT). Average water column dissolved MeHg was highest in the Delaware River ETM (0.5 to 0.7 pM; 16 to 24% of HgT) and lowest at Portsmouth (0.06 to 0.12 pM; 1 to 2% of HgT). Significant positive correlations were found between MeHg and HgT across multiple estuaries in both sediment and the water column in 2008 and 2009. In contrast, water column dissolved and suspended particle MeHg do not correlate well with sediment MeHg or HgT, pore water MeHg or methylation rates in sediment across estuaries, indicating that sediment is often not a good predictor of water MeHg levels. However, ratios of average dissolved:pore water MeHg and suspended particle:sediment MeHg are close to 1 in the Delaware River ETM, suggesting that sediment supplies MeHg to the water column in this turbulent region, but average pore water MeHg was uniformly elevated above water dissolved MeHg in the other estuaries studied. Several estuaries had higher MeHg at low tide suggesting input as water was delivered from the watersheds. We conclude that the relative importance of sources is dependent on the physical (water residence time, water depth) and chemical characteristics (sediment organic carbon content) of the estuary.

  1. The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley.

    PubMed

    Chauhan, Harsh; Boni, Rainer; Bucher, Rahel; Kuhn, Benjamin; Buchmann, Gabriele; Sucher, Justine; Selter, Liselotte L; Hensel, Goetz; Kumlehn, Jochen; Bigler, Laurent; Glauser, Gaëtan; Wicker, Thomas; Krattinger, Simon G; Keller, Beat

    2015-10-01

    The wheat gene Lr34 encodes an ABCG-type transporter which provides durable resistance against multiple pathogens. Lr34 is functional as a transgene in barley, but its mode of action has remained largely unknown both in wheat and barley. Here we studied gene expression in uninfected barley lines transgenic for Lr34. Genes from multiple defense pathways contributing to basal and inducible disease resistance were constitutively active in seedlings and mature leaves. In addition, the hormones jasmonic acid and salicylic acid were induced to high levels, and increased levels of lignin as well as hordatines were observed. These results demonstrate a strong, constitutive re-programming of metabolism by Lr34. The resistant Lr34 allele (Lr34res) encodes a protein that differs by two amino acid polymorphisms from the susceptible Lr34sus allele. The deletion of a single phenylalanine residue in Lr34sus was sufficient to induce the characteristic Lr34-based responses. Combination of Lr34res and Lr34sus in the same plant resulted in a reduction of Lr34res expression by 8- to 20-fold when the low-expressing Lr34res line BG8 was used as a parent. Crosses with the high-expressing Lr34res line BG9 resulted in an increase of Lr34sus expression by 13- to 16-fold in progenies that inherited both alleles. These results indicate an interaction of the two Lr34 alleles on the transcriptional level. Reduction of Lr34res expression in BG8 crosses reduced the negative pleiotropic effects of Lr34res on barley growth and vigor without compromising disease resistance, suggesting that transgenic combination of Lr34res and Lr34sus can result in agronomically useful resistance. PMID:26315512

  2. The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

    PubMed Central

    Bader, Joel S.; Friedmann, Theodore

    2013-01-01

    The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease. PMID:24130677

  3. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    NASA Technical Reports Server (NTRS)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  4. Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

    PubMed

    Chaabane, Wiem; Cie?lar-Pobuda, Artur; El-Gazzah, Mohamed; Jain, Mayur V; Rzeszowska-Wolny, Joanna; Rafat, Mehrdad; Stetefeld, Joerg; Ghavami, Saeid; Los, Marek J

    2014-09-01

    The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin-induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin-induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin-induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway. PMID:25246270

  5. A decision analysis approach to climate adaptation: comparing multiple pathways for multi-decadal decision making

    NASA Astrophysics Data System (ADS)

    Lin, B. B.; Little, L.

    2013-12-01

    Policy planners around the world are required to consider the implications of adapting to climatic change across spatial contexts and decadal timeframes. However, local level information for planning is often poorly defined, even though climate adaptation decision-making is made at this scale. This is especially true when considering sea level rise and coastal impacts of climate change. We present a simple approach using sea level rise simulations paired with adaptation scenarios to assess a range of adaptation options available to local councils dealing with issues of beach recession under present and future sea level rise and storm surge. Erosion and beach recession pose a large socioeconomic risk to coastal communities because of the loss of key coastal infrastructure. We examine the well-known adaptation technique of beach nourishment and assess various timings and amounts of beach nourishment at decadal time spans in relation to beach recession impacts. The objective was to identify an adaptation strategy that would allow for a low frequency of management interventions, the maintenance of beach width, and the ability to minimize variation in beach width over the 2010 to 2100 simulation period. 1000 replications of each adaptation option were produced against the 90 year simulation in order to model the ability each adaptation option to achieve the three key objectives. Three sets of adaptation scenarios were identified. Within each scenario, a number of adaptation options were tested. The three scenarios were: 1) Fixed periodic beach replenishment of specific amounts at 20 and 50 year intervals, 2) Beach replenishment to the initial beach width based on trigger levels of recession (5m, 10m, 20m), and 3) Fixed period beach replenishment of a variable amount at decadal intervals (every 10, 20, 30, 40, 50 years). For each adaptation option, we show the effectiveness of each beach replenishment scenario to maintain beach width and consider the implications of more frequent replenishment with that of implementation cost. We determine that a business as usual scenario, where no adaptation is implemented, would lead to an average beach recession of 12.02 meters and a maximum beach recession of 33.23 meters during the period of 2010-2100. The best adaptation option modeled was a fixed replenishment of 5 meters every 20 years leading to 4 replenishment events with an average beach recession of 2.99 meters and a maximum beach recession of 15.02 meters during the period of 2010-2100. The presented simulations explicitly address the uncertainty of future impacts due to sea level rise and storm surge and show a range of options that could be considered by a local council to meet their policy objectives. The simulation runs provide managers the ability to consider the utility of various adaptation options and the timing and costs of implementation. Such information provides an evidence-based practice to decision-making and allows policy makers to transparently make decisions based on best estimates of modeled climate change.

  6. Signaling of chloroquine-induced stress in the yeast Saccharomyces cerevisiae requires the Hog1 and Slt2 mitogen-activated protein kinase pathways.

    PubMed

    Baranwal, Shivani; Azad, Gajendra Kumar; Singh, Vikash; Tomar, Raghuvir S

    2014-09-01

    Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule. PMID:25022582

  7. Proper Actin Ring Formation and Septum Constriction Requires Coordinated Regulation of SIN and MOR Pathways through the Germinal Centre Kinase MST-1

    PubMed Central

    Heilig, Yvonne; Dettmann, Anne; Mouriño-Pérez, Rosa R.; Schmitt, Kerstin; Valerius, Oliver; Seiler, Stephan

    2014-01-01

    Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in ?mst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes. PMID:24762679

  8. VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection

    PubMed Central

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Wang, Qing; Lin, Ling

    2015-01-01

    In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection. PMID:26670613

  9. VASOACTIVE INTESTINAL PEPTIDE (VIP) ACTS VIA MULTIPLE SIGNAL PATHWAYS TO REGULATE HIPPOCAMPAL NMDA RECEPTORS AND SYNAPTIC TRANSMISSION

    PubMed Central

    Yang, Kai; Trepanier, Catherine H.; Li, Hongbin; Beazely, Michael A.; Lerner, Ethan A.; Jackson, Michael F.; MacDonald, John F.

    2009-01-01

    Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide which belongs to a superfamily of structurally related peptide hormones including pituitary adenylate cyclase-activating polypeptide (PACAP). Although several studies have identified the involvement of PACAP in learning and memory, little work has been done to investigate such a role for VIP. At least three receptors for VIP have been identified including the PACAP receptor (PAC1-R) and the two VIP receptors (VPAC receptors). VIP can activate the PAC1-R only if it is used at relatively high concentrations (e.g. 100 nM); however, at lower concentrations (e.g. 1 nM) it is selective for the VPAC receptors. Our lab has showed that PAC1-R activation signals through PKC/CAK?/Src pathway to regulate NMDA receptors, however there is little known about the potential regulation of NMDA receptors by VPAC receptors. Our studies demonstrated that application of 1nM VIP enhanced NMDA currents by stimulating VPAC receptors as the effect was blocked by VPAC receptor antagonist [Ac-Tyr1, D-Phe2]GRF (1–29). This enhancement of NMDA currents was blocked by both Rp-cAMPS and PKI14–22 (they are highly specific PKA inhibitors), but not by the specific PKC inhibitor, bisindolylmaleimide I. In addition, the VIP-induced enhancement of NMDA currents was accentuated by inhibition of phosphodiesterase 4, which inhibits the degradation of cAMP. This regulation of NMDA receptors also required the scaffolding protein AKAP. In contrast, the potentiation induced by high concentration of VIP (e.g. 100 nM) was mediated by PAC1-R as well as by Src kinase. Overall, these results show that VIP can regulate NMDA receptors through different receptors and signaling pathways. PMID:19173226

  10. Phase delays to light and gastrin-releasing peptide require the protein kinase A pathway.

    PubMed

    Sterniczuk, Roxanne; Yamakawa, Glenn R; Pomeroy, Tara; Antle, Michael C

    2014-01-24

    Daily photic resetting of the circadian system relies on the transmission of light information from the retina to retinorecipient cells within the ventrolateral suprachiasmatic nucleus (SCN) core, and subsequent activation of rhythmic clock cells in the dorsolateral region. Some neurochemicals such as gastrin-releasing peptide (GRP) mimic the phase shifting effects of light and induce Ca(2+)-dependent gene expression in the SCN. Activation of the cAMP-response element binding protein (CREB) is necessary for Ca(2+)-dependent transcription to occur and accompanies behavioral phase shifting; however, several biochemical cascades are involved in this phenomenon. One pathway that has been implicated in photic responses involves protein kinase A (PKA). It is not known if this pathway participates in mediating phase shifts to GRP. Here we show that preventing PKA activation attenuates both light- and GRP-induced phase shifts in locomotor behavior, but only during the early-subjective night. This finding demonstrates that activation of PKA is an important component in the photic signaling pathway and may mediate GRP output signaling from the SCN core to the shell; however, this effect appears to be temporally dependent. PMID:24287375

  11. TCR ITAM multiplicity is required for the generation of follicular helper T-cells

    PubMed Central

    Hwang, SuJin; Palin, Amy C.; Li, LiQi; Song, Ki-Duk; Lee, Jan; Herz, Jasmin; Tubo, Noah; Chu, Hamlet; Pepper, Marion; Lesourne, Renaud; Zvezdova, Ekaterina; Pinkhasov, Julia; Jenkins, Marc K.; McGavern, Dorian; Love, Paul E.

    2015-01-01

    The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3? subunits. We generated ‘knock-in' mice that express non-signalling CD3? chains in lieu of wild-type CD3?. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR–ligand interactions, but is not essential for ‘general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire. PMID:25959494

  12. TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

    PubMed

    Hwang, SuJin; Palin, Amy C; Li, LiQi; Song, Ki-Duk; Lee, Jan; Herz, Jasmin; Tubo, Noah; Chu, Hamlet; Pepper, Marion; Lesourne, Renaud; Zvezdova, Ekaterina; Pinkhasov, Julia; Jenkins, Marc K; McGavern, Dorian; Love, Paul E

    2015-01-01

    The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3? subunits. We generated 'knock-in' mice that express non-signalling CD3? chains in lieu of wild-type CD3?. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR-ligand interactions, but is not essential for 'general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire. PMID:25959494

  13. Spontaneous sternal fracture due to multiple myeloma requiring extensive surgical repair.

    PubMed

    Reuling, Ellen M Bp; Jakma, Tijs S C; Schnater, Johannes Marco; Westerweel, Peter E

    2015-01-01

    Spontaneous sternal fracture is a well-known complication of multiple myeloma due to osteolytic bone lesions. The possibility of a multiple myeloma should be thoroughly investigated in patients presenting with a spontaneous sternal fracture. This work up should go beyond protein electrophoresis alone as a monoclonal paraprotein is not always present. In some cases, the myeloma plasma cell clone produces only the free light chain (? or ?) or may even be non-secretory. The underlying plasma cell dyscrasia is treated with chemotherapy and, if needed, local radiotherapy. However, for patients with a fracture causing persistent pain and physical discomfort, internal fixation may be additionally required. We present a case of a patient who presented with a displaced pathological sternal fracture. She was treated with chemotherapy, radiotherapy and an open reduction and internal fixation with a Locking Compression Plate (LCP). This technique offers a feasible option for rigid fixation of pathological fractures. PMID:26621901

  14. Long and compact x-ray pathway for experiments requiring high coherent x-ray beams.

    PubMed

    Hönnicke, Marcelo G; Kakuno, Edson M; Kellerman, Guinther; Mazzaro, Irineu; Abler, Daniel; Cusatis, Cesar

    2008-06-23

    A long x-ray pathway based on an x-ray back-diffraction cavity for coherent x-ray beam experiments is presented. In the present work, such a setup was tested and used for propagation-based x-ray phase contrast imaging (PBI). This setup showed to be useful for PBI purposes, with the advantage of being compact (3 m long) when compared with long x-ray synchrotron beamlines with dimensions from tens to hundreds of meters. PMID:18575492

  15. Optimization of photosynthesis by multiple metabolic pathways involving interorganelle interactions: resource sharing and ROS maintenance as the bases.

    PubMed

    Sunil, Bobba; Talla, Sai K; Aswani, Vetcha; Raghavendra, Agepati S

    2013-11-01

    The bioenergetic processes of photosynthesis and respiration are mutually beneficial. Their interaction extends to photorespiration, which is linked to optimize photosynthesis. The interplay of these three pathways is facilitated by two major phenomena: sharing of energy/metabolite resources and maintenance of optimal levels of reactive oxygen species (ROS). The resource sharing among different compartments of plant cells is based on the production/utilization of reducing equivalents (NADPH, NADH) and ATP as well as on the metabolite exchange. The responsibility of generating the cellular requirements of ATP and NAD(P)H is mostly by the chloroplasts and mitochondria. In turn, besides the chloroplasts, the mitochondria, cytosol and peroxisomes are common sinks for reduced equivalents. Transporters located in membranes ensure the coordinated movement of metabolites across the cellular compartments. The present review emphasizes the beneficial interactions among photosynthesis, dark respiration and photorespiration, in relation to metabolism of C, N and S. Since the bioenergetic reactions tend to generate ROS, the cells modulate chloroplast and mitochondrial reactions, so as to ensure that the ROS levels do not rise to toxic levels. The patterns of minimization of ROS production and scavenging of excess ROS in intracellular compartments are highlighted. Some of the emerging developments are pointed out, such as model plants, orientation/movement of organelles and metabolomics. PMID:23881384

  16. Tumefactive multiple sclerosis requiring emergency craniotomy: case report and literature review.

    PubMed

    Munarriz, Pablo M; Castaño-Leon, Ana M; Martinez-Perez, Rafael; Hernandez-Lain, Aurelio; Ramos, Ana; Lagares, Alfonso

    2013-01-01

    Multiple sclerosis (MS) is a demyelinating disease of the central nervous system, characterized by focal neurological dysfunction with a relapsing and remitting course. Tumor-like presentation of MS (or "tumefactive"/"pseudotumoral" presentation) has been described before with a certain frequency; it consists of a large single plaque (>2cm) with presence of edema and mass effect and it is hard to distinguish from a brain tumor. However, we present a very rare case of a 53-year-old woman with a right temporal mass that turned out to be a MS plaque, who deteriorated within hours (brain herniation with loss of consciousness and unilateral mydriasis) and required an emergency craniotomy. We also present a review of the literature. It appears that only 4 cases of emergency craniotomy/craniectomy required in a patient with a tumor-like MS plaque have been reported before. PMID:23582489

  17. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    SciTech Connect

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  18. A Deployment Strategy for Multiple Types of Requirements in Wireless Sensor Networks.

    PubMed

    Liu, Xuxun

    2015-10-01

    Node deployment is one of the most crucial issues in wireless sensor networks, and it is of realistic significance to complete the deployment task with multiple types of application requirements. In this paper, we propose a deployment strategy for multiple types of requirements to solve the problem of deterministic and grid-based deployment. This deployment strategy consists of three deployment algorithms, which are for different deployment objectives. First, instead of general random search, we put forward a deterministic search mechanism and the related cost-based deployment algorithm, in which nodes are assigned to different groups which are connected by near-shortest paths, and realize significant reduction of path length and deployment cost. Second, rather than ordinary nondirection deployment, we present a notion of counterflow and the related delay-based deployment algorithm, in which the profit of deployment cost and loss of transmission delay are evaluated, and achieve much diminishing of transmission path length and transmission delay. Third, instead of conventional uneven deployment based on the distances to the sink, we propose a concept of node load level and the related lifetime-based deployment algorithm, in which node distribution is determined by the actual load levels and extra nodes are deployed only where really necessary. This contributes to great improvement of network lifetime. Last, extensive simulations are used to test and verify the effectiveness and superiority of our findings. PMID:26390178

  19. Sumoylation Pathway Is Required to Maintain the Basal Breast Cancer Subtype

    PubMed Central

    Bogachek, Maria V.; Chen, Yizhen; Kulak, Mikhail V.; Woodfield, George W.; Cyr, Anthony R.; Park, Jung Min; Spanheimer, Philip M.; Li, Yingyue; Li, Tiandao; Weigel, Ronald J.

    2014-01-01

    SUMMARY The TFAP2C/AP-2? transcription factor regulates luminal breast cancer genes and loss of TFAP2C induces epithelial-mesenchymal transition. By contrast, the highly homologous family member, TFAP2A, lacks transcriptional activity at luminal gene promoters. A detailed structure-function analysis identified that sumoylation of TFAP2A blocks its ability to induce the expression of luminal genes. Disruption of the sumoylation pathway by knockdown of sumoylation enzymes, mutation of the SUMO-target lysine of TFAP2A, or treatment with sumoylation inhibitors induced a basal to luminal transition, which was dependent upon TFAP2A. Sumoylation inhibitors cleared the CD44+/hi/CD24?/low cell population characterizing basal cancers and inhibited tumor outgrowth of basal cancer xenografts. These findings establish a critical role for sumoylation in regulating the transcriptional mechanisms that maintain the basal cancer phenotype. PMID:24835590

  20. Process and utility water requirements for cellulosic ethanol production processes via fermentation pathway

    EPA Science Inventory

    The increasing need of additional water resources for energy production is a growing concern for future economic development. In technology development for ethanol production from cellulosic feedstocks, a detailed assessment of the quantity and quality of water required, and the ...

  1. Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D.

    PubMed

    Dubey, Raghvendra K; Fingerle, Jürgen; Gillespie, Delbert G; Mi, Zaichuan; Rosselli, Marinella; Imthurn, Bruno; Jackson, Edwin K

    2015-12-01

    The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 ?mol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37?424±18?371 pixels; treated, 10?352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin that controls cell-cycle progression. PMID:26416848

  2. P38 AND EGF RECEPTOR KINASE-MEDIATED ACTIVATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE/AKT PATHWAY IS REQUIRED FOR ZN2+INDUCED CYCLOOXYGENASE-2 EXPRESSION

    EPA Science Inventory

    Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus- and cell type-specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction...

  3. Nuclear Import of the Yeast Hexokinase 2 Protein Requires ?/?-Importin-dependent Pathway*

    PubMed Central

    Peláez, Rafael; Fernández-García, Paula; Herrero, Pilar; Moreno, Fernando

    2012-01-01

    Hexokinase 2 (Hxk2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization and role, it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. However, the mechanism by which Hxk2 enters in the nucleus was unknown until now. Here, we report that the Hxk2 protein is an import substrate of the carriers ?-importin (Kap60 in yeast) and ?-importin (Kap95 in yeast). We also show that the Hxk2 nuclear import and the binding of Hxk2 with Kap60 are glucose-dependent and involve one lysine-rich nuclear localization sequence (NLS), located between lysine 6 and lysine 12. Moreover, Kap95 facilitates the recognition of the Hxk2 NLS1 motif by Kap60 and both importins are essential for Hxk2 nuclear import. It is also demonstrated that Hxk2 nuclear import and its binding to Kap95 and Kap60 depend on the Gsp1-GTP/GDP protein levels. Thus, our study uncovers Hxk2 as a new cargo for the ?/?-importin pathway of S. cerevisiae. PMID:22157003

  4. CAP defines a second signalling pathway required for insulin-stimulated glucose transport

    NASA Astrophysics Data System (ADS)

    Baumann, Christian A.; Ribon, Vered; Kanzaki, Makoto; Thurmond, Debbie C.; Mora, Silvia; Shigematsu, Satoshi; Bickel, Perry E.; Pessin, Jeffrey E.; Saltiel, Alan R.

    2000-09-01

    Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl protooncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

  5. Fluxes of Ca2+ and K+ Are Required for the Listeriolysin O-Dependent Internalization Pathway of Listeria monocytogenes

    PubMed Central

    Vadia, Stephen

    2014-01-01

    Listeria monocytogenes is responsible for the life-threatening food-borne disease listeriosis. This disease mainly affects elderly and immunocompromised individuals, causing bacteremia and meningoencephalitis. In pregnant women, L. monocytogenes infection leads to abortion and severe infection of the fetus or newborn. The L. monocytogenes intracellular life cycle is critical for pathogenesis. Previous studies have established that the major virulence factor of L. monocytogenes, the pore-forming toxin listeriolysin O (LLO), is sufficient to induce L. monocytogenes internalization into human epithelial cell lines. This internalization pathway strictly requires the formation of LLO pores in the plasma membrane and can be stimulated by the heterologous pore-forming toxin pneumolysin, suggesting that LLO acts nonspecifically by forming transmembrane pores. The present work tested the hypothesis that Ca2+ and K+ fluxes subsequent to perforation by LLO control L. monocytogenes internalization. We report that L. monocytogenes perforates the host cell plasma membrane in an LLO-dependent fashion at the early stage of invasion. In response to perforation, host cells undergo Ca2+-dependent but K+-independent resealing of their plasma membrane. In contrast to the plasma membrane resealing process, LLO-induced L. monocytogenes internalization requires both Ca2+ and K+ fluxes. Further linking ion fluxes to bacterial internalization, treating cells with a combination of Ca2+ and K+ ionophores but not with individual ionophores is sufficient to induce efficient internalization of large cargoes, such as 1-?m polystyrene beads and bacteria. We propose that LLO-induced L. monocytogenes internalization requires a Ca2+- and K+-dependent internalization pathway that is mechanistically distinct from the process of plasma membrane resealing. PMID:24366251

  6. Genetic dissection of TrkB activated signalling pathways required for specific aspects of the taste system

    PubMed Central

    2014-01-01

    Background Neurotrophin-4 (NT-4) and brain derived neurotrophic factor (BDNF) bind to the same receptor, Ntrk2/TrkB, but play distinct roles in the development of the rodent gustatory system. However, the mechanisms underlying these processes are lacking. Results Here, we demonstrate, in vivo, that single or combined point mutations in major adaptor protein docking sites on TrkB receptor affect specific aspects of the mouse gustatory development, known to be dependent on BDNF or NT-4. In particular, mice with a mutation in the TrkB-SHC docking site had reduced gustatory neuron survival at both early and later stages of development, when survival is dependent on NT-4 and BDNF, respectively. In addition, lingual innervation and taste bud morphology, both BDNF-dependent functions, were altered in these mutants. In contrast, mutation of the TrkB-PLC? docking site alone did not affect gustatory neuron survival. Moreover, innervation to the tongue was delayed in these mutants and taste receptor expression was altered. Conclusions We have genetically dissected pathways activated downstream of the TrkB receptor that are required for specific aspects of the taste system controlled by the two neurotrophins NT-4 and BDNF. In addition, our results indicate that TrkB also regulate the expression of specific taste receptors by distinct signalling pathways. These results advance our knowledge of the biology of the taste system, one of the fundamental sensory systems crucial for an organism to relate to the environment. PMID:25256039

  7. Oncogenic NRAS, required for pathogenesis of embryonic rhabdomyosarcoma, relies upon the HMGA2-IGF2BP2 pathway.

    PubMed

    Li, Zhizhong; Zhang, Yunyu; Ramanujan, Krishnan; Ma, Yan; Kirsch, David G; Glass, David J

    2013-05-15

    Embryonic rhabdomyosarcoma (ERMS) is the most common soft-tissue tumor in children. Here, we report the identification of the minor groove DNA-binding factor high mobility group AT-hook 2 (HMGA2) as a driver of ERMS development. HMGA2 was highly expressed in normal myoblasts and ERMS cells, where its expression was essential to maintain cell proliferation, survival in vitro, and tumor outgrowth in vivo. Mechanistic investigations revealed that upregulation of the insulin-like growth factor (IGF) mRNA-binding protein IGF2BP2 was critical for HMGA2 action. In particular, IGF2BP2 was essential for mRNA and protein stability of NRAS, a frequently mutated gene in ERMS. shRNA-mediated attenuation of NRAS or pharmacologic inhibition of the MAP-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) effector pathway showed that NRAS and NRAS-mediated signaling was required for tumor maintenance. Taken together, these findings implicate the HMGA2-IGFBP2-NRAS signaling pathway as a critical oncogenic driver in ERMS. PMID:23536553

  8. Proteomic-based identification of multiple pathways underlying n-butylidenephthalide-induced apoptosis in LNCaP human prostate cancer cells.

    PubMed

    Pang, Cheng-Yoong; Chiu, Sheng-Chun; Harn, Horng-Jyh; Zhai, Wei-Jun; Lin, Shinn-Zong; Yang, Hsueh-Hui

    2013-09-01

    Although numerous studies have shown the cancer-preventive properties of butylidenephthalide (BP), there is little report of BP affecting human prostate cancer cells. In the present study, proteomic-based approaches were used to elucidate the anticancer mechanism of BP in LNCaP human prostate cancer cells. BP treatment decreased the viability of LNCaP human prostate cancer cells in a concentration- and time-dependent manner, which was correlated with G0/G1 phase cell cycle arrest. Increased cell cycle arrest was associated with a decrease in the level of CCND1, CDK2, and PCNA proteins and an increase in the level of CDKN2A, CDKN1A, and SFN proteins. Proteomic studies revealed that among 48 differentially expressed proteins, 25 proteins were down-regulated and 23 proteins were up-regulated and these proteins fall into one large protein protein interaction network. Among these proteins, FAS, AIFM1, BIK, CYCS, SFN, PPP2R1A, CALR, HSPA5, DDIT3, and ERN1 are apoptosis and endoplasmic reticulum (ER) stress associated proteins. Proteomic data suggested that multiple signaling pathways including FAS-dependent pathway, mitochondrial pathway, and ER stress pathway are involved in the apoptosis induced by BP. PMID:23770345

  9. Renal Protective Role of Xiexin Decoction with Multiple Active Ingredients Involves Inhibition of Inflammation through Downregulation of the Nuclear Factor-?B Pathway in Diabetic Rats

    PubMed Central

    Wu, Jia-sheng; Shi, Rong; Zhong, Jie; Lu, Xiong; Ma, Bing-liang; Wang, Tian-ming; Zan, Bin; Ma, Yue-ming; Cheng, Neng-neng; Qiu, Fu-rong

    2013-01-01

    In Chinese medicine, Xiexin decoction (XXD) has been used for the clinical treatment of diabetes for at least 1700 years. The present study was conducted to investigate the effective ingredients of XXD and their molecular mechanisms of antidiabetic nephropathy in rats. Rats with diabetes induced by high-fat diet and streptozotocin were treated with XXD extract for 12 weeks. XXD significantly improved the glucolipid metabolism disorder, attenuated albuminuria and renal pathological changes, reduced renal advanced glycation end-products, inhibited receptor for advanced glycation end-product and inflammation factors expression, suppressed renal nuclear factor-?B pathway activity, and downregulated renal transforming growth factor-?1. The concentrations of multiple components in plasma from XXD were determined by liquid chromatography and tandem mass spectrometry. Pharmacokinetic/pharmacodynamic analysis using partial least square regression revealed that 8 ingredients of XXD were responsible for renal protective effects via actions on multiple molecular targets. Our study suggests that the renal protective role of XXD with multiple effective ingredients involves inhibition of inflammation through downregulation of the nuclear factor-?B pathway, reducing renal advanced glycation end-products and receptor for advanced glycation end-product in diabetic rats. PMID:23935673

  10. Higher biodiversity is required to sustain multiple ecosystem processes across temperature regimes

    PubMed Central

    Perkins, Daniel M; Bailey, R A; Dossena, Matteo; Gamfeldt, Lars; Reiss, Julia; Trimmer, Mark; Woodward, Guy

    2015-01-01

    Biodiversity loss is occurring rapidly worldwide, yet it is uncertain whether few or many species are required to sustain ecosystem functioning in the face of environmental change. The importance of biodiversity might be enhanced when multiple ecosystem processes (termed multifunctionality) and environmental contexts are considered, yet no studies have quantified this explicitly to date. We measured five key processes and their combined multifunctionality at three temperatures (5, 10 and 15 °C) in freshwater aquaria containing different animal assemblages (1–4 benthic macroinvertebrate species). For single processes, biodiversity effects were weak and were best predicted by additive-based models, i.e. polyculture performances represented the sum of their monoculture parts. There were, however, significant effects of biodiversity on multifunctionality at the low and the high (but not the intermediate) temperature. Variation in the contribution of species to processes across temperatures meant that greater biodiversity was required to sustain multifunctionality across different temperatures than was the case for single processes. This suggests that previous studies might have underestimated the importance of biodiversity in sustaining ecosystem functioning in a changing environment. PMID:25131335

  11. Discovery of chrysoeriol, a PI3K-AKT-mTOR pathway inhibitor with potent antitumor activity against human multiple myeloma cells in vitro.

    PubMed

    Yang, Yang; Zhou, Xiaoxi; Xiao, Min; Hong, Zhenya; Gong, Quan; Jiang, Lijun; Zhou, Jianfeng

    2010-12-01

    This study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3, and its related molecular mechanisms. Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay. CCK-8 assay was employed to examine the growth inhibition rate and IC(50) (48 h) in peripheral blood mononuclear cells (PBMNCs), RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations. Cells were labeled with 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software. The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC(50) concentration of chrysoeriol was adopted. The alterations in cell-cycle related proteins (Cyclin B1, Cyclin D1, p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis. The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol, resulting in cell cycle arrest in G(2)/M phase. Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein, meanwhile enhanced Cyclin B1 and p21 protein expression. Similar effects were not observed in PBMNCs from normal donors. It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor. It restrained the proliferation of human multiple myeloma cells, but didn't affect proliferation of PBMNCs from normal donors. It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway. PMID:21181363

  12. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages

    E-print Network

    Sarkies, Peter; Selkirk, Murray E.; Jones, John T; Blok, Vivian; Boothby, Thomas; Goldstein, Bob; Hanelt, Ben; Ardila-Garcia, Alex; M. Fast, Naomi; M. Schiffer, Phillip; Kraus, Christopher; Taylor, Mark J.; Koutsovoulos, Georgios; Blaxter, Mark L.; Miska, Eric A.

    2015-02-10

    .). Moreover, we identified a Piwi homologue from draft transcriptome data, suggesting that these small RNAs could be piRNAs (S1 Text). H. dujardini piRNAs were clustered on the genome, and showed a signature suggestive of ping-pong ampli- fication... RP activity by using a different amplification strategy (the ping- pong mechanism). In surveying the diversity of small RNAs within Nematoda, we found evi- dence of both the ancestral Dicer/RdRP pathway and the piRNA pathway. In addition a unique non...

  13. Molecular mechanism of anticancer effect of Sclerotium rolfsii lectin in HT29 cells involves differential expression of genes associated with multiple signaling pathways: A microarray analysis.

    PubMed

    Barkeer, Srikanth; Guha, Nilanjan; Hothpet, Vishwanathreddy; Saligrama Adavigowda, Deepak; Hegde, Prajna; Padmanaban, Arunkumar; Yu, Lu-Gang; Swamy, Bale M; Inamdar, Shashikala R

    2015-12-01

    Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Gal?1-3GalNAc-?-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin. PMID:26347523

  14. From a Subtractive to Multiplicative Approach: A Concept-Driven Interactive Pathway on the Selective Absorption of Light

    ERIC Educational Resources Information Center

    Viennot, Laurence; de Hosson, Cécile

    2015-01-01

    This research documents the aims and the impact of a teaching experiment on how the absorption of light depends on the thickness of the absorbing medium. This teaching experiment is more specifically characterized as bringing to bear a "concept-driven interactive pathway". It is designed to make students analyse the absorption of light…

  15. A Polyadenylation-Dependent 3' End Maturation Pathway Is Required for the Synthesis of the Human Telomerase RNA.

    PubMed

    Nguyen, Duy; Grenier St-Sauveur, Valérie; Bergeron, Danny; Dupuis-Sandoval, Fabien; Scott, Michelle S; Bachand, François

    2015-12-15

    Telomere maintenance by the telomerase reverse transcriptase requires a noncoding RNA subunit that acts as a template for the synthesis of telomeric repeats. In humans, the telomerase RNA (hTR) is a non-polyadenylated transcript produced from an independent transcriptional unit. As yet, the mechanism and factors responsible for hTR 3' end processing have remained largely unknown. Here, we show that hTR is matured via a polyadenylation-dependent pathway that relies on the nuclear poly(A)-binding protein PABPN1 and the poly(A)-specific RNase PARN. Depletion of PABPN1 and PARN results in telomerase RNA deficiency and the accumulation of polyadenylated precursors. Accordingly, a deficiency in PABPN1 leads to impaired telomerase activity and telomere shortening. In contrast, we find that hTRAMP-dependent polyadenylation and exosome-mediated degradation function antagonistically to hTR maturation, thereby limiting telomerase RNA accumulation. Our findings unveil a critical requirement for RNA polyadenylation in telomerase RNA biogenesis, providing alternative approaches for telomerase inhibition in cancer. PMID:26628368

  16. The Ess1 prolyl isomerase is required for transcription termination of small noncoding RNAs via the Nrd1 pathway.

    PubMed

    Singh, Navjot; Ma, Zhuo; Gemmill, Trent; Wu, Xiaoyun; Defiglio, Holland; Rossettini, Anne; Rabeler, Christina; Beane, Olivia; Morse, Randall H; Palumbo, Michael J; Hanes, Steven D

    2009-10-23

    Genome-wide studies have identified abundant small, noncoding RNAs, including small nuclear RNAs, small nucleolar RNAs (snoRNAs), cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs), that are transcribed by RNA polymerase II (pol II) and terminated by an Nrd1-dependent pathway. Here, we show that the prolyl isomerase Ess1 is required for Nrd1-dependent termination of noncoding RNAs. Ess1 binds the carboxy-terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of approximately 10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, stable unannotated transcripts, and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase. We also provide evidence for a competition between Nrd1 and Pcf11 for CTD binding that is regulated by Ess1. These data indicate that a prolyl isomerase is required for specifying the "CTD code." PMID:19854134

  17. The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

    SciTech Connect

    Hua, Fang; Wang, Jun; Sayeed, Iqbal; Ishrat, Tauheed; Atif, Fahim; Stein, Donald G.

    2009-12-18

    TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-{kappa}B). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-{kappa}B and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-{kappa}B activity and phosphorylation of the inhibitor of kappa B (I{kappa}B{alpha}) increased in ischemic brains, but IRF3, inhibitor of {kappa}B kinase complex-{epsilon} (IKK{epsilon}), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-{kappa}B activity or p-I{kappa}B{alpha} induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-{kappa}B signaling and brain injury after acute cerebral I/R.

  18. New Alleles of the Yeast MPS1 Gene Reveal Multiple Requirements in Spindle Pole Body Duplication

    PubMed Central

    Schutz, Amy R.; Winey, Mark

    1998-01-01

    In Saccharomyces cerevisiae, the Mps1p protein kinase is critical for both spindle pole body (SPB) duplication and the mitotic spindle assembly checkpoint. The mps1–1 mutation causes failure early in SPB duplication, and because the spindle assembly checkpoint is also compromised, mps1–1 cells proceed with a monopolar mitosis and rapidly lose viability. Here we report the genetic and molecular characterization of mps1–1 and five new temperature-sensitive alleles of MPS1. Each of the six alleles contains a single point mutation in the region of the gene encoding the protein kinase domain. The mutations affect several residues conserved among protein kinases, most notably the invariant glutamate in subdomain III. In vivo and in vitro kinase activity of the six epitope-tagged mutant proteins varies widely. Only two display appreciable in vitro activity, and interestingly, this activity is not thermolabile under the assay conditions used. While five of the six alleles cause SPB duplication to fail early, yielding cells with a single SPB, mps1–737 cells proceed into SPB duplication and assemble a second SPB that is structurally defective. This phenotype, together with the observation of intragenic complementation between this unique allele and two others, suggests that Mps1p is required for multiple events in SPB duplication. PMID:9529376

  19. ASIC-dependent LTP at multiple glutamatergic synapses in amygdala network is required for fear memory

    PubMed Central

    Chiang, Po-Han; Chien, Ta-Chun; Chen, Chih-Cheng; Yanagawa, Yuchio; Lien, Cheng-Chang

    2015-01-01

    Genetic variants in the human ortholog of acid-sensing ion channel-1a subunit (ASIC1a) gene are associated with panic disorder and amygdala dysfunction. Both fear learning and activity-induced long-term potentiation (LTP) of cortico-basolateral amygdala (BLA) synapses are impaired in ASIC1a-null mice, suggesting a critical role of ASICs in fear memory formation. In this study, we found that ASICs were differentially expressed within the amygdala neuronal population, and the extent of LTP at various glutamatergic synapses correlated with the level of ASIC expression in postsynaptic neurons. Importantly, selective deletion of ASIC1a in GABAergic cells, including amygdala output neurons, eliminated LTP in these cells and reduced fear learning to the same extent as that found when ASIC1a was selectively abolished in BLA glutamatergic neurons. Thus, fear learning requires ASIC-dependent LTP at multiple amygdala synapses, including both cortico-BLA input synapses and intra-amygdala synapses on output neurons. PMID:25988357

  20. Decursin and Doxorubicin Are in Synergy for the Induction of Apoptosis via STAT3 and/or mTOR Pathways in Human Multiple Myeloma Cells

    PubMed Central

    Jang, Jinsil; Jeong, Soo-Jin; Kwon, Hee-Young; Jung, Ji Hoon; Sohn, Eun Jung; Lee, Hyo-Jung; Kim, Ji-Hyun; Kim, Sun-Hee; Kim, Jin Hyoung; Kim, Sung-Hoon

    2013-01-01

    Background. Combination cancer therapy is one of the attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin from Angelica gigas and doxorubicin on the induction of apoptosis in three human multiple myeloma cells. Methodology/Principal Findings. Combined treatment of decursin and doxorubicin significantly exerted significant cytotoxicity compared to doxorubicin or decursin in U266, RPMI8226, and MM.1S cells. Furthermore, the combination treatment enhanced the activation of caspase-9 and -3, the cleavage of PARP, and the sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment downregulated the phosphorylation of mTOR and its downstream S6K1 and activated the phosphorylation of ERK in three multiple myeloma cells. Furthermore, the combined treatment reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3, and Src, activated SHP-2, and attenuated the expression of cyclind-D1 and survivin in U266 cells. Conversely, tyrosine phosphatase inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activation induced by combined treatment of doxorubicin and decursin in U266 cells. Conclusions/Significance. Overall, the combination treatment of decursin and doxorubicin can enhance apoptotic activity via mTOR and/or STAT3 signaling pathway in multiple myeloma cells. PMID:23818927

  1. Decursin and Doxorubicin Are in Synergy for the Induction of Apoptosis via STAT3 and/or mTOR Pathways in Human Multiple Myeloma Cells.

    PubMed

    Jang, Jinsil; Jeong, Soo-Jin; Kwon, Hee-Young; Jung, Ji Hoon; Sohn, Eun Jung; Lee, Hyo-Jung; Kim, Ji-Hyun; Kim, Sun-Hee; Kim, Jin Hyoung; Kim, Sung-Hoon

    2013-01-01

    Background. Combination cancer therapy is one of the attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin from Angelica gigas and doxorubicin on the induction of apoptosis in three human multiple myeloma cells. Methodology/Principal Findings. Combined treatment of decursin and doxorubicin significantly exerted significant cytotoxicity compared to doxorubicin or decursin in U266, RPMI8226, and MM.1S cells. Furthermore, the combination treatment enhanced the activation of caspase-9 and -3, the cleavage of PARP, and the sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment downregulated the phosphorylation of mTOR and its downstream S6K1 and activated the phosphorylation of ERK in three multiple myeloma cells. Furthermore, the combined treatment reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3, and Src, activated SHP-2, and attenuated the expression of cyclind-D1 and survivin in U266 cells. Conversely, tyrosine phosphatase inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activation induced by combined treatment of doxorubicin and decursin in U266 cells. Conclusions/Significance. Overall, the combination treatment of decursin and doxorubicin can enhance apoptotic activity via mTOR and/or STAT3 signaling pathway in multiple myeloma cells. PMID:23818927

  2. Low-Dose DMC Significantly Enhances the Effect of TMZ on Glioma Cells by Targeting Multiple Signaling Pathways Both In Vivo and In Vitro.

    PubMed

    Shi, Lei; Sun, Guan

    2015-12-01

    Temozolomide (TMZ) is widely used for treating glioblastoma (GBM), which can effectively inhibit the GBM growth for some months; however, it still cannot prevent the invariable recurrence of GBM. Improving the chemotherapeutic sensitization becomes an urgent agenda. In this study, we found low-dose demethoxycurcumin (DMC) could enhance the sensitivity of TMZ on glioma cells, and high-dose DMC has more significant effects on GBM cells compared with TMZ treatment alone both in vitro and in vivo. And co-administration of DMC and TMZ resulted in a significant increase in GBM apoptosis and a marked inhibition of cell growth pathogenesis of GBM. Mechanistically, DMC and TMZ synergistically increase intracellular level of reactive oxygen species (ROS) production, activate caspase-3-dependent apoptotic pathway, and inactivate of JAK/STAT3 signaling pathway in GBMs, which account for the cell apoptosis and proliferation inhibition. Together, these data implicate that low-dose DMC combined with TMZ represents an effective therapy regimen against GBMs by targeting multiple signaling pathways. PMID:26458914

  3. p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways

    PubMed Central

    Shatz, Maria; Shats, Igor; Menendez, Daniel; Resnick, Michael A.

    2015-01-01

    The p53 tumor suppressor regulates transcription of genes associated with diverse cellular functions including apoptosis, growth arrest, DNA repair and differentiation. Recently, we established that p53 can modulate expression of Toll-like receptor (TLR) innate immunity genes but the degree of cross-talk between p53 and TLR pathways remained unclear. Here, using gene expression profiling we characterize the global effect of p53 on the TLR5-mediated transcription in MCF7 cells. We found that combined activation of p53 and TLR5 pathways synergistically increases expression of over 200 genes, mostly associated with immunity and inflammation. The synergy was observed in several human cancer cells and primary lymphocytes. The p53-dependent amplification of transcriptional response to TLR5 activation required expression of NF?B subunit p65 and was mediated by several molecular mechanisms including increased phosphorylation of p38 MAP kinase, PI3K and STAT3 signaling. Additionally, p53 induction increased cytokine expression in response to TNF?, another activator of NF?B and MAP kinase pathways, suggesting a broad interaction between p53 and these signaling pathways. The expression of many synergistically induced genes is elevated in breast cancer patients responsive to chemotherapy. We suggest that p53's capacity to enhance immune response could be exploited to increase antitumor immunity and to improve cancer treatment. PMID:26220208

  4. Stress peptide PACAP engages multiple signaling pathways within the carotid body to initiate excitatory responses in respiratory and sympathetic chemosensory afferents.

    PubMed

    Roy, Arijit; Derakhshan, Fatemeh; Wilson, Richard J A

    2013-06-15

    Consistent with a critical role in respiratory and autonomic stress responses, the carotid bodies are strongly excited by pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide implicated in stress responses throughout the sympathetic nervous system. PACAP excites isolated carotid body glomus cells via activation of PAC1 receptors, with one study suggesting PAC1-induced excitation is due entirely to protein kinase A (PKA)-mediated inhibition of TASK channels. However, in other systems, PAC1 is known to be coupled to multiple intracellular signaling pathways, including PKA, phospholipase C (PLC), phospholipase D (PLD), and protein kinase C (PKC), that trigger multiple downstream effectors including increased Ca²? mobilization, inhibition of various K? channels, and activation of nonselective cation channels. This study tests if non-PKA/TASK channel signaling helps mediate the stimulatory effects of PACAP on the carotid body. Using an ex vivo arterially perfused rat carotid body preparation, we show that PACAP-38 stimulates carotid sinus nerve activity in a biphasic manner (peak response, falling to plateau). PKA blocker H-89 only reduced the plateau response (~41%), whereas the TASK-1-like K? channel blocker/transient receptor potential vanilloid 1 channel agonist anandamide only inhibited the peak response (~48%), suggesting involvement of additional pathways. The PLD blocker CAY10594 significantly inhibited both peak and plateau responses. The PLC blocker U73122 decimated both peak and plateau responses. Brefeldin A, a blocker of Epac (cAMP-activated guanine exchange factor, reported to link Gs-coupled receptors with PLC/PLD), also reduced both phases of the response, as did blocking signaling downstream of PLC/PLD with the PKC inhibitors chelerythrine chloride and GF109203X. Suggesting the involvement of non-TASK ion channels in the effects of PACAP, the A-type K? channel blocker 4-aminopyridine, and the putative transient receptor potential channel (TRPC)/T-type calcium channel blocker SKF96365 each significantly inhibited the peak and steady-state responses. These data suggest the stimulatory effect of PACAP-38 on carotid body sensory activity is mediated through multiple signaling pathways: the PLC-PKC pathways predominates, with TRPC and/or T-type channel activation and Kv channel inactivation; only partial involvement is attributable to PKA and PLD activation. PMID:23594614

  5. Interleukin-1? promotes the expression of monocyte chemoattractant protein-1 in human aorta smooth muscle cells via multiple signaling pathways

    PubMed Central

    Lim, Jun Hee; Um, Hee Jung; Park, Jong-Wook; Lee, In-Kyu

    2009-01-01

    Monocyte chemoattractant protein-1 (MCP1) plays a key role in monocyte/macrophage infiltration to the sub-endothelial space of the blood vessel wall, which is a critical initial step in atherosclerosis. In this study, we examined the intracellular signaling pathway of IL-1?-induced MCP1 expression using various chemical inhibitors. The pretreatment of a phosphatidylcholine (PC)-specific PLC (PC-PLC) inhibitor (D609), PKC inhibitors, or an NF-?B inhibitor completely suppressed the IL-1?-induced MCP1 expression through blocking NF-?B translocation to the nucleus. Pretreatment with inhibitors of tyrosine kinase or PLD partially suppressed MCP1 expression and failed to block nuclear NF-?B translocation. These results suggest that IL-1? induces MCP1 expression through activation of NF-?B via the PC-PLC/PKC signaling pathway. PMID:19561397

  6. Complementary transcriptomic and proteomic analyses of a chlorophyll-deficient tea plant cultivar reveal multiple metabolic pathway changes.

    PubMed

    Wang, Lu; Cao, Hongli; Chen, Changsong; Yue, Chuan; Hao, Xinyuan; Yang, Yajun; Wang, Xinchao

    2016-01-01

    To uncover the mechanisms that underlie the chlorina phenotype of the tea plant, this study employs morphological, biochemical, transcriptomic, and iTRAQ-based proteomic analyses to compare the green tea cultivar LJ43 and the yellow-leaf tea cultivar ZH1. ZH1 exhibited the chlorina phenotype, with significantly decreased chlorophyll content and abnormal chloroplast development compared with LJ43. ZH1 also displayed higher theanine and free amino acid content and lower carotenoid and catechin content. Microarray and iTRAQ analyses indicated that the differentially expressed genes and proteins could be mapped to the following pathways: 'phenylpropanoid biosynthesis,' 'glutathione metabolism,' 'phenylalanine metabolism,' 'photosynthesis,' and 'flavonoid biosynthesis.' Altered gene and protein levels in these pathways may account for the increased amino acid content and reduced chlorophyll and flavonoid content of ZH1. Altogether, this study combines transcriptomic and proteomic approaches to better understand the mechanisms responsible for the chlorina phenotype. PMID:26344129

  7. Source-pathway separation of multiple contaminants during a rainfall-runoff event in an artificially drained agricultural watershed.

    PubMed

    Tomer, M D; Wilson, C G; Moorman, T B; Cole, K J; Heer, D; Isenhart, T M

    2010-01-01

    A watershed's water quality is influenced by contaminant-transport pathways unique to each landscape. Accurate information on contaminant-pathways could provide a basis for mitigation through well-targeted approaches. This study determined dynamics of nitrate-N, total P, Escherichia coli, and sediment during a runoff event in Tipton Creek, Iowa. The watershed, under crop and livestock production, has extensive tile drainage discharging through an alluvial valley. A September 2006 storm yielded 5.9 mm of discharge during the ensuing 7 d, which was monitored at the outlet (19,850 ha), two tile-drainage outfalls (total 1856 ha), and a runoff flume (11 ha) within the sloped valley. Hydrograph separations indicated 13% of tile discharge was from surface intakes. Tile and outlet nitrate-N loads were similar, verifying subsurface tiles dominate nitrate delivery. On a unit-area basis, tile total P and E. coli loads, respectively, were about half and 30% of the outlet's; their rapid, synchronous timing showed surface intakes are an important pathway for both contaminants. Flume results indicated field runoff was a significant source of total P and E. coli loads, but not the dominant one. At the outlet, sediment, P, and E. coli were reasonably synchronous. Radionuclide activities of (7)Be and (210)Pb in suspended sediments showed sheet-and-rill erosion sourced only 22% of sediment contributions; therefore, channel sources dominated and were an important source of P and E. coli. The contaminants followed unique pathways, necessitating separate mitigation strategies. To comprehensively address water quality, erosion-control and nitrogen-management practices currently encouraged could be complemented by buffering surface intakes and stabilizing stream banks. PMID:20400584

  8. Lipopolysaccharide-induced activation of NF-{kappa}B non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations

    SciTech Connect

    Bhattacharyya, Sumit; Borthakur, Alip; Dudeja, Pradeep K.; Tobacman, Joanne K.

    2010-11-15

    Background and aims: B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-{kappa}B (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods: Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-{kappa}B activation, including nuclear RelB and p52 and phospho- and total NF-{kappa}B inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results: By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-I{kappa}B{alpha} of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions: LPS induces activation of both canonical and non-canonical pathways of NF-{kappa}B in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.

  9. Neurospora crassa Female Development Requires the PACC and Other Signal Transduction Pathways, Transcription Factors, Chromatin Remodeling, Cell-To-Cell Fusion, and Autophagy

    PubMed Central

    Chinnici, Jennifer L.; Fu, Ci; Caccamise, Lauren M.; Arnold, Jason W.; Free, Stephen J.

    2014-01-01

    Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus Neurospora crassa. We find that we can divide these genes into five general groups: 1) Genes encoding components of the PACC signal transduction pathway, 2) Other signal transduction pathway genes, including genes from the three N. crassa MAP kinase pathways, 3) Transcriptional factor genes, 4) Autophagy genes, and 5) Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes palA, palB, palC, palF, palH, and pacC were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The N. crassa PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in Aspergillus nidulans and Saccharomyces cerevisiae. In N. crassa the pathway plays a key role in regulating female development. PMID:25333968

  10. Neurospora crassa female development requires the PACC and other signal transduction pathways, transcription factors, chromatin remodeling, cell-to-cell fusion, and autophagy.

    PubMed

    Chinnici, Jennifer L; Fu, Ci; Caccamise, Lauren M; Arnold, Jason W; Free, Stephen J

    2014-01-01

    Using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus Neurospora crassa. We find that we can divide these genes into five general groups: 1) Genes encoding components of the PACC signal transduction pathway, 2) Other signal transduction pathway genes, including genes from the three N. crassa MAP kinase pathways, 3) Transcriptional factor genes, 4) Autophagy genes, and 5) Other miscellaneous genes. Complementation and RIP studies verified that these genes are needed for the formation of the female mating structure, the protoperithecium, and for the maturation of a fertilized protoperithecium into a perithecium. Perithecia grafting experiments demonstrate that the autophagy genes and the cell-to-cell fusion genes (the MAK-1 and MAK-2 pathway genes) are needed for the mobilization and movement of nutrients from an established vegetative hyphal network into the developing protoperithecium. Deletion mutants for the PACC pathway genes palA, palB, palC, palF, palH, and pacC were found to be defective in two aspects of female development. First, they were unable to initiate female development on synthetic crossing medium. However, they could form protoperithecia when grown on cellophane, on corn meal agar, or in response to the presence of nearby perithecia. Second, fertilized perithecia from PACC pathway mutants were unable to produce asci and complete female development. Protein localization experiments with a GFP-tagged PALA construct showed that PALA was localized in a peripheral punctate pattern, consistent with a signaling center associated with the ESCRT complex. The N. crassa PACC signal transduction pathway appears to be similar to the PacC/Rim101 pathway previously characterized in Aspergillus nidulans and Saccharomyces cerevisiae. In N. crassa the pathway plays a key role in regulating female development. PMID:25333968

  11. ATP Utilization by Yeast Replication Factor C II. MULTIPLE STEPWISE ATP BINDING EVENTS ARE REQUIRED TO LOAD PROLIFERATING CELL

    E-print Network

    Burgers, Peter M.

    ATP Utilization by Yeast Replication Factor C II. MULTIPLE STEPWISE ATP BINDING EVENTS ARE REQUIRED of adenosine (3-thiotriphosphate) (ATP S), a nonhydrolyzable analog of ATP, to replication factor C with a N-terminal truncation ( 2­273) of the Rfc1 sub- unit (RFC) was studied by filter binding. RFC alone bound 1.8 ATP

  12. Multiple WASP-interacting Protein Recognition Motifs Are Required for a Functional Interaction with N-WASP*

    E-print Network

    Prehoda, Ken

    Multiple WASP-interacting Protein Recognition Motifs Are Required for a Functional Interaction with N-WASP* Received for publication,October 23, 2006, and in revised form, December 27, 2006 Published Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom The WASP

  13. MicroRNA-155 tunes both the threshold and extent of NK cell activation via targeting of multiple signaling pathways.

    PubMed

    Sullivan, Ryan P; Fogel, Leslie A; Leong, Jeffrey W; Schneider, Stephanie E; Wong, Rachel; Romee, Rizwan; Thai, To-Ha; Sexl, Veronika; Matkovich, Scot J; Dorn, Gerald W; French, Anthony R; Fehniger, Todd A

    2013-12-15

    NK cells are innate lymphocytes important for host defense against viral infections and malignancy. However, the molecular programs orchestrating NK cell activation are incompletely understood. MicroRNA-155 (miR-155) is markedly upregulated following cytokine activation of human and mouse NK cells. Surprisingly, mature human and mouse NK cells transduced to overexpress miR-155, NK cells from mice with NK cell-specific miR-155 overexpression, and miR-155(-/-) NK cells all secreted more IFN-? compared with controls. Investigating further, we found that activated NK cells with miR-155 overexpression had increased per-cell IFN-? with normal IFN-?(+) percentages, whereas greater percentages of miR-155(-/-) NK cells were IFN-?(+). In vivo murine CMV-induced IFN-? expression by NK cells in these miR-155 models recapitulated the in vitro phenotypes. We performed unbiased RNA-induced silencing complex sequencing on wild-type and miR-155(-/-) NK cells and found that mRNAs targeted by miR-155 were enriched in NK cell activation signaling pathways. Using specific inhibitors, we confirmed these pathways were mechanistically involved in regulating IFN-? production by miR-155(-/-) NK cells. These data indicate that miR-155 regulation of NK cell activation is complex and that miR-155 functions as a dynamic tuner for NK cell activation via both setting the activation threshold as well as controlling the extent of activation in mature NK cells. In summary, miR-155(-/-) NK cells are more easily activated, through increased expression of proteins in the PI3K, NF-?B, and calcineurin pathways, and miR-155(-/-) and 155-overexpressing NK cells exhibit increased IFN-? production through distinct cellular mechanisms. PMID:24227772

  14. miR-155 tunes both the threshold and extent of NK cell activation via targeting of multiple signaling pathways

    PubMed Central

    Sullivan, Ryan P.; Fogel, Leslie A.; Leong, Jeffrey W.; Schneider, Stephanie E.; Wong, Rachel; Romee, Rizwan; Thai, To-Ha; Sexl, Veronika; Matkovich, Scot J.; Dorn, Gerald W.; French, Anthony R.; Fehniger, Todd A.

    2013-01-01

    NK cells are innate lymphocytes important for host defense against viral infections and malignancy. However, the molecular programs orchestrating NK cell activation are incompletely understood. miR-155 is markedly upregulated following cytokine activation of human and mouse NK cells. Surprisingly, mature human and mouse NK cells transduced to overexpress miR-155, NK cells from mice with NK cell-specific miR-155 overexpression, and miR-155?/? NK cells all secreted more IFN-? compared to controls. Investigating further, we found that activated NK cells with miR-155 overexpression had increased per cell IFN-? with normal IFN-?+ percentages, whereas greater percentages of miR-155?/? NK cells were IFN-?+. In vivo MCMV-induced IFN-? expression by NK cells in these miR-155 models recapitulated the in vitro phenotypes. We performed unbiased RISC-Seq on WT and miR-155?/? NK cells, and found that mRNAs targeted by miR-155 were enriched in NK cell activation signaling pathways. Using specific inhibitors, we confirmed these pathways were mechanistically involved in regulating IFN–? production by miR-155?/? NK cells. These data indicate that miR-155 regulation of NK cell activation is complex, and that miR-155 functions as a dynamic tuner for NK cell activation via both setting the activation threshold as well as controlling the extent of activation in mature NK cells. In summary, miR-155?/? NK cells are more easily activated, through increased expression of proteins in the PI3K, NF-?B, and calcineurin pathways, and miR-155?/? and 155-overexpressing NK cells exhibit increased IFN-? production through distinct cellular mechanisms. PMID:24227772

  15. Vegetables’ juice influences polyol pathway by multiple mechanisms in favour of reducing development of oxidative stress and resultant diabetic complications

    PubMed Central

    Tiwari, Ashok K.; Kumar, D. Anand; Sweeya, Pisupati S.; Chauhan, H. Anusha; Lavanya, V.; Sireesha, K.; Pavithra, K.; Zehra, Amtul

    2014-01-01

    Objective: Hyperglycemia induced generation of free radicals and consequent development of oxidative stress by polyol pathway is one of the crucial mechanisms stirring up development of diabetic complications. We evaluated influence of ten vegetables’ juice on polyol pathway along with their antioxidant and antioxidative stress potentials. Materials and Methods: Aldose reductase activity was determined utilising goat lens and human erythrocytes. In goat lens, utilization of nicotinamine adenine dinucleotide phosphate (NADPH) and aldose reductase inhibition was assayed. In human erythrocytes, sorbitol formation was measured as an index of aldose reductase activity under normoglycemic and hyperglycemic conditions. Ability of juices in inhibiting oxidative damage to deoxyribose sugar and calf thymus DNA and inhibitory activity against hydrogen peroxide induced hemolysis of erythrocytes was also analysed. Phytochemical contents like total polyphenol, total flavonoid and total protein were measured to find their influence on biological activities. Results: Vegetables’ juice displayed varying degrees of inhibitory potentials in mitigating NADPH dependent catalytic activity of aldose reductase in goat lens, accumulation of sorbitol in human erythrocytes under different glucose concentrations; Fenton-reaction induced oxidative damage to deoxyribose sugar, and calf thymus DNA. Substantial variations in vegetables phytochemicals content were also noticed in this study. Conclusions: Vegetables’ juice possesses potent activities in influencing polyol pathway by various mechanisms in favour of reducing development of oxidative stress independent of their inherent antioxidative properties. Juice of ivy gourd followed by green cucumber and ridge gourd were among the most potent for they displayed strong activities on various parameters analysed in this study. These vegetables’ juice may become part of mechanism-based complementary antioxidant therapy to prevent development of diabetic complications. PMID:24991118

  16. Gene Expression Profiles from Disease Discordant Twins Suggest Shared Antiviral Pathways and Viral Exposures among Multiple Systemic Autoimmune Diseases

    PubMed Central

    Gan, Lu; O’Hanlon, Terrance P.; Lai, Zhennan; Fannin, Rick; Weller, Melodie L.; Rider, Lisa G.; Chiorini, John A.; Miller, Frederick W.

    2015-01-01

    Viral agents are of interest as possible autoimmune triggers due to prior reported associations and widely studied molecular mechanisms of antiviral immune responses in autoimmunity. Here we examined new viral candidates for the initiation and/or promotion of systemic autoimmune diseases (SAID), as well as possible related signaling pathways shared in the pathogenesis of those disorders. RNA isolated from peripheral blood samples from 33 twins discordant for SAID and 33 matched, unrelated healthy controls was analyzed using a custom viral-human gene microarray. Paired comparisons were made among three study groups—probands with SAID, their unaffected twins, and matched, unrelated healthy controls—using statistical and molecular pathway analyses. Probands and unaffected twins differed significantly in the expression of 537 human genes, and 107 of those were associated with viral infections. These 537 differentially expressed human genes participate in overlapping networks of several canonical, biologic pathways relating to antiviral responses and inflammation. Moreover, certain viral genes were expressed at higher levels in probands compared to either unaffected twins or unrelated, healthy controls. Interestingly, viral gene expression levels in unaffected twins appeared intermediate between those of probands and the matched, unrelated healthy controls. Of the viruses with overexpressed viral genes, herpes simplex virus-2 (HSV-2) was the only human viral pathogen identified using four distinct oligonucleotide probes corresponding to three HSV-2 genes associated with different stages of viral infection. Although the effects from immunosuppressive therapy on viral gene expression remain unclear, this exploratory study suggests a new approach to evaluate shared viral agents and antiviral immune responses that may be involved in the development of SAID. PMID:26556803

  17. CCAAT/enhancer-binding protein ? is required for hepatic outgrowth via the p53 pathway in zebrafish

    PubMed Central

    Yuan, Hao; Wen, Bin; Liu, Xiaohui; Gao, Ce; Yang, Ruimeng; Wang, Luxiang; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

    2015-01-01

    CCAAT/enhancer-binding protein ? (C/ebp?) is a transcription factor that plays important roles in the regulation of hepatogenesis, adipogenesis and hematopoiesis. Disruption of the C/EBP? gene in mice leads to disturbed liver architecture and neonatal death due to hypoglycemia. However, the precise stages of liver development affected by C/ebp? loss are poorly studied. Using the zebrafish embryo as a model organism, we show that inactivation of the cebpa gene by TALENs results in a small liver phenotype. Further studies reveal that C/ebp? is distinctively required for hepatic outgrowth but not for hepatoblast specification. Lack of C/ebp? leads to enhanced hepatic cell proliferation and subsequent increased cell apoptosis. Additional loss of p53 can largely rescue the hepatic defect in cebpa mutants, suggesting that C/ebp? plays a role in liver growth regulation via the p53 pathway. Thus, our findings for the first time demonstrate a stage-specific role for C/ebp? during liver organogenesis. PMID:26511037

  18. CCAAT/enhancer-binding protein ? is required for hepatic outgrowth via the p53 pathway in zebrafish.

    PubMed

    Yuan, Hao; Wen, Bin; Liu, Xiaohui; Gao, Ce; Yang, Ruimeng; Wang, Luxiang; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

    2015-01-01

    CCAAT/enhancer-binding protein ? (C/ebp?) is a transcription factor that plays important roles in the regulation of hepatogenesis, adipogenesis and hematopoiesis. Disruption of the C/EBP? gene in mice leads to disturbed liver architecture and neonatal death due to hypoglycemia. However, the precise stages of liver development affected by C/ebp? loss are poorly studied. Using the zebrafish embryo as a model organism, we show that inactivation of the cebpa gene by TALENs results in a small liver phenotype. Further studies reveal that C/ebp? is distinctively required for hepatic outgrowth but not for hepatoblast specification. Lack of C/ebp? leads to enhanced hepatic cell proliferation and subsequent increased cell apoptosis. Additional loss of p53 can largely rescue the hepatic defect in cebpa mutants, suggesting that C/ebp? plays a role in liver growth regulation via the p53 pathway. Thus, our findings for the first time demonstrate a stage-specific role for C/ebp? during liver organogenesis. PMID:26511037

  19. Policy, Service Pathways and Mortality: A 10-Year Longitudinal Study of People with Profound Intellectual and Multiple Disabilities

    ERIC Educational Resources Information Center

    Hogg, James; Juhlberg, K.; Lambe, L.

    2007-01-01

    Background: One hundred and forty-two children and adults with profound intellectual and multiple disabilities were identified in 1993 in a single Scottish region on whom detailed information was collected via a postal questionnaire survey. Methods: They were followed up in 2003. The time spanned represented a period of significant policy change…

  20. Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

    PubMed Central

    Forrest, Marc P.; Waite, Adrian J.; Martin-Rendon, Enca; Blake, Derek J.

    2013-01-01

    Haploinsufficiency of TCF4 causes Pitt-Hopkins syndrome (PTHS): a severe form of mental retardation with phenotypic similarities to Angelman, Mowat-Wilson and Rett syndromes. Genome-wide association studies have also found that common variants in TCF4 are associated with an increased risk of schizophrenia. Although TCF4 is transcription factor, little is known about TCF4-regulated processes in the brain. In this study we used genome-wide expression profiling to determine the effects of acute TCF4 knockdown on gene expression in SH-SY5Y neuroblastoma cells. We identified 1204 gene expression changes (494 upregulated, 710 downregulated) in TCF4 knockdown cells. Pathway and enrichment analysis on the differentially expressed genes in TCF4-knockdown cells identified an over-representation of genes involved in TGF-? signaling, epithelial to mesenchymal transition (EMT) and apoptosis. Among the most significantly differentially expressed genes were the EMT regulators, SNAI2 and DEC1 and the proneural genes, NEUROG2 and ASCL1. Altered expression of several mental retardation genes such as UBE3A (Angelman Syndrome), ZEB2 (Mowat-Wilson Syndrome) and MEF2C was also found in TCF4-depleted cells. These data suggest that TCF4 regulates a number of convergent signaling pathways involved in cell differentiation and survival in addition to a subset of clinically important mental retardation genes. PMID:24058414

  1. Deoxyelephantopin impairs growth of cervical carcinoma SiHa cells and induces apoptosis by targeting multiple molecular signaling pathways.

    PubMed

    Farha, Arakkaveettil Kabeer; Dhanya, Sethumadhavannair Rajalekshmi; Mangalam, Sivasankaran Nair; Geetha, Balakrishnan Sreedevi; Latha, Panickamparambil Gopalakrishnan; Remani, Prathapan

    2014-12-01

    Deoxyelephantopin, a sesquiterpene lactone extracted and purified from Elephantopus scaber, has been shown to exhibit antitumor and hepatoprotective activities. The purpose of this study was to investigate the antiproliferative and apoptosis-inducing properties of deoxyelephantopin in SiHa cells and to elucidate the underlying molecular mechanisms. Deoxyelephantopin inhibited growth of SiHa cells and triggered apoptosis. Apoptosis was accompanied by sequential activation of caspases (8, 9, 3, and 7) and reactive oxygen species (ROS) production. Downregulation of antiapoptotic proteins (Bcl2 and Bcl-xL) and upregulation of apoptotic protein (bax) were also detected. Our results demonstrated that deoxyelephantopin-induced G2/M phase arrest was associated with a marked increase in the levels of p53 and p21 and a decrease in phospho-signal transducer and activator of transcription 3 (pSTAT3-Tyr705), cyclin-dependent kinase 1 (cdc2), and cyclin B1. The expression of p-Akt and p-mTOR was downregulated. p-ERK was inhibited while p-JNK and p-p38 was activated on deoxyelephantopin treatment. Our findings provided the first evidence that STAT3/p53/p21 signaling, MAPK pathway, PI3k/Akt/mTOR pathway, caspase cascades, and ROS play critical roles in deoxyelephantopin-induced G2/M phase arrest and apoptosis of SiHa cells. PMID:25260383

  2. Activation of complement by Schistosoma mansoni schistosomula: killing of parasites by the alternative pathway and requirement of IgG for classical pathway activation.

    PubMed

    Santoro, F; Lachmann, P J; Capron, A; Capron, M

    1979-10-01

    Living Schistosoma mansoni schistosomula incubated with normal chicken, guinea pig, human, and monkey sera were killed after 4 hr contact at 37 degrees C. The following data indicate that this action is dependent on the activation of the alternative complement pathway (AP): a) the inactivity of RB, RD, and zymosan-treated serum against schistosomula; b) the partial activity of RD restored in FD; c) the full effect of the C4-deficient guinea pig, C2-deficient human, and the agammaglobulinemic human sera; d) the consumption of both the AP and FB after the incubation of NHS with schistosomula; e) the detection of C3d breakdown product during the contact of the C2-deficient human serum with these young parasites. Killing by serum was decreased as the immature schistosomes developed and was completely absent against 4-day-old lung schistosomula (LS). In other experiments, it was demonstrated that schistosomula, in the presence of IgG, were able to initiate complement activation also through the classical pathway (CP). However, the CP does not appear to play a role in the schistosomulicidal activity of complement. The in vivo relevance of these observations is considered. PMID:113459

  3. FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway.

    PubMed

    Ali, Abdullah Mahmood; Pradhan, Arun; Singh, Thiyam Ramsingh; Du, Changhu; Li, Jie; Wahengbam, Kebola; Grassman, Elke; Auerbach, Arleen D; Pang, Qishen; Meetei, Amom Ruhikanta

    2012-04-01

    Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance. PMID:22343915

  4. Assessment of groundwater pathways and contaminant transport in Florida and Georgia using multiple chemical and microbiological indicators

    USGS Publications Warehouse

    Mahon, Gary L.

    2011-01-01

    The hydrogeology of Florida, especially in the northern part of the state, and southwestern Georgia is characterized by a predominance of limestone aquifers overlain by varying amounts of sands, silts, and clays. This karstic system of aquifers and their associated springs is particularly vulnerable to contamination from various anthropogenic activities at the land surface. Numerous sinkholes, disappearing streams, and conduit systems or dissolution pathways, often associated with large spring systems, allow rapid movement of contaminants from the land surface to the groundwater system with little or no attenuation or degradation. The fate of contaminants in the groundwater system is not fully understood, but traveltimes from sources are greatly reduced when conduits are intercepted by pumping wells and springs. Contaminant introduction to groundwater systems in Florida and Georgia is not limited to seepage from land surface, but can be associated with passive (drainage wells) and forced subsurface injection (aquifer storage and recovery, waste-water disposal).

  5. Targeting the miR-221-222/PUMA/BAK/BAX Pathway Abrogates Dexamethasone Resistance in Multiple Myeloma.

    PubMed

    Zhao, Jian-Jun; Chu, Zhang-Bo; Hu, Yu; Lin, Jianhong; Wang, Zhongqiu; Jiang, Meng; Chen, Ming; Wang, Xujun; Kang, Yue; Zhou, Yangsheng; Chonghaile, Triona Ni; Johncilla, Melanie E; Tai, Yu-Tzu; Cheng, Jin Q; Letai, Antony; Munshi, Nikhil C; Anderson, Kenneth C; Carrasco, Ruben D

    2015-10-15

    Despite recent therapeutic advances that have doubled the median survival time of patients with multiple myeloma, intratumor genetic heterogeneity contributes to disease progression and emergence of drug resistance. miRNAs are noncoding small RNAs that play important roles in the regulation of gene expression and have been implicated in cancer progression and drug resistance. We investigated the role of the miR-221-222 family in dexamethasone-induced drug resistance in multiple myeloma using the isogenic cell lines MM1R and MM1S, which represent models of resistance and sensitivity, respectively. Analysis of array comparative genome hybridization data revealed gain of chromosome X regions at band p11.3, wherein the miR-221-222 resides, in resistant MM1R cells but not in sensitive MM1S cells. DNA copy number gains in MM1R cells were associated with increased miR-221-222 expression and downregulation of p53-upregulated modulator of apoptosis (PUMA) as a likely proapoptotic target. We confirmed PUMA mRNA as a direct target of miR-221-222 in MM1S and MM1R cells by both gain-of-function and loss-of-function studies. In addition, miR-221-222 treatment rendered MM1S cells resistant to dexamethasone, whereas anti-miR-221-222 partially restored the dexamethasone sensitivity of MM1R cells. These studies have uncovered a role for miR-221-222 in multiple myeloma drug resistance and suggest a potential therapeutic role for inhibitors of miR-221-222 binding to PUMA mRNA as a means of overcoming dexamethasone resistance in patients. The clinical utility of this approach is predicated on the ability of antisense miR-221-222 to increase survival while reducing tumor burden and is strongly supported by the metastatic propensity of MM1R cells in preclinical mouse xenograft models of multiple myeloma. Moreover, our observation of increased levels of miR-221-222 with decreased PUMA expression in multiple myeloma cells from patients at relapse versus untreated controls suggests an even broader role for miR-221-222 in drug resistance and provides a rationale for the targeting of miR-221-222 as a means of improving patient outcomes. Cancer Res; 75(20); 4384-97. ©2015 AACR. PMID:26249174

  6. Multiple IMU system test plan, volume 4. [subroutines for space shuttle requirements

    NASA Technical Reports Server (NTRS)

    Landey, M.; Vincent, K. T., Jr.; Whittredge, R. S.

    1974-01-01

    Operating procedures for this redundant system are described. A test plan is developed with two objectives. First, performance of the hardware and software delivered is demonstrated. Second, applicability of multiple IMU systems to the space shuttle mission is shown through detailed experiments with FDI algorithms and other multiple IMU software: gyrocompassing, calibration, and navigation. Gimbal flip is examined in light of its possible detrimental effects on FDI and navigation. For Vol. 3, see N74-10296.

  7. Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

    PubMed Central

    Balmer, Jasmin; Zulliger, Rahel; Roberti, Stefano; Enzmann, Volker

    2015-01-01

    Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3) both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg). Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE) cells, primary retinal cells, and the cone photoreceptor (PRC) cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1) was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3. PMID:26151844

  8. Smooth muscle cell surface tissue factor pathway activation by oxidized low-density lipoprotein requires cellular lipid peroxidation.

    PubMed

    Penn, M S; Cui, M Z; Winokur, A L; Bethea, J; Hamilton, T A; DiCorleto, P E; Chisolm, G M

    2000-11-01

    Tissue factor, which is expressed in vascular lesions, increases thrombin production, blood coagulation, and smooth muscle cell proliferation. We demonstrate that oxidized low-density lipoprotein (LDL) induces surface tissue factor pathway activity (ie, activity of the tissue factor:factor VIIa complex) on human and rat smooth muscle cells. Tissue factor messenger RNA (mRNA) was induced by oxidized LDL or native LDL; however, native LDL did not markedly increase tissue factor activity. We hypothesized that oxidized LDL mediated the activation of the tissue factor pathway via an oxidant-dependent mechanism, because antioxidants blocked the enhanced tissue factor pathway activity by oxidized LDL, but not the increased mRNA or protein induction. We separated total lipid extracts of oxidized LDL using high-performance liquid chromatography (HPLC). This yielded 2 major peaks that induced tissue factor activity. Of the known oxysterols contained in the first peak, 7alpha- or 7beta-hydroxy or 7-ketocholesterol had no effect on tissue factor pathway activity; however, 7beta-hydroperoxycholesterol increased tissue factor pathway activity without induction of tissue factor mRNA. Tertiary butyl hydroperoxide also increased tissue factor pathway activity, suggesting that lipid hydroperoxides, some of which exist in atherosclerotic lesions, activate the tissue factor pathway. We speculate that thrombin production could be elevated via a mechanism involving peroxidation of cellular lipids, contributing to arterial thrombosis after plaque rupture. Our data suggest a mechanism by which antioxidants may offer a clinical benefit in acute coronary syndrome and restenosis. PMID:11049984

  9. [123I]-IBVM SPECT imaging of cholinergic systems in multiple system atrophy: A specific alteration of the ponto-thalamic cholinergic pathways (Ch5–Ch6)?

    PubMed Central

    Mazere, Joachim; Meissner, Wassilios. G.; Sibon, Igor; Lamare, Frédéric; Tison, François; Allard, Michèle; Mayo, Willy

    2013-01-01

    We evaluated in vivo the integrity of brain cholinergic pathways in Multiple System Atrophy (MSA) and the relationship between cholinergic dysfunction and motor disturbances, by measuring the vesicular acetylcholine transporter (VAChT) expression using Single Photon Emission Computed Tomography (SPECT) and [123I]-iodobenzovesamicol ([123I]-IBVM). Methods Nine patients with probable MSA and 12 healthy volunteers underwent a dynamic [123I]-IBVM SPECT–CT scan and a magnetic resonance imaging (MRI) scan. All patients were examined with the Unified MSA Rating Scale (UMSARS; subscale I = activities of daily living (ADL), II = motor and IV = disability). CT and MRI images were used to register the dynamic SPECT image to the Montreal Neurological Institute brain template, which includes the regions of interest (ROI) of striatum and Ch1 (medial septum nucleus-hippocampus), Ch4 (nucleus basalis of Meynert-cortex) and Ch5–Ch6 (pedunculopontine and laterodorsal tegmental nuclei-thalamus) cholinergic pathways. For each ROI, pharmacokinetic modeling of regional time activity curves led to the calculation of [123I]-IBVM to VAChT binding potential (BPND) value, proportional to VAChT expression. Results When compared to controls, BPND values for MSA in Ch5–Ch6 were significantly decreased in both the pedunculopontine–laterodorsal nuclei and the thalamus (p = 0.004 and p = 0.006, respectively). Additionally, thalamus BPND values were correlated with UMSARS ADL (p = 0.006), motor (p = 0.002) and disability (p = 0.02) sub-scores. UMSARS motor subscale items 13 (postural instability) and 14 (gait) were also correlated with thalamus BPND values (p = 0.04). Conclusion Ch5–Ch6 are the most affected cholinergic pathways in MSA at both cell bodies and thalamic cholinergic terminals. These results underscore the relevant role of [123I]-IBVM SPECT for improving our understanding of the pathophysiology in MSA. PMID:24179865

  10. A highly conserved family of domains related to the DNA-glycosylase fold helps predict multiple novel pathways for RNA modifications

    PubMed Central

    Burroughs, A Maxwell; Aravind, L

    2014-01-01

    A protein family including mammalian NEMF, Drosophila caliban, yeast Tae2, and bacterial FpbA-like proteins was first defined over a decade ago and found to be universally distributed across the three domains/superkingdoms of life. Since its initial characterization, this family of proteins has been tantalizingly linked to a wide range of biochemical functions. Tapping the enormous wealth of genome information that has accumulated since the initial characterization of these proteins, we perform a detailed computational analysis of the family, identifying multiple conserved domains. Domains identified include an enzymatic domain related to the formamidopyrimidine (Fpg), MutM, and Nei/EndoVIII family of DNA glycosylases, a novel, predicted RNA-binding domain, and a domain potentially mediating protein–protein interactions. Through this characterization, we predict that the DNA glycosylase-like domain catalytically operates on double-stranded RNA, as part of a hitherto unknown base modification mechanism that probably targets rRNAs. At least in archaea, and possibly eukaryotes, this pathway might additionally include the AMMECR1 family of proteins. The predicted RNA-binding domain associated with this family is also observed in distinct architectural contexts in other proteins across phylogenetically diverse prokaryotes. Here it is predicted to play a key role in a new pathway for tRNA 4-thiouridylation along with TusA-like sulfur transfer proteins. PMID:24646681

  11. SC06, a novel small molecule compound, displays preclinical activity against multiple myeloma by disrupting the mTOR signaling pathway

    PubMed Central

    Han, Kunkun; Xu, Xin; Xu, Zhuan; Chen, Guodong; Zeng, Yuanying; Zhang, Zubin; Cao, Biyin; Kong, Yan; Tang, Xiaowen; Mao, Xinliang

    2015-01-01

    The mammalian target of rapamycin (mTOR) is extensively involved in multiple myeloma (MM) pathophysiology. In the present study, we reported a novel small molecule SC06 that induced MM cell apoptosis and delayed MM xenograft growth in vivo. Oral administration of SC06 to mice bearing human MM xenografts resulted in significant inhibition of tumor growth at doses that were well tolerated. Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK. Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex. SC06 also suppressed the phosphorylation of 4E-BP1 and P70S6K, two typical substrates in the mTORC1 signaling pathway. Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies. Therefore, given the potency and low toxicity, SC06 could be developed as a potential anti-MM drug candidate by disrupting the mTOR signaling. PMID:26329846

  12. Intraperitoneal Administration of a Novel TAT-BDNF Peptide Ameliorates Cognitive Impairments via Modulating Multiple Pathways in Two Alzheimer’s Rodent Models

    PubMed Central

    Wu, Yuanyuan; Luo, Xiaobin; Liu, Xinhua; Liu, Deyi; Wang, Xiong; Guo, Ziyuan; Zhu, Lingqiang; Tian, Qing; Yang, Xifei; Wang, Jian-Zhi

    2015-01-01

    Although Alzheimer’s disease (AD) has been reported for more than 100 years, there is still a lack of effective cures for this devastating disorder. Among the various obstacles that hold back drug development, the blood-brain barrier (BBB) is one of them. Here, we constructed a novel fusion peptide by linking the active domain of brain-derived neurotrophic factor (BDNF) with an HIV-encoded transactivator of transcription (TAT) that has a strong membrane-penetrating property. After intraperitoneal injection, the eGFP-TAT could be robustly detected in different brain regions. By using scopolamine-induced rats and APPswe mice representing AD-like cholinergic deficits and amyloidosis, respectively, we found that intraperitoneal administration of the peptide significantly improved spatial memory with activation of the TrkB/ERK1/2/Akt pathway and restoration of several memory-associated proteins in both models. Administration of the peptide also modulated ?-amyloid and tau pathologies in APPswe mice, and it increased the amount of M receptor with modulation of acetylcholinesterase in scopolamine-induced rats. We conclude that intraperitoneal administration of our TAT-BDNF peptide could efficiently target multiple molecular pathways in the brain and improve the cognitive functions in AD-like rodent models. PMID:26463268

  13. The Shh Signaling Pathway Is Upregulated in Multiple Cell Types in Cortical Ischemia and Influences the Outcome of Stroke in an Animal Model

    PubMed Central

    Jin, Yongmin; Raviv, Nataly; Barnett, Austin; Bambakidis, Nicholas C.; Filichia, Emily; Luo, Yu

    2015-01-01

    Recently the sonic hedgehog (shh) signaling pathway has been shown to play an important role in regulating repair and regenerative responses after brain injury, including ischemia. However, the precise cellular components that express and upregulate the shh gene and the cellular components that respond to shh signaling remain to be identified. In this study, using a distal MCA occlusion model, our data show that the shh signal is upregulated both at the cortical area near the injury site and in the adjacent striatum. Multiple cell types upregulate shh signaling in ischemic brain, including neurons, reactive astrocytes and nestin-expressing cells. The shh signaling pathway genes are also expressed in the neural stem cells (NSCs) niche in the subventricular zone (SVZ). Conditional deletion of the shh gene in nestin-expressing cells both at the SVZ niche and at the ischemic site lead to significantly more severe behavioral deficits in these shh iKO mice after cortical stroke, measured using an automated open field locomotion apparatus (Student’s t-test, p<0.05). In contrast, animals given post-stroke treatment with the shh signaling agonist (SAG) demonstrated less deficits in behavioral function, compared to vehicle-treated mice. At 7 days after stroke, SAG-treated mice showed higher values in multiple horizontal movement parameters compared to vehicle treated mice (Student’s t-test, p<0.05) whereas there were no differences in pre-stroke measurements, (Student’s t-test, p>0.05). In summary, our data demonstrate that shh signaling plays critical and ongoing roles in response to ischemic injury and modulation of shh signaling in vivo alters the functional outcome after cortical ischemic injury. PMID:25927436

  14. Targeting Vascular Endothelial Growth Factor Receptor 2 and Protein Kinase D1 Related Pathways by a Multiple Kinase Inhibitor in Angiogenesis and Inflammation Related Processes In Vitro

    PubMed Central

    Varga, Attila; Gyulavári, Pál; Greff, Zoltán; Futosi, Krisztina; Németh, Tamás; Simon-Szabó, Laura; Kerekes, Krisztina; Szántai-Kis, Csaba; Brauswetter, Diána; Kokas, Márton; Borbély, Gábor; Erdei, Anna; Mócsai, Attila; Kéri, György; Vántus, Tibor

    2015-01-01

    Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-? -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways. PMID:25874616

  15. A novel regulatory pathway in granulosa cells, the LH/human chorionic gonadotropin-microRNA-125a-3p-Fyn pathway, is required for ovulation.

    PubMed

    Grossman, Hadas; Chuderland, Dana; Ninio-Many, Lihi; Hasky, Noa; Kaplan-Kraicer, Ruth; Shalgi, Ruth

    2015-08-01

    Granulosa cells support the developing oocytes and serve as transducers of the ovulatory stimulus induced by LH surge. Fyn kinase is expressed in granulosa cells, though its role in these cells has not been studied. In human embryonic kidney 293T cells, microRNA (miR)-125a-3p down-regulates Fyn expression, causing a decrease in cells' migratory ability. Our aim was to explore the role of miR-125a-3p and Fyn in granulosa cells toward ovulation, focusing on migration as a possible mechanism. We demonstrate expression of miR-125a-3p and Fyn in mouse mural granulosa cells of preovulatory follicles and miR-125a-3p-induced down-regulation of Fyn expression in a granulosa cell line (rat). Administration of human chorionic gonadotropin (hCG; LH analog) caused a 75% decrease in the in vivo miR-125a-3p:Fyn mRNA ratio, followed by a 2-fold increased migratory ability of mural granulosa cells. In the hCG-treated granulosa cell line, miR-125a-3p expression was decreased, followed by Fyn up-regulation and phosphorylation of focal adhesion kinase and paxillin, enabling cell migration. An in vivo interference with miR-125a-3p:Fyn mRNA ratio in granulosa cells by intrabursal injections of Fyn small interfering RNA or miR-125a-3p mimic caused a 33 or 55% decrease in the number of ovulated oocytes, respectively. These observations reveal a new regulatory pathway in mural granulosa cells under the regulation of LH/hCG. Modulation of cell migration may account for the significance of the LH/hCG-miR-125a-3p-Fyn pathway to ovulation. PMID:25921829

  16. Novel carbocyclic curcumin analog CUR3d modulates genes involved in multiple apoptosis pathways in human hepatocellular carcinoma cells.

    PubMed

    Bhullar, Khushwant S; Jha, Amitabh; Rupasinghe, H P Vasantha

    2015-12-01

    Anticancer activity of a novel curcumin analog (E)-2-(4-hydroxy-3-methoxybenzylidene)-5-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)cyclopentanone (CUR3d) was studied using a human hepatocellular carcinoma cell line (HepG2). The results showed that CUR3d completely inhibits the tumor cell proliferation in a dose- and time-dependent manner. CUR3d at 100 ?mol/L activated the pro-apoptotic caspase-3 along with downregulation of anti-apoptotic BIRC5 and Bcl2. CUR3d treatment controlled the cancer cell growth by downregulating the expression of PI3K/Akt (Akt1, Akt2) pathway along with NF-?B. CUR3d down-regulated the members of epidermal growth receptor family (EGFR, ERBB3, ERBB2) and insulin like growth receptors (IGF1, IGF-1R, IGF2). This correlated with the downregulation of G-protein (RHOA, RHOB) and RAS (ATF2, HRAS, KRAS, NRAS) pathway signaling. CUR3d also arrested cell cycle via inhibition of CDK2, CDK4, CDK5, CDK9, MDM2, MDM4 and TERT genes. Cell cycle essential aurora kinases (AURK?, AURK?) and polo-like kinases (PLK1, PLK2, PLK3) were also modulated by CUR3d. Topoisomerases (TOP2?, TOP2?), important factors in cancer cell immortality, as well as HIF-1? were downregulated following CUR3d treatment. The expression of protein kinase-C family (PRKC-A, PRKC-D, PRKC-E) was also attenuated by CUR3d. The downregulation of histone deacetylases (Class I, II, IV) and PARP I further strengthened the anticancer efficacy of CUR3d. Downregulation of carcinogenic cathepsins (CTSB, CTSD) and heat shock proteins exhibited CUR3d's potency as a potential immunological adjuvant. Finally, the non-toxic manifestation of CUR3d in healthy liver and lung cells along with downregulation of drug resistant gene ABCC1 further warrant need for advance investigations. PMID:26409325

  17. Embryonic stem cell-specific microRNAs contribute to pluripotency by inhibiting regulators of multiple differentiation pathways

    PubMed Central

    Gruber, Andreas J.; Grandy, William A.; Balwierz, Piotr J.; Dimitrova, Yoana A.; Pachkov, Mikhail; Ciaudo, Constance; van Nimwegen, Erik; Zavolan, Mihaela

    2014-01-01

    The findings that microRNAs (miRNAs) are essential for early development in many species and that embryonic miRNAs can reprogram somatic cells into induced pluripotent stem cells suggest that these miRNAs act directly on transcriptional and chromatin regulators of pluripotency. To elucidate the transcription regulatory networks immediately downstream of embryonic miRNAs, we extended the motif activity response analysis approach that infers the regulatory impact of both transcription factors (TFs) and miRNAs from genome-wide expression states. Applying this approach to multiple experimental data sets generated from mouse embryonic stem cells (ESCs) that did or did not express miRNAs of the ESC-specific miR-290-295 cluster, we identified multiple TFs that are direct miRNA targets, some of which are known to be active during cell differentiation. Our results provide new insights into the transcription regulatory network downstream of ESC-specific miRNAs, indicating that these miRNAs act on cell cycle and chromatin regulators at several levels and downregulate TFs that are involved in the innate immune response. PMID:25030899

  18. Embryonic stem cell-specific microRNAs contribute to pluripotency by inhibiting regulators of multiple differentiation pathways.

    PubMed

    Gruber, Andreas J; Grandy, William A; Balwierz, Piotr J; Dimitrova, Yoana A; Pachkov, Mikhail; Ciaudo, Constance; Nimwegen, Erik van; Zavolan, Mihaela

    2014-08-01

    The findings that microRNAs (miRNAs) are essential for early development in many species and that embryonic miRNAs can reprogram somatic cells into induced pluripotent stem cells suggest that these miRNAs act directly on transcriptional and chromatin regulators of pluripotency. To elucidate the transcription regulatory networks immediately downstream of embryonic miRNAs, we extended the motif activity response analysis approach that infers the regulatory impact of both transcription factors (TFs) and miRNAs from genome-wide expression states. Applying this approach to multiple experimental data sets generated from mouse embryonic stem cells (ESCs) that did or did not express miRNAs of the ESC-specific miR-290-295 cluster, we identified multiple TFs that are direct miRNA targets, some of which are known to be active during cell differentiation. Our results provide new insights into the transcription regulatory network downstream of ESC-specific miRNAs, indicating that these miRNAs act on cell cycle and chromatin regulators at several levels and downregulate TFs that are involved in the innate immune response. PMID:25030899

  19. Signal transducer and activator of transcription 3 pathway mediates genipin-induced apoptosis in U266 multiple myeloma cells.

    PubMed

    Lee, Jang Choon; Ahn, Kwang Seok; Jeong, Soo-Jin; Jung, Ji Hoon; Kwon, Tae-Rin; Rhee, Yun-Hee; Kim, Sun-Hee; Kim, Si-Young; Yoon, Hwi-Joong; Zhu, Shudong; Chen, Chang-Yan; Kim, Sung-Hoon

    2011-06-01

    It has drawn a lot of attention to target signal transducer and activator of transcription 3 (STAT3) as a potential strategy for cancer therapeutics. Using several myelogenous cell lines, the effect of genipin (an active compound of Gardenia fruit) on the STAT3 pathway and apoptosis was investigated. Genipin suppressed the constitutive STAT3 activation in U266 and U937 cells and stimulated Src homology 2 domain-containing phosphatase 1 (SHP-1), which dephosphorylates and inactivates STAT3. Specifically, genipin blocked STAT3 activation via repressing the activation of c-Src, but not Janus kinase 1 (JAK1). Genipin also downregulated the expression of STAT3 target genes including Bcl-2, Bcl-x(L) , Survivin, Cyclin D1, and VEGF. Conversely, protein tyrosine phosphatase inhibitor pervanadate blocked genipin induced STAT3 inactivation. Using DNA fragmentation or TUNEL assays, we demonstrated the apoptotic effect of genipin on U266, MM.1S, and U937 cells. Furthermore, genipin effectively potentiated the cytotoxic effect of chemotherapeutic agents, such as bortezomib, thalidomide, and paclitaxel in U266 cells. Our data suggest that through regulation of Src and SHP-1, genipin antagonizes STAT3 for the induction of apoptosis in myeloma cells. PMID:21344490

  20. Global phosphotyrosine survey in triple-negative breast cancer reveals activation of multiple tyrosine kinase signaling pathways.

    PubMed

    Wu, Xinyan; Zahari, Muhammad Saddiq; Ma, Binyun; Liu, Ren; Renuse, Santosh; Sahasrabuddhe, Nandini A; Chen, Lily; Chaerkady, Raghothama; Kim, Min-Sik; Zhong, Jun; Jelinek, Christine; Barbhuiya, Mustafa A; Leal-Rojas, Pamela; Yang, Yi; Kashyap, Manoj Kumar; Marimuthu, Arivusudar; Ling, Min; Fackler, Mary Jo; Merino, Vanessa; Zhang, Zhen; Zahnow, Cynthia A; Gabrielson, Edward; Stearns, Vered; Roa, Juan Carlos; Sukumar, Saraswati; Gill, Parkash S; Pandey, Akhilesh

    2015-10-01

    Breast cancer is the most prevalent cancer in women worldwide. About 15-20% of all breast cancers are triple negative breast cancer (TNBC) and are often highly aggressive when compared to other subtypes of breast cancers. To better characterize the biology that underlies the TNBC phenotype, we profiled the phosphotyrosine proteome of a panel of twenty-six TNBC cell lines using quantitative high resolution Fourier transform mass spectrometry. A heterogeneous pattern of tyrosine kinase activation was observed based on 1,789 tyrosine-phosphorylated peptides identified from 969 proteins. One of the tyrosine kinases, AXL, was found to be activated in a majority of aggressive TNBC cell lines and was accompanied by a higher level of AXL expression. High levels of AXL expression are correlated with a significant decrease in patient survival. Treatment of cells bearing activated AXL with a humanized AXL antibody inhibited cell proliferation and migration in vitro, and tumor growth in mice. Overall, our global phosphoproteomic analysis provided new insights into the heterogeneity in the activation status of tyrosine kinase pathways in TNBCs. Our approach presents an effective means of identifying important novel biomarkers and targets for therapy such as AXL in TNBC. PMID:26356563

  1. Artesunate alleviates hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-?B signaling pathway in rats.

    PubMed

    Lai, Lina; Chen, Yunxia; Tian, Xiaoxia; Li, Xujiong; Zhang, Xiaojing; Lei, Jingwen; Bi, Yanghui; Fang, Buwu; Song, Xiaoliang

    2015-10-15

    The current study was performed in order to explore the effect of artesunate (Art) on experimental hepatic fibrosis and the potential mechanism involved. Art, a water-soluble hemisuccinate derivative of artemisinin extracted from the Chinese herb Artemisia Annua, is a safe and effective antimalarial drug. Hepatic fibrosis was induced in SD rats by multiple pathogenic factors. Rats were treated concurrently with Art (28.8mg/kg) given daily by oral gavage for 6 or 8 weeks to evaluate its protective effects. Our data demonstrated that Art treatment obviously attenuated hepatic fibrosis, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM). Art remarkably decreased endotoxin, tumor necrosis factor-? (TNF-?), and interleukin-6 (IL-6) levels as well. Art significantly downregulated protein and mRNA expression of ?-smooth muscle actin (?-SMA), toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88) and transforming growth factor beta 1 (TGF-?1). Art also significantly inhibited the nuclear transcription factor kappa B p65 (NF-?B p65) translocation into the nucleus. In addition, there were no remarkable differences between the N group and the NA group. In conclusion, we found that Art could alleviate hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-?B signaling pathway in rats, suggesting that Art may be a potential candidate for the therapy of hepatic fibrosis. PMID:26318197

  2. The Aids' Requirements of Children with Severe Multiple Handicaps and the People Looking after Them.

    ERIC Educational Resources Information Center

    Anden, Gerd

    The report presents findings from interviews with 10 families with children (4-19 years old) with severe mental retardation and multiple disabilities regarding the need for technical aids and adaptations in their homes. The following areas are addressed and examples of solutions proposed: hygienic aids (hot water adaptations, travel adaptations,…

  3. The Yeast Prion [SWI(+)] Abolishes Multicellular Growth by Triggering Conformational Changes of Multiple Regulators Required for Flocculin Gene Expression.

    PubMed

    Du, Zhiqiang; Zhang, Ying; Li, Liming

    2015-12-29

    Although transcription factors are prevalent among yeast prion proteins, the role of prion-mediated transcriptional regulation remains elusive. Here, we show that the yeast prion [SWI(+)] abolishes flocculin (FLO) gene expression and results in a complete loss of multicellularity. Further investigation demonstrates that besides Swi1, multiple other proteins essential for FLO expression, including Mss11, Sap30, and Msn1 also undergo conformational changes and become inactivated in [SWI(+)] cells. Moreover, the asparagine-rich region of Mss11 can exist as prion-like aggregates specifically in [SWI(+)] cells, which are SDS resistant, heritable, and curable, but become metastable after separation from [SWI(+)]. Our findings thus reveal a prion-mediated mechanism through which multiple regulators in a biological pathway can be inactivated. In combination with the partial loss-of-function phenotypes of [SWI(+)] cells on non-glucose sugar utilization, our data therefore demonstrate that a prion can influence distinct traits differently through multi-level regulations, providing insights into the biological roles of prions. PMID:26711350

  4. Differential effects of TR ligands on hormone dissociation rates: evidence for multiple ligand entry/exit pathways.

    PubMed

    Cunha Lima, Suzana T; Nguyen, Ngoc-Ha; Togashi, Marie; Apriletti, James W; Nguyen, Phuong; Polikarpov, Igor; Scanlan, Thomas S; Baxter, John D; Webb, Paul

    2009-11-01

    Some nuclear receptor (NR) ligands promote dissociation of radiolabeled bound hormone from the buried ligand binding cavity (LBC) more rapidly than excess unlabeled hormone itself. This result was interpreted to mean that challenger ligands bind allosteric sites on the LBD to induce hormone dissociation, and recent findings indicate that ligands bind weakly to multiple sites on the LBD surface. Here, we show that a large fraction of thyroid hormone receptor (TR) ligands promote rapid dissociation (T(1/2)<2h) of radiolabeled T(3) vs. T(3) (T(1/2) approximately 5-7h). We cannot discern relationships between this effect and ligand size, activity or affinity for TRbeta. One ligand, GC-24, binds the TR LBC and (weakly) to the TRbeta-LBD surface that mediates dimer/heterodimer interaction, but we cannot link this interaction to rapid T(3) dissociation. Instead, several lines of evidence suggest that the challenger ligand must interact with the buried LBC to promote rapid T(3) release. Since previous molecular dynamics simulations suggest that TR ligands leave the LBC by several routes, we propose that a subset of challenger ligands binds and stabilizes a partially unfolded intermediate state of TR that arises during T(3) release and that this effect enhances hormone dissociation. PMID:19729063

  5. Sexual behavior, risk perception, and HIV transmission can respond to HIV antiviral drugs and vaccines through multiple pathways.

    PubMed

    Tully, Stephen; Cojocaru, Monica; Bauch, Chris T

    2015-01-01

    There has been growing use of highly active antiretroviral treatment (HAART) for HIV and significant progress in developing prophylactic HIV vaccines. The simplest theories of counterproductive behavioral responses to such interventions tend to focus on single feedback mechanisms: for instance, HAART optimism makes infection less scary and thus promotes risky sexual behavior. Here, we develop an agent based, age-structured model of HIV transmission, risk perception, and partner selection in a core group to explore behavioral responses to interventions. We find that interventions can activate not one, but several feedback mechanisms that could potentially influence decision-making and HIV prevalence. In the model, HAART increases the attractiveness of unprotected sex, but it also increases perceived risk of infection and, on longer timescales, causes demographic impacts that partially counteract HAART optimism. Both HAART and vaccination usually lead to lower rates of unprotected sex on the whole, but intervention effectiveness depends strongly on whether individuals over- or under-estimate intervention coverage. Age-specific effects cause sexual behavior and HIV prevalence to change in opposite ways in old and young age groups. For complex infections like HIV-where interventions influence transmission, demography, sexual behavior and risk perception-we conclude that evaluations of behavioral responses should consider multiple feedback mechanisms. PMID:26507957

  6. Defining Human Pathways of Drug Metabolism In Vivo through the Development of a Multiple Humanized Mouse Model.

    PubMed

    Scheer, Nico; Kapelyukh, Yury; Rode, Anja; Oswald, Stefan; Busch, Diana; McLaughlin, Lesley A; Lin, De; Henderson, Colin J; Wolf, C Roland

    2015-11-01

    Variability in drug pharmacokinetics is a major factor in defining drug efficacy and side effects. There remains an urgent need, particularly with the growing use of polypharmacy, to obtain more informative experimental data predicting clinical outcomes. Major species differences in multiplicity, substrate specificity, and regulation of enzymes from the cytochrome P450-dependent mono-oxygenase system play a critical role in drug metabolism. To develop an in vivo model for predicting human responses to drugs, we generated a mouse, where 31 P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene families were exchanged for their relevant human counterparts. The model has been improved through additional humanization for the nuclear receptors constitutive androgen receptor and pregnane X receptor that control the expression of key drug metabolizing enzymes and transporters. In this most complex humanized mouse model reported to date, the cytochromes P450 function as predicted and we illustrate how these mice can be applied to predict drug-drug interactions in humans. PMID:26265742

  7. Sexual behavior, risk perception, and HIV transmission can respond to HIV antiviral drugs and vaccines through multiple pathways

    PubMed Central

    Tully, Stephen; Cojocaru, Monica; Bauch, Chris T.

    2015-01-01

    There has been growing use of highly active antiretroviral treatment (HAART) for HIV and significant progress in developing prophylactic HIV vaccines. The simplest theories of counterproductive behavioral responses to such interventions tend to focus on single feedback mechanisms: for instance, HAART optimism makes infection less scary and thus promotes risky sexual behavior. Here, we develop an agent based, age-structured model of HIV transmission, risk perception, and partner selection in a core group to explore behavioral responses to interventions. We find that interventions can activate not one, but several feedback mechanisms that could potentially influence decision-making and HIV prevalence. In the model, HAART increases the attractiveness of unprotected sex, but it also increases perceived risk of infection and, on longer timescales, causes demographic impacts that partially counteract HAART optimism. Both HAART and vaccination usually lead to lower rates of unprotected sex on the whole, but intervention effectiveness depends strongly on whether individuals over- or under-estimate intervention coverage. Age-specific effects cause sexual behavior and HIV prevalence to change in opposite ways in old and young age groups. For complex infections like HIV—where interventions influence transmission, demography, sexual behavior and risk perception—we conclude that evaluations of behavioral responses should consider multiple feedback mechanisms. PMID:26507957

  8. 78 FR 13781 - Filings Required of Multiple Employer Welfare Arrangements and Certain Other Related Entities

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-01

    ...reduce MEWA fraud and abuse, protecting consumers from unpaid medical...requiring MEWAs that are not group health plans (non-plan MEWAs...requirements to MEWAs which are group health plans (plan MEWAs...are within the same control group. The term ``control...

  9. Multiple Wnt genes are required for segmentation in the short-germ embryo of Tribolium castaneum.

    PubMed

    Bolognesi, Renata; Farzana, Laila; Fischer, Tamara D; Brown, Susan J

    2008-10-28

    wingless (wg)/Wnt family are essential to development in virtually all metazoans. In short-germ insects, including the red flour beetle (Tribolium castaneum), the segment-polarity function of wg is conserved [1]. Wnt signaling is also implicated in posterior patterning and germband elongation [2-4], but despite its expression in the posterior growth zone, Wnt1/wg alone is not responsible for these functions [1-3]. Tribolium contains additional Wnt family genes that are also expressed in the growth zone [5]. After depleting Tc-WntD/8 we found a small percentage of embryos lacking abdominal segments. Additional removal of Tc-Wnt1 significantly enhanced the penetrance of this phenotype. Seeking alternative methods to deplete Wnt signal, we performed RNAi with other components of the Wnt pathway including wntless (wls), porcupine (porc), and pangolin (pan). Tc-wls RNAi caused segmentation defects similar to Tc-Wnt1 RNAi, but not Tc-WntD/8 RNAi, indicating that Tc-WntD/8 function is Tc-wls independent. Depletion of Tc-porc and Tc-pan produced embryos resembling double Tc-Wnt1,Tc-WntD/8 RNAi embryos, suggesting that Tc-porc is essential for the function of both ligands, which signal through the canonical pathway. This is the first evidence of functional redundancy between Wnt ligands in posterior patterning in short-germ insects. This Wnt function appears to be conserved in other arthropods [6] and vertebrates [7-9]. PMID:18926702

  10. MULTIPLE PROTEIN KINASE PATHWAYS MEDIATE AMPLIFIED IL-6 RELEASE BY HUMAN LUNG FIBROBLASTS CO-EXPOSED TO NICKEL AND TLR-2 AGONIST, MALP-2

    PubMed Central

    Gao, Fei; Brant, Kelly A.; Ward, Rachel M.; Cattley, Richard T.; Barchowsky, Aaron; Fabisiak, James P.

    2010-01-01

    Microbial stimuli and atmospheric particulate matter (PM) interact to amplify the release of inflammatory and immune-modulating cytokines. The basis of this interaction, however, is not known. Cultured human lung fibroblasts (HLF) were used to determine whether various protein kinase pathways were involved in the release of IL-6 following combined exposure to the PM-derived metal, Ni, and M. fermentans-derived macrophage-activating lipopeptide 2 (MALP-2), a toll-like receptor 2 agonist. Synergistic release of IL-6 by MALP-2 and NiSO4 was obvious after 8 h of co-stimulation and correlated with a late phase accumulation of IL-6 mRNA. Ni and MALP-2, alone or together, all lead to rapid and transient phosphorylations of ERK1/2 and JNK/SAPK of similar magnitude. p38 phosphorylation, however, was observed only after prolonged treatment of cells with both stimuli together. A constitutive level of PI3K-dependent Akt phosphorylation remained unchanged by Ni and/or MALP-2 exposure. IL-6 induced by Ni/MALP-2 co-exposure was partially dependent on activity of HIF-1? and COX-2 as shown by targeted knockdown using siRNA. IL-6 release in response to Ni/MALP-2 was partially sensitive to pharmacological inhibition of ERK1/2, p38, and PI3K signaling. The protein kinase inhibitors had minimal or no effects on Ni/MALP-2-induced accumulation of HIF-1? protein, however, COX-2 expression and, more markedly PGE2 production, were suppressed by LY294002, SB203580, and U0126. Thus, Ni/MALP-2 interactions involve multiple protein kinase pathways (ERK1/2, p38, and PI3K) that modulate events downstream from the early accumulation of HIF-1? to promote IL-6 gene expression directly or secondarily, through COX-2-derived autocrine products like PGE2. PMID:20600219

  11. Inhibition of Multiple Protective Signaling Pathways and Ad.5/3 Delivery Enhances mda-7/IL-24 Therapy of Malignant Glioma

    PubMed Central

    Hamed, Hossein A; Yacoub, Adly; Park, Margaret A; Eulitt, Patrick J; Dash, Rupesh; Sarkar, Devanand; Dmitriev, Igor P; Lesniak, Maciej S; Shah, Khalid; Grant, Steven; Curiel, David T; Fisher, Paul B; Dent, Paul

    2010-01-01

    We have explored the mechanism by which inhibition of multiple cytoprotective cell-signaling pathways enhance melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) toxicity toward invasive primary human glioblastoma multiforme (GBM) cells, and whether improving adenoviral infectivity/delivery of mda-7/IL-24 enhances therapeutic outcome in animals containing orthotopic xenografted GBM cells. The toxicity of a serotype 5 recombinant adenovirus to express MDA-7/IL-24 (Ad.5-mda-7) was enhanced by combined molecular or small molecule inhibition of mitogen-activated extracellular regulated kinase (MEK)1/2 and phosphatidyl inositol 3-kinase (PI3K) or AKT; inhibition of mammalian target of rapamycin (mTOR) and MEK1/2; and the HSP90 inhibitor 17AAG. Molecular inhibition of mTOR/PI3K/MEK1 signaling in vivo also enhanced Ad.5-mda-7 toxicity. In GBM cells of diverse genetic backgrounds, inhibition of cytoprotective cell-signaling pathways enhanced MDA-7/IL-24–induced autophagy, mitochondrial dysfunction and tumor cell death. Due partly to insufficient adenovirus serotype 5 gene delivery this therapeutic approach has shown limited success in GBM. To address this problem, we employed a recombinant adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7. Ad.5/3-mda-7 more effectively infected and killed GBM cells in vitro and in vivo than Ad.5-mda-7. Future combinations of these approaches hold promise for developing an effective therapy for GBM. PMID:20179672

  12. Panobinostat synergizes with zoledronic acid in prostate cancer and multiple myeloma models by increasing ROS and modulating mevalonate and p38-MAPK pathways.

    PubMed

    Bruzzese, F; Pucci, B; Milone, M R; Ciardiello, C; Franco, R; Chianese, M I; Rocco, M; Di Gennaro, E; Leone, A; Luciano, A; Arra, C; Santini, D; Caraglia, M; Budillon, A

    2013-01-01

    Patients with advanced prostate cancer (PCa) and multiple myeloma (MM) have limited long-term responses to available therapies. The histone deacetylase inhibitor panobinostat has shown significant preclinical and clinical anticancer activity in both hematological and solid malignancies and is currently in phase III trials for relapsed MM. Bisphosphonates (BPs), such as zoledronic acid (ZOL), inhibit osteoclast-mediated bone resorption and are indicated for the treatment of bone metastasis. BPs, including ZOL, have also shown anticancer activity in several preclinical and clinical studies. In the present report, we found a potent synergistic antiproliferative effect of panobinostat/ZOL treatment in three PCa and three MM cell lines as well as in a PCa ZOL-resistant subline, independently of p53/KRAS status, androgen dependency, or the schedule of administration. The synergistic effect was also observed in an anchorage-independent agar assay in both ZOL-sensitive and ZOL-resistant cells and was confirmed in vivo in a PCa xenograft model. The co-administration of the antioxidant N-acetyl-L-cysteine blocked the increased reactive oxygen species generation and apoptosis observed in the combination setting compared with control or single-agent treatments, suggesting that oxidative injury plays a functional role in the synergism. Proapoptotic synergy was also partially antagonized by the addition of geranyl-geraniol, which bypasses the inhibition of farnesylpyrophosphate synthase by ZOL in the mevalonate pathway, supporting the involvement of this pathway in the synergy. Finally, at the molecular level, the inhibition of basal and ZOL-induced activation of p38-MAPK by panobinostat in sensitive and ZOL-resistant cells and in tumor xenografts could explain, at least in part, the observed synergism. PMID:24157872

  13. Requirements and potential development pathways for fission energy supply infrastructures of the 21st century - a systems viewpoint.

    SciTech Connect

    Wade, D. C.

    1999-06-14

    Using an energy supply systems approach, we envision attributes and characteristic needs of a future global fission-based energy supply infrastructure, enumerate potential pathways for meeting those needs, and identify the underlying enabling science and technology developments for R and D efforts to meet the needs.

  14. 77 FR 12927 - Federal Acquisition Regulation: Requirements for Acquisitions Pursuant to Multiple-Award Contracts

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-02

    ...establishing BPAs and allowing flexibility in establishing BPA ordering procedures; (6) BPA requirements and health-care programs; (7) Competition...statement of work is being evaluated when placed under a BPA with hourly rate services. This language is...

  15. Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages.

    PubMed Central

    Marra, A; Blander, S J; Horwitz, M A; Shuman, H A

    1992-01-01

    The legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular parasite. Its interaction with phagocytes has characteristics in common with several other intracellular parasites. Critical aspects of L. pneumophila intracellular multiplication are evasion of lysosomal host cell defenses and the presence of a nutritionally appropriate environment. Following phagocytosis, wild-type L. pneumophila multiply within a specialized phagosome which does not fuse with secondary lysosomes. Mutants which have lost the ability to grow within phagocytes no longer cause disease in animals, indicating that the capacity to multiply intracellularly is important for pathogenesis. One such mutant, 25D, has been shown to be defective in inhibiting phagosome-lysosome fusion. This phagolysosomal environment is not conducive to Legionella growth. We report the isolation of a region of the L. pneumophila genome (icm, intracellular multiplication) which restores the capacity of 25D to multiply in human macrophages. The complemented mutants also regain the capacity to interfere with phagosome-lysosome fusion and to cause lethal pneumonia in guinea pigs. Images PMID:1409673

  16. Multiple PLDs required for high salinity and water deficit tolerance in plants.

    PubMed

    Bargmann, Bastiaan O R; Laxalt, Ana M; ter Riet, Bas; van Schooten, Bas; Merquiol, Emmanuelle; Testerink, Christa; Haring, Michel A; Bartels, Dorothea; Munnik, Teun

    2009-01-01

    High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plant's response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plant's response to high salinity and dehydration. A tomato (Lycopersicon esculentum) alpha-class PLD, LePLDalpha1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDalpha1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDalpha1 and AtPLDdelta were found to be activated in response to salt stress. Moreover, pldalpha1 and plddelta single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth. PMID:19017627

  17. Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis

    PubMed Central

    Li, Shijie; Brown, Michael S.; Goldstein, Joseph L.

    2010-01-01

    The livers of insulin-resistant, diabetic mice manifest selective insulin resistance, suggesting a bifurcation in the insulin signaling pathway: Insulin loses its ability to block glucose production (i.e., it fails to suppress PEPCK and other genes of gluconeogenesis), yet it retains its ability to stimulate fatty acid synthesis (i.e., continued enhancement of genes of lipogenesis). Enhanced lipogenesis is accompanied by an insulin-stimulated increase in the mRNA encoding SREBP-1c, a transcription factor that activates the entire lipogenic program. Here, we report a branch point in the insulin signaling pathway that may account for selective insulin resistance. Exposure of rat hepatocytes to insulin produced a 25-fold increase in SREBP-1c mRNA and a 95% decrease in PEPCK mRNA. Insulin-mediated changes in both mRNAs were blocked by inhibitors of PI3K and Akt, indicating that these kinases are required for both pathways. In contrast, subnanomolar concentrations of rapamycin, an inhibitor of the mTORC1 kinase, blocked insulin induction of SREBP-1c, but had no effect on insulin suppression of PEPCK. We observed a similar selective effect of rapamycin in livers of rats and mice that experienced an insulin surge in response to a fasting-refeeding protocol. A specific inhibitor of S6 kinase, a downstream target of mTORC1, did not block insulin induction of SREBP-1c, suggesting a downstream pathway distinct from S6 kinase. These results establish mTORC1 as an essential component in the insulin-regulated pathway for hepatic lipogenesis but not gluconeogenesis, and may help to resolve the paradox of selective insulin resistance in livers of diabetic rodents. PMID:20133650

  18. 78 FR 13781 - Filings Required of Multiple Employer Welfare Arrangements and Certain Other Related Entities

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-01

    ... compliance. \\4\\ 65 FR 7152 (02/11/2000) and 68 FR 17494 (04/09/2003). The Form M-1 is reissued each year in... (76 FR 76222) implementing the new reporting requirements for MEWAs and ECEs. The Department received... medical care (within the meaning of section 733(a)(2) of ERISA and 29 CFR 2590.701-2) for employees of...

  19. 76 FR 14548 - Federal Acquisition Regulation; Requirements for Acquisitions Pursuant to Multiple-Award Contracts

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-16

    ... documented in the acquisition plan and contract file, (ii) the estimated value of the BPA does not exceed... the ordering activity to (i) provide a Request for Quotation (RFQ) to all BPA holders offering the required supplies or services under the BPA for orders over the SAT that includes a description of...

  20. FCAT Retakes: Trends in Multiple Attempts at Satisfying FCAT Graduation Requirements. Research Brief. Volume 0805

    ERIC Educational Resources Information Center

    Froman, Terry; Brown, Shelly

    2009-01-01

    According to Florida Law, students must pass the Grade 10 FCAT, among other academic requirements, in order to receive a standard high school diploma. Specifically, students must achieve a "passing" score of 300 or above on both the FCAT SSS Reading and the FCAT SSS Mathematics tests. Technically, students can retake the FCAT as many times as they…

  1. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... comply with the anti-fraud provisions of 24 CFR 1710.4 (b) and (c) and the sales practices and standards in 24 CFR 1715.10 through 1715.28. (11) A written Lot Information Statement must be delivered to, and... all of the requirements of 24 CFR 1710.15. I further affirm that the statements contained in...

  2. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... comply with the anti-fraud provisions of 24 CFR 1710.4 (b) and (c) and the sales practices and standards in 24 CFR 1715.10 through 1715.28. (11) A written Lot Information Statement must be delivered to, and... all of the requirements of 24 CFR 1710.15. I further affirm that the statements contained in...

  3. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... comply with the anti-fraud provisions of 24 CFR 1710.4 (b) and (c) and the sales practices and standards in 24 CFR 1715.10 through 1715.28. (11) A written Lot Information Statement must be delivered to, and... all of the requirements of 24 CFR 1710.15. I further affirm that the statements contained in...

  4. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... comply with the anti-fraud provisions of 24 CFR 1710.4 (b) and (c) and the sales practices and standards in 24 CFR 1715.10 through 1715.28. (11) A written Lot Information Statement must be delivered to, and... all of the requirements of 24 CFR 1710.15. I further affirm that the statements contained in...

  5. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... comply with the anti-fraud provisions of 24 CFR 1710.4 (b) and (c) and the sales practices and standards in 24 CFR 1715.10 through 1715.28. (11) A written Lot Information Statement must be delivered to, and... all of the requirements of 24 CFR 1710.15. I further affirm that the statements contained in...

  6. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...other requirements for water, sewer and electrical...sewage system and/or water system, you should...disposal system and/or well and whether there is an adequate supply of water. You should also become... Street Address: City: State:...

  7. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...other requirements for water, sewer and electrical...sewage system and/or water system, you should...disposal system and/or well and whether there is an adequate supply of water. You should also become... Street Address: City: State:...

  8. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...other requirements for water, sewer and electrical...sewage system and/or water system, you should...disposal system and/or well and whether there is an adequate supply of water. You should also become... Street Address: City: State:...

  9. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...other requirements for water, sewer and electrical...sewage system and/or water system, you should...disposal system and/or well and whether there is an adequate supply of water. You should also become... Street Address: City: State:...

  10. 24 CFR 1710.15 - Regulatory exemption-multiple site subdivision-determination required.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...other requirements for water, sewer and electrical...sewage system and/or water system, you should...disposal system and/or well and whether there is an adequate supply of water. You should also become... Street Address: City: State:...

  11. Targeting NAD+ salvage pathway induces autophagy in multiple myeloma cells via mTORC1 and extracellular signal-regulated kinase (ERK1/2) inhibition.

    PubMed

    Cea, Michele; Cagnetta, Antonia; Fulciniti, Mariateresa; Tai, Yu-Tzu; Hideshima, Teru; Chauhan, Dharminder; Roccaro, Aldo; Sacco, Antonio; Calimeri, Teresa; Cottini, Francesca; Jakubikova, Jana; Kong, Sun-Young; Patrone, Franco; Nencioni, Alessio; Gobbi, Marco; Richardson, Paul; Munshi, Nikhil; Anderson, Kenneth C

    2012-10-25

    Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach. PMID:22955917

  12. Biochemical pathways in seed oil synthesis.

    PubMed

    Bates, Philip D; Stymne, Sten; Ohlrogge, John

    2013-06-01

    Oil produced in plant seeds is utilized as a major source of calories for human nutrition, as feedstocks for non-food uses such as soaps and polymers, and can serve as a high-energy biofuel. The biochemical pathways leading to oil (triacylglycerol) synthesis in seeds involve multiple subcellular organelles, requiring extensive lipid trafficking. Phosphatidylcholine plays a central role in these pathways as a substrate for acyl modifications and likely as a carrier for the trafficking of acyl groups between organelles and membrane subdomains. Although much has been clarified regarding the enzymes and pathways responsible for acyl-group flux, there are still major gaps in our understanding. These include the identity of several key enzymes, how flux between alternative pathways is controlled and the specialized cell biology leading to biogenesis of oil bodies that store up to 80% of carbon in seeds. PMID:23529069

  13. JC Virus Multiplication in Human Hematopoietic Progenitor Cells Requires the NF-1 Class D Transcription Factor

    PubMed Central

    Monaco, Maria Chiara G.; Sabath, Bruce F.; Durham, Linda C.; Major, Eugene O.

    2001-01-01

    JCV, a small DNA virus of the polyomavirus family, has been shown to infect glial cells of the central nervous system, hematopoietic progenitor cells, and immune system lymphocytes. A family of DNA binding proteins called nuclear factor-1 (NF-1) has been linked with site-coding specific transcription of cellular and viral genes and replication of some viruses, including JC virus (JCV). It is unclear which NF-1 gene product must be expressed by cells to promote JCV multiplication. Previously, it was shown that elevated levels of NF-1 class D mRNA were expressed by human brain cells that are highly susceptible to JCV infection but not by JCV nonpermissive HeLa cells. Recently, we reported that CD34+ precursor cells of the KG-1 line, when treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), differentiated to cells with macrophage-like characteristics and lost susceptibility to JCV infection. These studies have now been extended by asking whether loss of JCV susceptibility by PMA-treated KG-1 cells is linked with alterations in levels of NF-1 class D expression. Using reverse transcription-PCR, we have found that PMA-treated KG-1 cells express mRNA that codes for all four classes of NF-1 proteins, although different levels of RNA expression were observed in the hematopoietic cells differentiated into macrophages. Northern hybridization confirms that the expression of NF-1 class D gene is lower in JCV nonpermissive PMA-treated KG-1 cells compared with non-PMA-treated cells. Further, using gel mobility shift assays, we were able to show the induction of specific NF-1–DNA complexes in KG-1 cells undergoing PMA treatment. The binding increases in direct relation to the duration of PMA treatment. These results suggest that the binding pattern of NF-1 class members may change in hematopoietic precursor cells, such as KG-1, as they undergo differentiation to macrophage-like cells. Transfection of PMA-treated KG-1 cells with an NF-1 class D expression vector restored the susceptibility of these cells to JCV infection, while the transfection of PMA-treated KG-1 cells with NF-1 class A, B, and C vectors was not able to restore JCV susceptibility. These data collectively suggest that selective expression of NF-1 class D has a regulatory role in JCV multiplication. PMID:11559801

  14. Requirement for safety monitoring for approved multiple sclerosis therapies: an overview

    PubMed Central

    Rommer, P S; Zettl, U K; Kieseier, B; Hartung, H-P; Menge, T; Frohman, E; Greenberg, B M; Hemmer, B; Stüve, O

    2014-01-01

    During the last two decades, treatment options for patients with multiple sclerosis (MS) have broadened tremendously. All agents that are currently approved for clinical use have potential side effects, and a careful risk–benefit evaluation is part of a decision algorithm to identify the optimal treatment choice for an individual patient. Whereas glatiramer acetate and interferon beta preparations have been used in MS for decades and have a proven safety record, more recently approved drugs appear to be more effective, but potential risks might be more severe. The potential complications of some novel therapies might not even have been identified to their full extent. This review is aimed at the clinical neurologist in that it offers insights into potential adverse events of each of the approved MS therapeutics: interferon beta, glatiramer acetate, mitoxantrone, natalizumab, fingolimod and teriflunomide, as well as recently approved therapeutics such as dimethyl fumarate and alemtuzumab. It also provides recommendations for monitoring the different drugs during therapy in order to avoid common side effects. PMID:24102425

  15. Recombinant factor VIIa enhances platelet deposition from flowing haemophilic blood but requires the contact pathway to promote fibrin deposition.

    PubMed

    Li, R; Panckeri, K A; Fogarty, P F; Diamond, S L

    2015-03-01

    In prior microfluidic studies with haemophilic blood perfused over collagen, we found that a severe deficiency (<1% factor level) reduced platelet and fibrin deposition, while a moderate deficiency (1-5%) only reduced fibrin deposition. We investigated: (i) the differential effect of rFVIIa (0.04-20 nm) on platelet and fibrin deposition, and (ii) the contribution of the contact pathway to rFVIIa-induced haemophilic blood clotting. Haemophilic or healthy blood with low and high corn trypsin inhibitor (CTI, 4 or 40 ?g mL(-1) ) was perfused over collagen at an initial venous wall shear rate of 100 s(-1) . At 100 s(-1) wall shear rate, where FXIIa leads to thrombin production without added tissue factor, FXI-deficient blood (3%) or severely FVIII-deficient blood (<1%) produced no fibrin at either CTI level. Whereas rFVIIa potently enhanced platelet deposition, fibrin generation was not rescued. Distinct from the high CTI condition, engagement of the contact pathway (low CTI) in moderately FVIII-deficient (3%) or moderately FIX-deficient blood (5%) resulted in enhanced platelet and fibrin deposition following 4 nm rFVIIa supplementation. In mildly FVIII-deficient blood (15%) at <24 h since haemostatic therapy, rFVIIa enhanced both platelet and fibrin generation in either CTI condition although fibrin was produced more quickly and abundantly in low CTI. For tissue factor-free conditions of severe haemophilic blood clotting, we conclude that rFVIIa reliably generates low levels of 'signaling' thrombin sufficient to enhance platelet deposition on collagen, but is insufficient to drive fibrin polymerization unless potentiated by the contact pathway. PMID:25311576

  16. Multiple protein kinase pathways mediate amplified IL-6 release by human lung fibroblasts co-exposed to nickel and TLR-2 agonist, MALP-2

    SciTech Connect

    Gao Fei; Brant, Kelly A.; Ward, Rachel M.; Cattley, Richard T.; Barchowsky, Aaron; Fabisiak, James P.

    2010-09-01

    Microbial stimuli and atmospheric particulate matter (PM) interact to amplify the release of inflammatory and immune-modulating cytokines. The basis of this interaction, however, is not known. Cultured human lung fibroblasts (HLF) were used to determine whether various protein kinase pathways were involved in the release of IL-6 following combined exposure to the PM-derived metal, Ni, and M. fermentans-derived macrophage-activating lipopeptide 2 (MALP-2), a toll-like receptor 2 agonist. Synergistic release of IL-6 by MALP-2 and NiSO{sub 4} was obvious after 8 h of co-stimulation and correlated with a late phase accumulation of IL-6 mRNA. Ni and MALP-2, alone or together, all led to rapid and transient phosphorylations of ERK{sub 1/2} and JNK/SAPK of similar magnitude. p38 phosphorylation, however, was observed only after prolonged treatment of cells with both stimuli together. A constitutive level of PI3K-dependent Akt phosphorylation remained unchanged by Ni and/or MALP-2 exposure. IL-6 induced by Ni/MALP-2 co-exposure was partially dependent on activity of HIF-1{alpha} and COX-2 as shown by targeted knockdown using siRNA. IL-6 release in response to Ni/MALP-2 was partially sensitive to pharmacological inhibition of ERK{sub 1/2}, p38, and PI3K signaling. The protein kinase inhibitors had minimal or no effects on Ni/MALP-2-induced accumulation of HIF-1{alpha} protein, however, COX-2 expression and, more markedly PGE{sub 2} production, were suppressed by LY294002, SB203580, and U0126. Thus, Ni/MALP-2 interactions involve multiple protein kinase pathways (ERK{sub 1/2}, p38, and PI3K) that modulate events downstream from the early accumulation of HIF-1{alpha} to promote IL-6 gene expression directly or secondarily, through COX-2-derived autocrine products like PGE{sub 2}.

  17. A Region of the Nucleosome Required for Multiple Types of Transcriptional Silencing in Saccharomyces cerevisiae

    PubMed Central

    Prescott, Eugenia T.; Safi, Alexias; Rusche, Laura N.

    2011-01-01

    Extended heterochromatin domains, which are repressive to transcription and help define centromeres and telomeres, are formed through specific interactions between silencing proteins and nucleosomes. This study reveals that in Saccharomyces cerevisiae, the same nucleosomal surface is critical for the formation of multiple types of heterochromatin, but not for local repression mediated by a related transcriptional repressor. Thus, this region of the nucleosome may be generally important to long-range silencing. In S. cerevisiae, the Sir proteins perform long-range silencing, whereas the Sum1 complex acts locally to repress specific genes. A mutant form of Sum1p, Sum1-1p, achieves silencing in the absence of Sir proteins. A genetic screen identified mutations in histones H3 and H4 that disrupt Sum1-1 silencing and fall in regions of the nucleosome previously known to disrupt Sir silencing and rDNA silencing. In contrast, no mutations were identified that disrupt wild-type Sum1 repression. Mutations that disrupt silencing fall in two regions of the nucleosome, the tip of the H3 tail and a surface of the nucleosomal core (LRS domain) and the adjacent base of the H4 tail. The LRS/H4 tail region interacts with the Sir3p bromo-adjacent homology (BAH) domain to facilitate Sir silencing. By analogy, this study is consistent with the LRS/H4 tail region interacting with Orc1p, a paralog of Sir3p, to facilitate Sum1-1 silencing. Thus, the LRS/H4 tail region of the nucleosome may be relatively accessible and facilitate interactions between silencing proteins and nucleosomes to stabilize long-range silencing. PMID:21546544

  18. FvBck1, a component of cell wall integrity MAP kinase pathway, is required for virulence and oxidative stress response in sugarcane Pokkah Boeng pathogen

    PubMed Central

    Zhang, Chengkang; Wang, Jianqiang; Tao, Hong; Dang, Xie; Wang, Yang; Chen, Miaoping; Zhai, Zhenzhen; Yu, Wenying; Xu, Liping; Shim, Won-Bo; Lu, Guodong; Wang, Zonghua

    2015-01-01

    Fusarium verticillioides (formerly F. moniliforme) is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a possibly carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane. PMID:26500635

  19. Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

    SciTech Connect

    Belakavadi, Madesh . E-mail: belakama@umdnj.edu; Prabhakar, B.T.; Salimath, Bharathi P.

    2005-10-07

    Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.

  20. Chemopreventive efficacy of hesperidin against chemically induced nephrotoxicity and renal carcinogenesis via amelioration of oxidative stress and modulation of multiple molecular pathways.

    PubMed

    Siddiqi, Aisha; Hasan, Syed Kazim; Nafees, Sana; Rashid, Summya; Saidullah, Bano; Sultana, Sarwat

    2015-12-01

    In the present study, chemopreventive efficacy of hesperidin was evaluated against ferric nitrilotriacetate (Fe-NTA) induced renal oxidative stress and carcinogenesis in wistar rats. Nephrotoxicity was induced by single intraperitoneal injection of Fe-NTA (9mg Fe/kg b.wt). Renal cancer was initiated by the administration of N-nitrosodiethylamine (DEN 200mg/kg b.wt ip) and promoted by Fe-NTA (9mg Fe/kg b.wt ip) twice weekly for 16weeks. Efficacy of hesperidin against Fe-NTA-induced nephrotoxicity was assessed in terms of biochemical estimation of antioxidant enzyme activities viz. reduced renal GSH, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, catalase, superoxide dismutase and renal toxicity markers (BUN, Creatinine, KIM-1). Administration of Fe-NTA significantly depleted antioxidant renal armory, enhanced renal lipid peroxidation as well as the levels of BUN, creatinine and KIM-1. However, simultaneous pretreatment of hesperidin restored their levels in a dose dependent manner. Expression of apoptotic markers caspase-3, caspase-9, bax, bcl-2 and proliferative marker PCNA along with inflammatory markers (NF?B, iNOS, TNF-?) were also analysed to assess the chemopreventive potential of hesperidin in two-stage renal carcinogenesis model. Hesperidin was found to induce caspase-3, caspase-9, bax expression and downregulate bcl-2, NF?B, iNOS, TNF-?, PCNA expression. Histopathological findings further revealed hesperidin's chemopreventive efficacy by restoring the renal morphology. Our results provide a powerful evidence suggesting hesperidin to be a potent chemopreventive agent against renal carcinogenesis possibly by virtue of its antioxidant properties and by modulation of multiple molecular pathways. PMID:26551080

  1. Multiple lesions in receptor tyrosine kinase pathway determine glioblastoma response to pan-ERBB inhibitor PF-00299804 and PI3K/mTOR dual inhibitor PF-05212384

    PubMed Central

    Zhu, Yanni; Shah, Khalid

    2014-01-01

    A novel pan ERBB inhibitor PF-00299804 (dacomitinib) is currently in phase II clinical trials in glioblastoma multiforme (GBM) patients; however its pre-clinical efficacy in GBMs has not been tested. In this study, we evaluated the efficacy of dacomitinib alone or in combination with PI3K/mTOR dual inhibitor PF-05212384 in GBM and assessed the mechanisms of resistance and the molecular determinants of response. A panel of established and patient derived primary GBM lines that present different molecular profiles and also the GBM lines engineered to express EGFRvIII mutant or PTEN were treated with either dacomitinib, PF-05212384, or combination and assessed for their viability and changes in EGFR/PI3K/mTOR signaling. We show that dacomitinib significantly reduced phosphorylated EGFR in all the GBM lines but did not show a dose-dependent response on cell viability in a majority of the lines tested. Multiple lesions in the receptor tyrosine kinases (RTKs) pathway including PTEN mutation, co-activation of RTKs, and EGFRvIII mutation resulted in unaltered active status of PI3K/mTOR in the GBM lines even in the presence of EGFR inhibition. Blocking PI3K/mTOR dramatically inhibited cell proliferation in most GBM lines and enhanced dacomitinib induction of apoptosis in a GBM line that has both EGFR amplification and EGFR-independent PI3K activation. These data suggest molecular profiling of EGFR/PI3K/PTEN status to select GBM patients for EGFR or/and PI3K/mTOR targeted therapies. PMID:24658109

  2. Multiple lesions in receptor tyrosine kinase pathway determine glioblastoma response to pan-ERBB inhibitor PF-00299804 and PI3K/mTOR dual inhibitor PF-05212384.

    PubMed

    Zhu, Yanni; Shah, Khalid

    2014-06-01

    A novel pan ERBB inhibitor PF-00299804 (dacomitinib) is currently in phase II clinical trials in glioblastoma multiforme (GBM) patients; however its pre-clinical efficacy in GBMs has not been tested. In this study, we evaluated the efficacy of dacomitinib alone or in combination with PI3K/mTOR dual inhibitor PF-05212384 in GBM and assessed the mechanisms of resistance and the molecular determinants of response. A panel of established and patient derived primary GBM lines that present different molecular profiles and also the GBM lines engineered to express EGFRvIII mutant or PTEN were treated with either dacomitinib, PF-05212384, or combination and assessed for their viability and changes in EGFR/PI3K/mTOR signaling. We show that dacomitinib significantly reduced phosphorylated EGFR in all the GBM lines but did not show a dose-dependent response on cell viability in a majority of the lines tested. Multiple lesions in the receptor tyrosine kinases (RTKs) pathway including PTEN mutation, co-activation of RTKs, and EGFRvIII mutation resulted in unaltered active status of PI3K/mTOR in the GBM lines even in the presence of EGFR inhibition. Blocking PI3K/mTOR dramatically inhibited cell proliferation in most GBM lines and enhanced dacomitinib induction of apoptosis in a GBM line that has both EGFR amplification and EGFR-independent PI3K activation. These data suggest molecular profiling of EGFR/PI3K/PTEN status to select GBM patients for EGFR or/and PI3K/mTOR targeted therapies. PMID:24658109

  3. Costimulatory pathways in multiple sclerosis: distinctive expression of PD-1 and PD-L1 in patients with different patterns of disease.

    PubMed

    Trabattoni, Daria; Saresella, Marina; Pacei, Michela; Marventano, Ivana; Mendozzi, Laura; Rovaris, Marco; Caputo, Domenico; Borelli, Manuela; Clerici, Mario

    2009-10-15

    T lymphocytes costimulatory molecules, including CD80, CD86, CD28, CTLA4, PD-1, PD-L1, and B7-H3, are associated with the preferential production of pro- or anti-inflammatory cytokines. We analyzed the expression of these molecules and myelin basic protein (MBP)-specific IL-10 and IFN-gamma production in patients with multiple sclerosis (MS) with relapsing-remitting acute (AMS, n = 40) or stable (SMS, n = 38). Twenty-two patients successfully undergoing therapy with glatimer acetate (n = 12) or IFNbeta (n = 10) were also analyzed. MBP-specific and PD-1-expressing T lymphocytes, PD-L1-expressing CD19(+) cells, and PD-L1(+)/IL-10(+)/CD14(+) and CD19(+) cells were significantly augmented in SMS patients. Additionally, MBP-specific and annexin V-expressing CD4(+) and CD8(+) (apoptotic) T lymphocytes were augmented and pAkt-positive (proliferating) cells were decreased in SMS compared with AMS patients. PD-1 ligation resulted in the increase of pAkt(+) lymphocytes in AMS patients alone. B7-H3 expression and IFN-gamma production were comparable in all individuals but the PD-L1(+)/IL-10(+) over B7-H3(+)/IFN-gamma(+) ratio was significantly lower in AMS compared with SMS patients. Finally, PD-L1 expression on immune cells was reduced in treated patients, suggesting that therapy-induced disease remission is not associated with the modulation of the expression of this molecule. The PD-1/PD-L1 pathway plays an important role in modulating immune functions in MS patients; monitoring and targeting these proteins could offer diagnostic and therapeutic advantages. PMID:19794071

  4. Multiple Facets of Arabidopsis Seedling Development Require ?Indole-3-Butyric Acid–Derived Auxin[W

    PubMed Central

    Strader, Lucia C.; Wheeler, Dorthea L.; Christensen, Sarah E.; Berens, John C.; Cohen, Jerry D.; Rampey, Rebekah A.; Bartel, Bonnie

    2011-01-01

    Levels of auxin, which regulates both cell division and cell elongation in plant development, are controlled by synthesis, inactivation, transport, and the use of storage forms. However, the specific contributions of various inputs to the active auxin pool are not well understood. One auxin precursor is indole-3-butyric acid (IBA), which undergoes peroxisomal ?-oxidation to release free indole-3-acetic acid (IAA). We identified ENOYL-COA HYDRATASE2 (ECH2) as an enzyme required for IBA response. Combining the ech2 mutant with previously identified iba response mutants resulted in enhanced IBA resistance, diverse auxin-related developmental defects, decreased auxin-responsive reporter activity in both untreated and auxin-treated seedlings, and decreased free IAA levels. The decreased auxin levels and responsiveness, along with the associated developmental defects, uncover previously unappreciated roles for IBA-derived IAA during seedling development, establish IBA as an important auxin precursor, and suggest that IBA-to-IAA conversion contributes to the positive feedback that maintains root auxin levels. PMID:21406624

  5. Multiple regulatory elements are required to direct trophoblast interferon gene expression in choriocarcinoma cells and trophectoderm.

    PubMed

    Leaman, D W; Cross, J C; Roberts, R M

    1994-04-01

    Interferon-tau (IFN tau) is produced exclusively by the trophectoderm during the peri-implantation stage of pregnancy in ruminant ungulate species. Human choriocarcinoma cells (Jar) stably transfected with 1.8 kilobases of promoter from a bovine IFN tau gene ahead of a human GH (hGH) reporter gene constitutively synthesize hGH, but expression is not increased further by exposure to Newcastle disease virus. This and earlier experiments suggest that the transcriptional cues regulating IFN tau expression are distinct from those operating on other type I IFN genes. Transient transfection experiments reveal that two distinct promoter regions are required for full constitutive expression: one proximal (to position -126), which directs basal expression, and a more distal promoter region (positions -280 to -400), which acts as an enhancer. Nuclear extracts prepared from ovine conceptuses during the period of IFN tau expression interact with the proximal promoter region (positions -34 to -126) to form several complexes of high electrophoretic mobility. Although nucleotide sequence motifs potentially capable of binding the transcription factor IRF-1 are present in this region, IRF-1 does not transactivate the IFN tau gene. The distal part of the promoter contains only one region (-322 to -358) that forms a complex with these conceptus nuclear extracts. Both proximal and distal gel shift patterns become dramatically different when IFN tau gene expression ceases, perhaps reflecting the appearance of transcriptional repressors. Together these experiments support the conclusion that the control of IFN tau gene expression is very different from that of other type I IFN genes and that trophoblast-specific expression depends upon distal as well as proximal promoter regulatory elements. PMID:8052267

  6. Wnt/?-catenin pathway is required for epithelial to mesenchymal transition in CXCL12 over expressed breast cancer cells

    PubMed Central

    Shan, Shumei; Lv, Qiang; Zhao, Yiling; Liu, Chunfeng; Sun, Yingyan; Xi, Kemin; Xiao, Jiayi; Li, Caijuan

    2015-01-01

    CXCL12 is positively associated with the metastasis and prognosis of various human malignancies. Cancer-associated fibroblasts (CAFs), the main cells secreting CXCL12, are capable of inducing epithelial to mesenchymal transition (EMT) of breast cancer cells. However, it has not been completely understood whether CXCL12 is involved in EMT of breast cancer cells and the underlying mechanisms. The present study aimed to investigate the effects of CXCL12 on the EMT and cancer stem cell (CSC)-like phenotypes formation by transfecting pEGFP-N1-CXCL12 plasmid into MCF-7 cells. Real time-PCR and Western blot analysis demonstrated the successful over expression of CXCL12 in MCF-7 cells. Cell counting kit-8 assay, wound healing assay and Transwell invasion analysis confirmed that over expression of CXCL12 significantly promoted the proliferation, migration and invasion in MCF-7 cells (P<0.05). In addition, ALDH activity was dramatically enhanced compared with parental (P<0.001), accompanied by the notably elevated mRNA and protein levels of OCT-4, Nanog, and SOX2 in CXCL12 overexpressed-MCF-7 cells (P<0.001). Furthermore, we observed the down regulation of E-cadherin and up regulation of vimentin, N-cadherin, and ?-SMA in CXCL12 overexpressed-MCF-7 cells (P<0.01). Meanwhile, western blot and immunofluorescence assay showed that over expression of CXCL12 activated Wnt/?-catenin pathway to induce EMT of MCF-7 cells, as evidenced by the increased expression of E-cadherin after silencing ?-catenin by siRNA interference (P<0.001). Collectively, our findings suggested that over expression of CXCL12 could trigger EMT by activating Wnt/?-catenin pathway and induce CSC-like phenotypes formation to promote the proliferation and metastasis in MCF-7. Hence, CXCL12 may become a promising candidate for breast cancer therapy. PMID:26722422

  7. Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc

    NASA Technical Reports Server (NTRS)

    Chen, C.; Sytkowski, A. J.

    2001-01-01

    Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

  8. Considering the chemical energy requirements of the tri-n-propylamine co-reactant pathways for the judicious design of new electrogenerated chemiluminescence detection systems.

    PubMed

    Kerr, Emily; Doeven, Egan H; Wilson, David J D; Hogan, Conor F; Francis, Paul S

    2016-01-01

    The introduction of a 'co-reactant' was a critical step in the evolution of electrogenerated chemiluminescence (ECL) from a laboratory curiosity to a widely utilised detection system. In conjunction with a suitable electrochemiluminophore, the co-reactant enables generation of both the oxidised and reduced precursors to the emitting species at a single electrode potential, under the aqueous conditions required for most analytical applications. The most commonly used co-reactant is tri-n-propylamine (TPrA), which was developed for the classic tris(2,2'-bipyridine)ruthenium(ii) ECL reagent. New electrochemiluminophores such as cyclometalated iridium(iii) complexes are also evaluated with this co-reactant. However, attaining the excited states in these systems can require much greater energy than that of tris(2,2'-bipyridine)ruthenium(ii), which has implications for the co-reactant reaction pathways. In this tutorial review, we describe a simple graphical approach to characterise the energetically feasible ECL pathways with TPrA, as a useful tool for the development of new ECL detection systems. PMID:26525583

  9. Psychiatric genome-wide association study analyses implicate neuronal, immune and histone pathways

    PubMed Central

    2015-01-01

    Genome-wide association studies (GWAS) of psychiatric disorders have identified multiple genetic associations with such disorders, but better methods are needed to derive the underlying biological mechanisms that these signals indicate. We sought to identify biological pathways in GWAS data from over 60,000 participants from the Psychiatric Genomics Consortium. We developed an analysis framework to rank pathways that requires only summary statistics. We combined this score across disorders to find common pathways across three adult psychiatric disorders: schizophrenia, major depression and bipolar disorder. Histone methylation processes showed the strongest association, and we also found statistically significant evidence for associations with multiple immune and neuronal signaling pathways and with the postsynaptic density. Our study indicates that risk variants for psychiatric disorders aggregate in particular biological pathways and that these pathways are frequently shared between disorders. Our results confirm known mechanisms and suggest several novel insights into the etiology of psychiatric disorders. PMID:25599223

  10. GENETIC EVIDENCE FOR THE REQUIREMENT OF THE ENDOCYTIC PATHWAY IN THE UPTAKE OF COENZYME Q6 IN SACCHAROMYCES CEREVISIAE

    PubMed Central

    Padilla-López, Sergio; Jiménez-Hidalgo, María; Martín-Montalvo, Alejandro; F. Clarke, Catherine; Navas, Plácido; Santos-Ocaña, Carlos

    2011-01-01

    SUMMARY Coenzyme Q is an isoprenylated benzoquinone lipid that functions in respiratory electron transport and as a lipid antioxidant. Dietary supplementation with Q is increasingly used as a therapeutic for treatment of mitochondrial and neurodegenerative diseases, yet little is known regarding the mechanism of its uptake. As opposed to other yeast backgrounds, EG103 strains are unable to import exogenous Q6 to the mitochondria. Furthermore, the distribution of exogenous Q6 among endomembranes suggests an impairment of the membrane traffic at the level of the endocytic pathway. This fact was confirmed after the detection of defects in the incorporation of FM4-64 marker and CPY delivery to the vacuole. A similar effect was demonstrated in double mutant strains in Q6 synthesis and several steps of endocytic process; those cells are unable to uptake exogenous Q6 to the mitochondria and restore the growth on non-fermentable carbon sources. Additional data about the positive effect of peptone presence for exogenous Q6 uptake support the hypothesis that Q6 is transported to mitochondria through an endocytic-based system. PMID:19345667

  11. Activation of the Wnt/Planar Cell Polarity Pathway Is Required for Pericyte Recruitment during Pulmonary Angiogenesis

    PubMed Central

    Yuan, Ke; Orcholski, Mark E.; Panaroni, Cristina; Shuffle, Eric M.; Huang, Ngan F.; Jiang, Xinguo; Tian, Wen; Vladar, Eszter K.; Wang, Lingli; Nicolls, Mark R.; Wu, Joy Y.; de Jesus Perez, Vinicio A.

    2016-01-01

    Pericytes are perivascular cells localized to capillaries that promote vessel maturation, and their absence can contribute to vessel loss. Whether impaired endothelial–pericyte interaction contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Using 3G5-specific, immunoglobulin G–coated magnetic beads, we isolated pericytes from the lungs of healthy subjects and PAH patients, followed by lineage validation. PAH pericytes seeded with healthy pulmonary microvascular endothelial cells failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those with healthy pericytes. After the demonstration of abnormal polarization toward endothelium via live-imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. PAH pericytes had reduced expression of frizzled 7 (Fzd7) and cdc42, genes crucial for Wnt/PCP activation. With simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes in vitro and in a murine model of angiogenesis, motility and polarization toward pulmonary microvascular endothelial cells were reduced, whereas with restoration of both genes in PAH pericytes, endothelial–pericyte association was improved, with larger vascular networks. These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis are regulated by Wnt/PCP activation, which can be targeted to prevent vessel loss in PAH. PMID:25447046

  12. A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements.

    PubMed

    Serra-Soriano, Marta; Pallás, Vicente; Navarro, José A

    2014-03-01

    Viral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus p7B, a type II transmembrane protein, leaves the endoplasmic reticulum (ER) through the COPII-dependent Golgi pathway to reach the plasmodesmata. Here we investigated the sequence requirements and putative mechanisms governing p7B transport through the early secretory pathway. Deletion of either the cytoplasmic N-terminal region (CR) or the luminal C-terminal region (LR) led to ER retention, suggesting that they are both essential for ER export. Through alanine-scanning mutagenesis, we identified residues in the CR and LR that are critical for both ER export and for viral cell-to-cell movement. Within the CR, alanine substitution of aspartic and proline residues in the DSSP ?-turn motif (D7 AP10 A) led to movement of discrete structures along the cortical ER in an actin-dependent manner. In contrast, alanine substitution of a lysine residue in the LR (K49 A) resulted in a homogenous ER distribution of the movement protein and inhibition of ER-Golgi traffic. Moreover, the ability of p7B to recruit Sar1 to the ER membrane is lost in the D7 AP10 A mutant, but enhanced in the K49 A mutant. In addition, fluorescence recovery after photobleaching revealed that K49 A but not D7 AP10 A dramatically diminished protein lateral mobility. From these data, we propose a model whereby the LR directs actin-dependent mobility toward the cortical ER, where the cytoplasmic DSSP ?-turn favors assembly of COPII vesicles for export of p7B from the ER. PMID:24438546

  13. Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

    PubMed Central

    Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

    2014-01-01

    Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. PMID:25411845

  14. Sequential Activation of Two Pathogen-Sensing Pathways Required for Type I Interferon Expression and Resistance to an Acute DNA Virus Infection.

    PubMed

    Xu, Ren-Huan; Wong, Eric B; Rubio, Daniel; Roscoe, Felicia; Ma, Xueying; Nair, Savita; Remakus, Sanda; Schwendener, Reto; John, Shinu; Shlomchik, Mark; Sigal, Luis J

    2015-12-15

    Toll-like receptor 9 (TLR9), its adaptor MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7), and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c(+) cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMos) to the draining lymph node (dLN). In the dLN, the major producers of IFN-I were infected iMos, which used the DNA sensor-adaptor STING to activate IRF7 and nuclear factor ?B (NF-?B) signaling to induce the expression of IFN-? and IFN-?, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease. PMID:26682986

  15. Cell wall integrity and high osmolarity glycerol pathways are required for adaptation of Alternaria brassicicola to cell wall stress caused by brassicaceous indolic phytoalexins.

    PubMed

    Joubert, Aymeric; Bataille-Simoneau, Nelly; Campion, Claire; Guillemette, Thomas; Hudhomme, Piétrick; Iacomi-Vasilescu, Béatrice; Leroy, Thibault; Pochon, Stéphanie; Poupard, Pascal; Simoneau, Philippe

    2011-01-01

    Camalexin, the characteristic phytoalexin of Arabidopsis thaliana, inhibits growth of the fungal necrotroph Alternaria brassicicola. This plant metabolite probably exerts its antifungal toxicity by causing cell membrane damage. Here we observed that activation of a cellular response to this damage requires cell wall integrity (CWI) and the high osmolarity glycerol (HOG) pathways. Camalexin was found to activate both AbHog1 and AbSlt2 MAP kinases, and activation of the latter was abrogated in a AbHog1 deficient strain. Mutant strains lacking functional MAP kinases showed hypersensitivity to camalexin and brassinin, a structurally related phytoalexin produced by several cultivated Brassica species. Enhanced susceptibility to the membrane permeabilization activity of camalexin was observed for MAP kinase deficient mutants. These results suggest that the two signalling pathways have a pivotal role in regulating a cellular compensatory response to preserve cell integrity during exposure to camalexin. AbHog1 and AbSlt2 deficient mutants had reduced virulence on host plants that may, at least for the latter mutants, partially result from their inability to cope with defence metabolites such as indolic phytoalexins. This constitutes the first evidence that a phytoalexin activates fungal MAP kinases and that outputs of activated cascades contribute to protecting the fungus against antimicrobial plant metabolites. PMID:20812995

  16. Gibberellic acid nitrite stimulates germination of two species of light-requiring seeds via the nitric oxide pathway.

    PubMed

    Jovanovi?, Vladan; Giba, Zlatko; Djokovi?, Dejan; Milosavljevi?, Slobodan; Grubisi?, Dragoljub; Konjevi?, Radomir

    2005-06-01

    We used two species of light-requiring seeds, Paulownia tomentosa, which have absolute light requirement (no germination in darkness), and Stellaria media seeds, which germinate in darkness to a certain extent because of presence of preformed active phytochrome, to obtain results strongly suggesting that gibberellic acid nitrite stimulates seed germination via its capability as a functional NO donor. Exogenous application of gibberellic acid nitrite stimulates gibberellin-insensitive Stellaria media seed germination in darkness as do a wide variety of NO donors. Pure gibberellic acid could replace the light requirement of P. tomentosa seeds, thus enabling them to germinate in darkness. Gibberellic acid nitrite did not have this effect. A stimulative effect from gibberellic acid nitrite could be detected only after exposure of these seeds to short, 10 min, pulse of red light. Taken together, these results suggest that gibberellic activity of gibberellic acid nitrite is lost after nitrosation but, regarding to the presence of -O-NO moiety in the molecule, gibberellic acid nitrite shares stimulative properties in seed germination with other compounds with NO-releasing properties. PMID:16154981

  17. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  18. Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase C Terminus Is Required for Nuclear Localization and Viral DNA Replication

    PubMed Central

    Feng, Guozhong

    2014-01-01

    ABSTRACT The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. IMPORTANCE The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication. PMID:25008932

  19. Functional and Structural Characterization of Polysaccharide Co-polymerase Proteins Required for Polymer Export in ATP-binding Cassette Transporter-dependent Capsule Biosynthesis Pathways*

    PubMed Central

    Larue, Kane; Ford, Robert C.; Willis, Lisa M.; Whitfield, Chris

    2011-01-01

    Neisseria meningitidis serogroup B and Escherichia coli K1 bacteria produce a capsular polysaccharide (CPS) that is composed of ?2,8-linked polysialic acid (PSA). Biosynthesis of PSA in these bacteria occurs via an ABC (ATP-binding cassette) transporter-dependent pathway. In N. meningitidis, export of PSA to the surface of the bacterium requires two proteins that form an ABC transporter (CtrC and CtrD) and two additional proteins, CtrA and CtrB, that are proposed to form a cell envelope-spanning export complex. CtrA is a member of the outer membrane polysaccharide export (OPX) family of proteins, which are proposed to form a pore to mediate export of CPSs across the outer membrane. CtrB is an inner membrane protein belonging to the polysaccharide co-polymerase (PCP) family. PCP proteins involved in other bacterial polysaccharide assembly systems form structures that extend into the periplasm from the inner membrane. There is currently no structural information available for PCP or OPX proteins involved in an ABC transporter-dependent CPS biosynthesis pathway to support their proposed roles in polysaccharide export. Here, we report cryo-EM images of purified CtrB reconstituted into lipid bilayers. These images contained molecular top and side views of CtrB and showed that it formed a conical oligomer that extended ?125 Å from the membrane. This structure is consistent with CtrB functioning as a component of an envelope-spanning complex. Cross-complementation of CtrA and CtrB in E. coli mutants with defects in genes encoding the corresponding PCP and OPX proteins show that PCP-OPX pairs require interactions with their cognate partners to export polysaccharide. These experiments add further support for the model of an ABC transporter-PCP-OPX multiprotein complex that functions to export CPS across the cell envelope. PMID:21454677

  20. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.

    PubMed Central

    Goruppi, S; Ruaro, E; Varnum, B; Schneider, C

    1997-01-01

    Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival. PMID:9234702

  1. Updating the Wnt pathways

    PubMed Central

    Yu, Jia; Virshup, David M.

    2014-01-01

    In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF (epidermal growth factor) and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases. PMID:25208913

  2. Reactive oxygen species homeostasis and virulence of the fungal pathogen Cryptococcus neoformans requires an intact proline catabolism pathway.

    PubMed

    Lee, I Russel; Lui, Edmund Y L; Chow, Eve W L; Arras, Samantha D M; Morrow, Carl A; Fraser, James A

    2013-06-01

    Degradation of the multifunctional amino acid proline is associated with mitochondrial oxidative respiration. The two-step oxidation of proline is catalyzed by proline oxidase and ?(1)-pyrroline-5-carboxylate (P5C) dehydrogenase, which produce P5C and glutamate, respectively. In animal and plant cells, impairment of P5C dehydrogenase activity results in P5C-proline cycling when exogenous proline is supplied via the actions of proline oxidase and P5C reductase (the enzyme that converts P5C to proline). This proline is oxidized by the proline oxidase-FAD complex that delivers electrons to the electron transport chain and to O2, leading to mitochondrial reactive oxygen species (ROS) overproduction. Coupled activity of proline oxidase and P5C dehydrogenase is therefore important for maintaining ROS homeostasis. In the genome of the fungal pathogen Cryptococcus neoformans, there are two paralogs (PUT1 and PUT5) that encode proline oxidases and a single ortholog (PUT2) that encodes P5C dehydrogenase. Transcription of all three catabolic genes is inducible by the presence of proline. However, through the creation of deletion mutants, only Put5 and Put2 were found to be required for proline utilization. The put2? mutant also generates excessive mitochondrial superoxide when exposed to proline. Intracellular accumulation of ROS is a critical feature of cell death; consistent with this fact, the put2? mutant exhibits a slight, general growth defect. Furthermore, Put2 is required for optimal production of the major cryptococcal virulence factors. During murine infection, the put2? mutant was discovered to be avirulent; this is the first report highlighting the importance of P5C dehydrogenase in enabling pathogenesis of a microorganism. PMID:23564202

  3. Low Piconewton Towing of CNS Axons against Diffusing and Surface-Bound Repellents Requires the Inhibition of Motor Protein-Associated Pathways

    NASA Astrophysics Data System (ADS)

    Kilinc, Devrim; Blasiak, Agata; O'Mahony, James J.; Lee, Gil U.

    2014-11-01

    Growth cones, dynamic structures at axon tips, integrate chemical and physical stimuli and translate them into coordinated axon behaviour, e.g., elongation or turning. External force application to growth cones directs and enhances axon elongation in vitro; however, direct mechanical stimulation is rarely combined with chemotactic stimulation. We describe a microfluidic device that exposes isolated cortical axons to gradients of diffusing and substrate-bound molecules, and permits the simultaneous application of piconewton (pN) forces to multiple individual growth cones via magnetic tweezers. Axons treated with Y-27632, a RhoA kinase inhibitor, were successfully towed against Semaphorin 3A gradients, which repel untreated axons, with less than 12 pN acting on a small number of neural cell adhesion molecules. Treatment with Y-27632 or monastrol, a kinesin-5 inhibitor, promoted axon towing on substrates coated with chondroitin sulfate proteoglycans, potent axon repellents. Thus, modulating key molecular pathways that regulate contractile stress generation in axons counteracts the effects of repellent molecules and promotes tension-induced growth. The demonstration of parallel towing of axons towards inhibitory environments with minute forces suggests that mechanochemical stimulation may be a promising therapeutic approach for the repair of the damaged central nervous system, where regenerating axons face repellent factors over-expressed in the glial scar.

  4. Genetic Variations in Multiple Drug Action Pathways and Survival in Advanced-Stage Non-small Cell Lung Cancer Treated with Chemotherapy

    PubMed Central

    Li, Yafei; Sun, Zhifu; Cunningham, Julie M; Aubry, Marie C.; Wampfler, Jason A.; Croghan, Gary A.; Johnson, Cassandra; Wu, Danli; Aakre, Jeremiah A.; Molina, Julian; Wang, Liewei; Pankratz, V. Shane; Yang, Ping

    2011-01-01

    Purpose Variations in genes related to anticancer drugs' biologic activity could influence treatment responses and lung cancer prognosis. Genetic variants in four biological pathways, i.e., glutathione metabolism, DNA repair, cell cycle, and EGFR, were systematically investigated to examine their association with survival in advanced-stage NSCLC treated with chemotherapy. Experimental Design A total of 894 tagging single-nucleotide polymorphisms (tagSNPs) in 70 genes from the four pathways were genotyped and analyzed in a 1076-patient cohort. Association with overall survival was analyzed at single-SNP and whole-gene levels within all patients and major chemotherapy agent combination groups. Results A poorer overall survival was observed in patients with genetic variations in GSS (glutathione pathway) and MAP3K1 (EGFR pathway) (HR=1.45, 95% CI=1.20–1.70 and HR=1.25, 95% CI=1.05–1.50, respectively). In stratified analysis on patients receiving platinum plus taxane treatment, we observed a hazardous effect on overall survival by MAP3K1 variant (HR=1.38, 95% CI =1.11–1.72) and a protective effect by RAF1 (HR=0.64, 95% CI=0.5–0.82) in the EGFR pathway. In patients receiving platinum plus gemcitabine treatment, RAF and GPX5 (glutathione pathway) genetic variations showed protective effects on survival (HR=0.54, 95% CI=0.38–0.77; HR=0.67, 95% CI=0.52–0.85, respectively); in contrast, NRAS (EGFR pathway) and GPX7 (glutathione pathway) variations showed hazardous effects on overall survival (HR=1.91, 95% CI=1.30–2.80; HR=1.83, 95% CI=1.27–2.63, respectively). All genes that harbored these significant SNPs remained significant by whole-gene analysis. Conclusion Common genetic variations in genes of EGFR and glutathione pathways may be associated with overall survival among patients with advanced-stage NSCLC treated with platinum, taxane, and/or gemicitabine combinations. PMID:21636554

  5. Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis.

    PubMed

    Ren, Qiansheng; Wimmer, Christian; Chicka, Michael C; Ye, Shaojing; Ren, Yi; Hughson, Frederick M; Whiteheart, Sidney W

    2010-08-12

    Activation-dependent platelet granule release is mediated by integral membrane proteins called soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) and their regulators; however, the mechanisms for this process are ill-defined. To further characterize platelet secretion, we analyzed the function of platelets from Unc13d(Jinx) mice. Platelets from these animals lack the putative vesicle priming factor, Munc13-4, and have a severe secretion defect. Release from dense granules was completely ablated and that from alpha-granules and lysosomes was severely compromised. Unc13d(Jinx) platelets showed attenuated aggregation and, consequently, Unc13d(Jinx) mice had prolonged tail-bleeding times. The secretion defect was not due to altered expression of SNAREs or SNARE regulators, defective granule biogenesis, or faulty platelet activation. The defective release could be rescued by adding recombinant Munc13-4 to permeabilized Unc13d(Jinx) platelets. In wild-type mouse platelets, Munc13-4 levels were lower than those of SNAREs suggesting that Munc13-4 could be a limiting component of the platelets' secretory machinery. Consistently, Munc13-4 levels directly correlated with the extent of granule release from permeabilized platelets and from intact, heterozygous Unc13d(Jinx) platelets. These data highlight the importance of Munc13-4 in platelets and indicate that it is a limiting factor required for platelet secretion and hemostasis. PMID:20435885

  6. Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis

    PubMed Central

    Ren, Qiansheng; Wimmer, Christian; Chicka, Michael C.; Ye, Shaojing; Ren, Yi; Hughson, Frederick M.

    2010-01-01

    Activation-dependent platelet granule release is mediated by integral membrane proteins called soluble N-ethylmaleimide–sensitive fusion protein attachment protein receptors (SNAREs) and their regulators; however, the mechanisms for this process are ill-defined. To further characterize platelet secretion, we analyzed the function of platelets from Unc13dJinx mice. Platelets from these animals lack the putative vesicle priming factor, Munc13-4, and have a severe secretion defect. Release from dense granules was completely ablated and that from ?-granules and lysosomes was severely compromised. Unc13dJinx platelets showed attenuated aggregation and, consequently, Unc13dJinx mice had prolonged tail-bleeding times. The secretion defect was not due to altered expression of SNAREs or SNARE regulators, defective granule biogenesis, or faulty platelet activation. The defective release could be rescued by adding recombinant Munc13-4 to permeabilized Unc13dJinx platelets. In wild-type mouse platelets, Munc13-4 levels were lower than those of SNAREs suggesting that Munc13-4 could be a limiting component of the platelets' secretory machinery. Consistently, Munc13-4 levels directly correlated with the extent of granule release from permeabilized platelets and from intact, heterozygous Unc13dJinx platelets. These data highlight the importance of Munc13-4 in platelets and indicate that it is a limiting factor required for platelet secretion and hemostasis. PMID:20435885

  7. Local Kinetic Measures of Macromolecular Structure Reveal Partitioning Among Multiple Parallel Pathways from the Earliest Steps in the Folding of a Large RNA Molecule

    SciTech Connect

    Laederach,A.; Shcherbakova, I.; Liang, M.; Brenowitz, M.; Altman, R.

    2006-01-01

    At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical ({center_dot}OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg{sup 2+} and Na{sup +}-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg{sup 2+}-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na{sup +}-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure from ensembles of local measures that will illuminate the chemical and physical characteristics of each step in the process. The applicability of this analysis approach to other macromolecules is discussed.

  8. Glucose Uptake Is Limiting in T Cell Activation and Requires CD28-Mediated Akt-Dependent and Independent Pathways1

    PubMed Central

    Jacobs, Sarah R.; Herman, Catherine E.; MacIver, Nancie J.; Wofford, Jessica A.; Wieman, Heather L.; Hammen, Jeremy J.; Rathmell, Jeffrey C.

    2008-01-01

    T cell activation potently stimulates cellular metabolism to support the elevated energetic and biosynthetic demands of growth, proliferation, and effector function. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required to allow maximal glucose uptake following TCR stimulation by up-regulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active myristoylated Akt increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent up-regulation of Glut1 and Akt-dependent Glut1 cell surface trafficking. In support of this model, coexpression of Glut1 and myristoylated Akt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism for T cell activation and proliferation. PMID:18354169

  9. Transmembrane-Bound IL-15–Promoted Epithelial-Mesenchymal Transition in Renal Cancer Cells Requires the Src-Dependent Akt/GSK-3?/?-Catenin Pathway12

    PubMed Central

    Yuan, Huaqin; Meng, Xiaoxin; Guo, Wenjie; Cai, Peifen; Li, Wanshuai; Li, Qian; Wang, Weicheng; Sun, Yang; Xu, Qiang; Gu, Yanhong

    2015-01-01

    Intrarenal interleukin-15 (IL-15) plays a major role controlling epithelial survival and polarization both in physiological and pathologic conditions. Herein, we confirmed that human renal cell carcinomas (RCCs) express a membrane-bound IL-15 isoform displaying an unusual molecular weight of 27 kDa. Its stimulation with soluble IL-15 receptor ? chain (s-IL-15R?) triggers epithelial-mesenchymal transition (EMT) process as shown by the down-regulation of E-cadherin and zona occludens 1 and the up-regulation of vimentin and N-cadherin and promotes the migratory and invasive properties of RCC. S-IL-15R? treatment triggered the Src/PI3K/Akt/GSK-3? pathway and promoted ?-catenin nuclei translocation. Deactivation of this pathway by using Src-specific inhibitor PP2, PI3K inhibitor LY294002, and AKT inhibitor MK2206 hampered ?-catenin nuclei translocation and suppressed EMT, migration, and invasion of RCC. S-IL-15R? treatment also enhanced Src-dependent phosphorylation of focal adhesion kinase (FAK) and extracellular signal–regulated kinase (Erk1/2). FAK knockdown significantly decreased the migration and invasion of RCC, which suggest that Src-FAK signaling was involved in s-IL-15R?–favored migration and invasion of RCC. At the same time, inhibitors of Erk1/2 also significantly decreased the migration and invasion of RCC but could not reverse s-IL-15R?–induced EMT. Taken together, our results reveal that Src-dependent PI3K/Akt/GSK3b/?-catenin pathway is required for s-IL-15Ra–dependent induction of EMT in RCC, while Src-FAK and Src-Erk1/2 signaling were involved in s-IL-15R?–promoted migration and invasion properties of RCC. Our study provides a better understanding of IL-15 signaling in RCC tumor progression, which may lead to novel targeted therapies and provide some suggestions when using IL-15 in clinic. PMID:26025664

  10. A Src-family-tyrosine kinase, Lyn, is required for efficient IFN-? expression in pattern recognition receptor, RIG-I, signal pathway by interacting with IPS-1.

    PubMed

    Lim, Young Ju; Koo, Jung Eun; Hong, Eun-Hee; Park, Zee-Yong; Lim, Kyung-Min; Bae, Ok-Nam; Lee, Joo Young

    2015-03-01

    Retinoic acid-inducible gene I (RIG-I) plays an important role in antiviral immunity as a cytosolic receptor recognizing invading viruses. The activation of downstream signaling pathways led by IFN-? promoter stimulator-1 (IPS-1), an adaptor, is known to culminate in the activation of IRFs and the expression of type I interferons. However, the role of Src-family-tyrosine kinases (STKs) in the RIG-I signaling pathway has not been fully evaluated. Through a combined approach of immunoprecipitation and micro reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis, we established that Lyn, one of the STKs, is associated with RIG-I in macrophages. The association of Lyn and RIG-I was confirmed by co-immunoprecipitation study with 293T cells overexpressing Lyn and RIG-I. Suppression of Lyn by siRNA knockdown or a pharmacological inhibitor (PP2) resulted in the attenuation of IRF3 activation and IFN-? expression induced by short poly I:C, a RIG-I agonist, in macrophages. Lyn activation, as determined by phosphorylation of Tyr396 residue, was observed upon short poly I:C stimulation in the mitochondria of macrophages. Short poly I:C induced the formation of speckle-like aggregates of Lyn, which are prominent in mitochondria. Lyn associated with IPS-1, an adaptor protein of RIG-I, which resides on mitochondria membrane. Helicase domain of RIG-I and CARD of IPS-1 are responsible for the interaction with Lyn while SH3 and SH2 domains in Lyn are required for the association with RIG-I and IPS-1. Collectively, our results indicate that Lyn plays a positive regulatory role in RIG-I-mediated interferon expression as a downstream component of IPS-1. They provide further information as to how tyrosine kinases such as STKs play a role in the regulation of antiviral immunity. PMID:25585356

  11. Analysis of the multiple roles of gld-1 in germline development: Interactions with the sex determination cascade and the glp-1 signaling pathway

    SciTech Connect

    Francis, R.; Schedl, T.; Maine, E.

    1995-02-01

    The Caenorhabditis elegans gene gld-1 is essential for oocyte development; in gld-1 (null) hermaphrodites, a tumor forms where oogenesis would normally occur. We use genetic epistasis analysis to demonstrate that tumor formation is dependent on the sexual fate of the germline. When the germline sex determination pathway is set in the female mode (terminal fem/fog genes inactive), gld-1 (null) germ cells exit meiotic prophase and proliferate to form a tumor, but when the pathway is et in the male mode, they develop into sperm. We conclude that the gld-1 (null) phenotype is cell-type specific and that gld-1(+) acts at the end of the cascade to direct oogenesis. We also use cell ablation and epistasis analysis to examine the dependence of tumor formation on the glp-1 signaling pathway. Although glp-1 activity promotes tumor growth, it is not essential for tumor formation by gld-1 (null) germ cells. These data also reveal that gld-1(+) plays a nonessential (and sex nonspecific) role in regulating germ cell proliferation before their entry into meiosis. Thus gld-1(+) may negatively regulate proliferation at two distinct points in germ cell development: before entry into meiotic prophase in both sexes (nonessential premeiotic gld-1 function) and during meiotic prophase when the sex determination pathway is set in the female mode (essential meiotic gld-1 function). 46 refs., 9 figs., 4 tabs.

  12. Symptoms of major depressive disorder subsequent to child maltreatment: Examining change across multiple levels of analysis to identify transdiagnostic risk pathways.

    PubMed

    Shenk, Chad E; Griffin, Amanda M; O'Donnell, Kieran J

    2015-11-01

    Major depressive disorder (MDD) is a prevalent psychiatric condition in the child maltreatment population. However, not all children who have been maltreated will develop MDD or MDD symptoms, suggesting the presence of unique risk pathways that explain how certain children develop MDD symptoms when others do not. The current study tested several candidate risk pathways to MDD symptoms following child maltreatment: neuroendocrine, autonomic, affective, and emotion regulation. Female adolescents (N = 110; age range = 14-19) were recruited into a substantiated child maltreatment or comparison condition and completed a laboratory stressor, saliva samples, and measures of emotion regulation, negative affect, and MDD symptoms. MDD symptoms were reassessed 18 months later. Mediational modeling revealed that emotion regulation was the only significant indirect effect of the relationship between child maltreatment and subsequent MDD symptoms, demonstrating that children exposed to maltreatment had greater difficulties managing affective states that in turn led to more severe MDD symptoms. These results highlight the importance of emotion dysregulation as a central risk pathway to MDD following child maltreatment. Areas of future research and implications for optimizing prevention and clinical intervention through the direct targeting of transdiagnostic risk pathways are discussed. PMID:26535940

  13. MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-? signalling pathway.

    PubMed

    Lynch, Jennifer; Fay, Joanna; Meehan, Maria; Bryan, Kenneth; Watters, Karen M; Murphy, Derek M; Stallings, Raymond L

    2012-05-01

    Transforming growth factor-? (TGF-?) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-? non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-? pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-? signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-? non-canonical signaling by miR-335, which in turn is downregulated by MYCN. PMID:22382496

  14. Multiple genes of mevalonate and non-mevalonate pathways contribute to high aconites content in an endangered medicinal herb, Aconitum heterophyllum Wall.

    PubMed

    Malhotra, Nikhil; Kumar, Varun; Sood, Hemant; Singh, Tiratha Raj; Chauhan, Rajinder Singh

    2014-12-01

    Aconitum heterophyllum Wall, popularly known as Atis or Patis, is an important medicinal herb of North-Western and Eastern Himalayas. No information exists on molecular aspects of aconites biosynthesis, including atisine- the major chemical constituent of A. heterophyllum. Atisine content ranged from 0.14% to 0.37% and total alkaloids (aconites) from 0.20% to 2.49% among 14 accessions of A. heterophyllum. Two accessions contained the highest atisine content with 0.30% and 0.37% as well as the highest alkaloids content with 2.22% and 2.49%, respectively. No atisine was detected in leaves and shoots of A. heterophyllum, thereby, suggesting that the biosynthesis and accumulation of aconite alkaloids occur mainly in roots. Quantitative expression analysis of 15 genes of MVA/MEP pathways in roots versus shoots, differing for atisine content (0-2.2 folds) showed 11-100 folds increase in transcript amounts of 4 genes of MVA pathway; HMGS, HMGR, PMK, IPPI, and 4 genes of MEP pathway; DXPS, ISPD, HDS, GDPS, respectively. The overall expression of 8 genes decreased to 5-12 folds after comparative expression analysis between roots of high (0.37%) versus low (0.14%) atisine content accessions, but their relative transcript amounts remained higher in high content accessions, thereby implying their role in atisine biosynthesis and accumulation. PCA analysis revealed a positive correlation between MVA/MEP pathways genes and alkaloids content. The current study provides first report wherein partial sequences of 15 genes of MVA/MEP pathways have been cloned and studied for their possible role in aconites biosynthesis. The outcome of study has potential applications in the genetic improvement of A. heterophyllum. PMID:25239552

  15. DEPTOR Is an mTOR Inhibitor Frequently Overexpressed in Multiple Myeloma Cells and Required for Their Survival

    E-print Network

    Peterson, Timothy R.

    The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates ...

  16. Tomato fruits expressing a bacterial feedback-insensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase of the shikimate pathway possess enhanced levels of multiple specialized metabolites and upgraded aroma

    PubMed Central

    Tzin, Vered; Moyal Ben Zvi, Michal; Galili, Gad

    2013-01-01

    Tomato (Solanum lycopersicum) fruit contains significant amounts of bioactive compounds, particularly multiple classes of specialized metabolites. Enhancing the synthesis and accumulation of these substances, specifically in fruits, are central for improving tomato fruit quality (e.g. flavour and aroma) and could aid in elucidate pathways of specialized metabolism. To promote the production of specialized metabolites in tomato fruit, this work expressed under a fruit ripening-specific promoter, E8, a bacterial AroG gene encoding a 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is feedback-insensitive to phenylalanine inhibition. DAHPS, the first enzyme of the shikimate pathway, links between the primary and specialized metabolism derived from aromatic amino acids. AroG expression influenced the levels of number of primary metabolites, such as shikimic acid and aromatic amino acids, as well as multiple volatile and non-volatile phenylpropanoids specialized metabolites and carotenoids. An organoleptic test, performed by trained panellists, suggested that the ripe AroG-expressing tomato fruits had a preferred floral aroma compare with fruits of the wild-type line. These results imply that fruit-specific manipulation of the conversion of primary to specialized metabolism is an attractive approach for improving fruit aroma and flavour qualities as well as discovering novel fruit-specialized metabolites. PMID:24006429

  17. Combination of a Selective HSP90?/? Inhibitor and a RAS-RAF-MEK-ERK Signaling Pathway Inhibitor Triggers Synergistic Cytotoxicity in Multiple Myeloma Cells

    PubMed Central

    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 HSP90?/? 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 HSP90?/? inhibitor and RAS-RAF-MEK-ERK signaling pathway inhibitors to improve outcomes in patients with in RAS- or BRAF-mutated MM. PMID:26630652

  18. Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/?-catenin pathway during brain angiogenesis.

    PubMed

    Vanhollebeke, Benoit; Stone, Oliver A; Bostaille, Naguissa; Cho, Chris; Zhou, Yulian; Maquet, Emilie; Gauquier, Anne; Cabochette, Pauline; Fukuhara, Shigetomo; Mochizuki, Naoki; Nathans, Jeremy; Stainier, Didier Yr

    2015-01-01

    Despite the critical role of endothelial Wnt/?-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/?-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/?-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/?-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function. PMID:26051822

  19. Reconstitution of nuclear factor kappa B activation induced by tumor necrosis factor requires membrane-associated components. Comparison with pathway activated by ceramide.

    PubMed

    Reddy, S A; Chaturvedi, M M; Darnay, B G; Chan, H; Higuchi, M; Aggarwal, B B

    1994-10-14

    Tumor necrosis factor (TNF) is known to induce the activation of a nuclear transcription factor, nuclear factor kappa B (NF-kappa B), in a wide variety of cell types. The post-receptor binding events that culminate in TNF-dependent NF-kappa B activation are not understood. To dissect this pathway, we developed a reconstitution system consisting of membrane, cytosolic, and post-nuclear fractions. Our results indicate that when incubated with the post-nuclear fraction derived from TNF-untreated cells, the membrane fraction from TNF-treated cells causes the activation of NF-kappa B with kinetics similar to that observed in intact cells. Under these conditions, the cytosolic fraction has no effect. This activation is tyrosine kinase-dependent since erbstatin completely abolished the effect. Furthermore, as revealed by immunoblotting, no degradation of the inhibitory subunit of NF-kappa B was observed. In this reconstitution system, we can also demonstrate the activation of NF-kappa B by ceramide, but this activation is not tyrosine kinase-dependent. Overall, our results indicate that intermediates required for NF-kappa B activation by TNF or ceramide are membrane-bound, but the mechanism of activation by TNF is most likely different from that of ceramide. PMID:7929233

  20. The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway.

    PubMed Central

    Chapman, R E; Munro, S

    1994-01-01

    Mnt1p is an alpha 1.2-mannosyltransferase which resides in an early compartment of the Saccharomyces cerevisiae Golgi apparatus. We have shown that the signal-anchor region is sufficient, and the transmembrane domain necessary, for its normal Golgi localization. This is similar to the transmembrane domain-mediated retention of mammalian glycosyltransferases, and distinct from the tail-mediated recycling retention of certain mammalian and yeast trans-Golgi proteins. To examine the mechanism involved in transmembrane domain-mediated retention, we have isolated six classes of mutants which fail to retain Mnt1p-reporter fusions in the early Golgi. These mutants all show additional phenotypes which are consistent with alterations in Golgi function. We have called the mutant classes 'gem', for Golgi enzyme maintenance. GEM3 is identical to the previously cloned gene ANP1, and homologous to VAN1 and MNN9. Together, these define a new class of proteins involved in the organization and functioning of the secretory pathway. Interestingly, Anp1p is localized to the endoplasmic reticulum (ER), implying that some function of the ER is required to maintain a functional Golgi apparatus. Images PMID:7957057

  1. Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway

    PubMed Central

    Crossland, Hannah; Kazi, Abid A.; Lang, Charles H.; Timmons, James A.; Pierre, Philippe; Wilkinson, Daniel J.; Smith, Kenneth; Szewczyk, Nathaniel J.

    2013-01-01

    Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C2C12 skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (±IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 ± 7% vs. non-IGF-I-treated) and total protein (+44 ± 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr397 phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding. PMID:23695213

  2. An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.

    PubMed

    Contreras-Ferrat, A E; Toro, B; Bravo, R; Parra, V; Vásquez, C; Ibarra, C; Mears, D; Chiong, M; Jaimovich, E; Klip, A; Lavandero, S

    2010-10-01

    Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected G?? peptidic inhibitor ?ARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and ?ARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase ? inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium. PMID:20685879

  3. Activation of intracellular calcium by multiple Wnt ligands and translocation of ?-catenin into the nucleus: a convergent model of Wnt/Ca2+ and Wnt/?-catenin pathways.

    PubMed

    Thrasivoulou, Christopher; Millar, Michael; Ahmed, Aamir

    2013-12-13

    Ca(2+) and ?-catenin, a 92-kDa negatively charged transcription factor, transduce Wnt signaling via the non-canonical, Wnt/Ca(2+) and canonical, Wnt/?-catenin pathways independently. The nuclear envelope is a barrier to large protein entry, and this process is regulated by intracellular calcium [Ca(2+)]i and trans-nuclear potential. How ?-catenin traverses the nuclear envelope is not well known. We hypothesized that Wnt/Ca(2+) and Wnt/?-catenin pathways act in a coordinated manner and that [Ca(2+)]i release facilitates ?-catenin entry into the nucleus in mammalian cells. In a live assay using calcium dyes in PC3 prostate cancer cells, six Wnt peptides (3A, 4, 5A, 7A, 9B, and 10B) mobilized [Ca(2+)]i but Wnt11 did not. Based upon dwell time (range = 15-30 s) of the calcium waveform, these Wnts could be classified into three classes: short, 3A and 5A; long, 7A and 10B; and very long, 4 and 9B. Wnt-activated [Ca(2+)]i release was followed by an increase in intranuclear calcium and the depolarization of both the cell and nuclear membranes, determined by using FM4-64. In cells treated with Wnts 5A, 9B, and 10B, paradigm substrates for each Wnt class, increased [Ca(2+)]i was followed by ?-catenin translocation into the nucleus in PC3, MCF7, and 253J, prostate, breast, and bladder cancer cell lines; both the increase in Wnt 5A, 9B, and 10B induced [Ca(2+)]i release and ?-catenin translocation are suppressed by thapsigargin in PC3 cell line. We propose a convergent model of Wnt signaling network where Ca(2+) and ?-catenin pathways may act in a coordinated, interdependent, rather than independent, manner. PMID:24158438

  4. Protein kinase CK2 inhibition down modulates the NF-?B and STAT3 survival pathways, enhances the cellular proteotoxic stress and synergistically boosts the cytotoxic effect of bortezomib on multiple myeloma and mantle cell lymphoma cells.

    PubMed

    Manni, Sabrina; Brancalion, Alessandra; Mandato, Elisa; Tubi, Laura Quotti; Colpo, Anna; Pizzi, Marco; Cappellesso, Rocco; Zaffino, Fortunato; Di Maggio, Speranza Antonia; Cabrelle, Anna; Marino, Filippo; Zambello, Renato; Trentin, Livio; Adami, Fausto; Gurrieri, Carmela; Semenzato, Gianpietro; Piazza, Francesco

    2013-01-01

    CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-?B and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-?B p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1? were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2? levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies. PMID:24086494

  5. Protein Kinase CK2 Inhibition Down Modulates the NF-?B and STAT3 Survival Pathways, Enhances the Cellular Proteotoxic Stress and Synergistically Boosts the Cytotoxic Effect of Bortezomib on Multiple Myeloma and Mantle Cell Lymphoma Cells

    PubMed Central

    Manni, Sabrina; Brancalion, Alessandra; Mandato, Elisa; Tubi, Laura Quotti; Colpo, Anna; Pizzi, Marco; Cappellesso, Rocco; Zaffino, Fortunato; Di Maggio, Speranza Antonia; Cabrelle, Anna; Marino, Filippo; Zambello, Renato; Trentin, Livio; Adami, Fausto; Gurrieri, Carmela; Semenzato, Gianpietro; Piazza, Francesco

    2013-01-01

    CK2 is a pivotal pro-survival protein kinase in multiple myeloma that may likely impinge on bortezomib-regulated cellular pathways. In the present study, we investigated CK2 expression in multiple myeloma and mantle cell lymphoma, two bortezomib-responsive B cell tumors, as well as its involvement in bortezomib-induced cytotoxicity and signaling cascades potentially mediating bortezomib resistance. In both tumors, CK2 expression correlated with that of its activated targets NF-?B and STAT3 transcription factors. Bortezomib-induced proliferation arrest and apoptosis were significantly amplified by the simultaneous inhibition of CK2 with two inhibitors (CX-4945 and K27) in multiple myeloma and mantle cell lymphoma cell lines, in a model of multiple myeloma bone marrow microenvironment and in cells isolated from patients. CK2 inhibition empowered bortezomib-triggered mitochondrial-dependent cell death. Phosphorylation of NF-?B p65 on Ser529 (a CK2 target site) and rise of the levels of the endoplasmic reticulum stress kinase/endoribonuclease Ire1? were markedly reduced upon CK2 inhibition, as were STAT3 phospho Ser727 levels. On the contrary, CK2 inhibition increased phospho Ser51 eIF2? levels and enhanced the bortezomib-dependent accumulation of poly-ubiquitylated proteins and of the proteotoxic stress-associated chaperone Hsp70. Our data suggest that CK2 over expression in multiple myeloma and mantle cell lymphoma cells might sustain survival signaling cascades and can antagonize bortezomib-induced apoptosis at different levels. CK2 inhibitors could be useful in bortezomib-based combination therapies. PMID:24086494

  6. Multiple Pathways for Benzyl Alcohol Oxidation by RuV=O3+ and RuIV=O2+

    SciTech Connect

    Paul, Amit; Hull, Jonathan F.; Norris, Michael R.; Chen, Zuofeng; Ess, Daniel H.; Concepcion, Javier J.; Meyer, Thomas J.

    2011-01-20

    Significant rate enhancements are found for benzyl alcohol oxidation by the RuV=O3+ form of the water oxidation catalyst [Ru(Mebimpy)(bpy)(OH2)]2+ [Mebimpy = 2,6-bis(1-methylbenzimidazol-2-yl)pyridine; bpy = 2,2'-bipyridine] compared to RuIV=O2+ and for the RuIV=O2+ form with added bases due to a new pathway, concerted hydride proton transfer (HPT).

  7. Multiple Pathways for Benzyl Alcohol Oxidation by RuV=O3+ and RuIV=O2+

    SciTech Connect

    Paul, Amit; Hull, Jonathan F.; Norris, Michael R.; Chen, Zuofeng; Ess, Daniel H.; Concepcion, Javier J.; Meyer, Thomas J.

    2011-01-20

    Significant rate enhancements are found for benzyl alcohol oxidation by the RuV=O3+ form of the water oxidation catalyst [Ru(Mebimpy)(bpy)(OH2)]2+ [Mebimpy = 2,6-bis(1-methylbenzimidazol-2-yl)pyridine; bpy = 2,2'-bipyridine] compared to RuIV=O2+ and for the RuIV=O2+ form with added bases due to a new pathway involving concerted hydride proton transfer (HPT).

  8. Oncogenic NRAS hyper-activates multiple pathways in human cord blood stem/progenitor cells and promotes myelomonocytic proliferation in vivo

    PubMed Central

    Wang, Tongjie; Li, Chen; Xia, Chengxiang; Dong, Yong; Yang, Dan; Geng, Yang; Cai, Jizhen; Zhang, Jing; Zhang, Xiangzhong; Wang, Jinyong

    2015-01-01

    Oncogenic NRAS mutations are prevalent in human myeloid leukemia, especially in chronic myelomonocytic leukemia (CMML). NrasG12D mutation at its endogenous locus in murine hematopoietic stem cells (HSCs) leads to CMML and acute T-cell lymphoblastic lymphoma/leukemia in a dose-dependent manner. Hyper-activated MAPK and STAT5 pathways by oncogenic Nras contribute to the leukemogenesis in vivo. However, it is unclear whether these conclusions remain true in a more human relevant model. Here, we evaluated the effects of NRASG12D on human hematopoiesis and leukemogenesis in vitro and in vivo by ectopically expressing NRASG12D in human cord blood stem/progenitor cells (hSPCs). NRASG12D expressing hSPCs preferentially differentiated into myelomonocytic lineage cells, demonstrated by forming more colony forming unit-macrophages than control hSPCs in cultures. Transplantation of NRASG12D expressing hSPCs initiated myeloproliferative neoplasm in immune deficiency mice. All the recipient mice died of myeloid tumor burdens in spleens and bone marrows and none developed lymphoid leukemia. Phospho-flow analysis of CD34+ CD38- hSPCs confirmed that NRASG12D hyper-activated MAPK, AKT and STAT5 pathways. Our study provides the strong evidence that NRASG12D mutation mainly targets monocytic lineage cells and leads to myelomonocytic proliferation in vivo in a highly human relevant context.

  9. The Shared Crosstalk of Multiple Pathways Involved in the Inflammation between Rheumatoid Arthritis and Coronary Artery Disease Based on a Digital Gene Expression Profile

    PubMed Central

    Zhang, Zhiguo; Jiang, Miao; He, Dan; Bian, Yanqin; Zhang, Ge; Bian, Zhaoxiang; Lu, Aiping

    2014-01-01

    Rheumatoid arthritis (RA) and coronary artery disease (CAD) are both complex inflammatory diseases, and an increased prevalence of CAD and a high rate of mortality have been observed in RA patients. But the molecular mechanism of inflammation that is shared between the two disorders is unclear. High-throughput techniques, such as transcriptome analysis, are becoming important tools for genetic biomarker discovery in highly complex biological samples, which is critical for the diagnosis, prognosis, and treatment of disease. In the present study, we reported one type of transcriptome analysis method: digital gene expression profiling of peripheral blood mononuclear cells of 10 RA patients, 10 CAD patients and 10 healthy people. In all, 213 and 152 differently expressed genes (DEGs) were identified in RA patients compared with normal controls (RA vs. normal) and CAD patients compared with normal controls (CAD vs. normal), respectively, with 73 shared DEGs between them. Using this technique in combination with Ingenuity Pathways Analysis software, the effects on inflammation of four shared canonical pathways, three shared activated predicted upstream regulators and three shared molecular interaction networks were identified and explored. These shared molecular mechanisms may provide the genetic basis and potential targets for optimizing the application of current drugs to more effectively treat these diseases simultaneously and for preventing one when the other is diagnosed. PMID:25514790

  10. Origin of product selectivity in a prenyl transfer reaction from the same intermediate: exploration of multiple FtmPT1-catalyzed prenyl transfer pathways.

    PubMed

    Pan, Li-Li; Yang, Yue; Merz, Kenneth M

    2014-09-30

    FtmPT1 is a fungal indole prenyltransferase that catalyzes the reaction of tryptophan derivatives with dimethylallyl pyrophosphate to form various biologically active compounds. Herein, we describe detailed studies of FtmPT1 catalysis involving dimethylallyl pyrophosphate and Brevianamide F following the native pathway (yielding Tryprostatin B) and an alternate pathway observed in the Gly115Thr mutant of FtmPT1 yielding a novel cyclized product. Importantly, these two products arise from the same intermediate state, meaning that a step other than the cleavage of the dimethylallyl pyrophosphate (DMAPP; C-O) bond is differentiating between the two product reaction channels. From detailed potential of mean force (PMF) and two-dimensional PMF analyses, we conclude that the rate-limiting step is the cleavage of the C-O bond in DMAPP, while the deprotonation/cyclization step determines the final product distribution. Hence, in the case of FtmPT1, the optimization of the necessary catalytic machinery guides the generation of the final product after formation of the intermediate carbocation. PMID:25188320

  11. Mechanical motion promotes expression of Prg4 in articular cartilage via multiple CREB-dependent, fluid flow shear stress-induced signaling pathways

    PubMed Central

    Ogawa, Hiroyasu; Kozhemyakina, Elena; Hung, Han-Hwa; Grodzinsky, Alan J.; Lassar, Andrew B.

    2014-01-01

    Lubricin is a secreted proteoglycan encoded by the Prg4 locus that is abundantly expressed by superficial zone articular chondrocytes and has been noted to both be sensitive to mechanical loading and protect against the development of osteoarthritis. In this study, we document that running induces maximal expression of Prg4 in the superficial zone of knee joint articular cartilage in a COX-2-dependent fashion, which correlates with augmented levels of phospho-S133 CREB and increased nuclear localization of CREB-regulated transcriptional coactivators (CRTCs) in this tissue. Furthermore, we found that fluid flow shear stress (FFSS) increases secretion of extracellular PGE2, PTHrP, and ATP (by epiphyseal chondrocytes), which together engage both PKA- and Ca++-regulated signaling pathways that work in combination to promote CREB-dependent induction of Prg4, specifically in superficial zone articular chondrocytes. Because running and FFSS both boost Prg4 expression in a COX-2-dependent fashion, our results suggest that mechanical motion may induce Prg4 expression in the superficial zone of articular cartilage by engaging the same signaling pathways activated in vitro by FFSS that promote CREB-dependent gene expression in this tissue. PMID:24449269

  12. Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory.

    PubMed

    Shuai, Yichun; Hirokawa, Areekul; Ai, Yulian; Zhang, Min; Li, Wanhe; Zhong, Yi

    2015-12-01

    Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-?'1) and a pair of glutamatergic neurons (MBON-?4>?1?2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts. PMID:26627257

  13. PJ34, a poly(ADP-ribose) polymerase (PARP) inhibitor, reverses melphalan-resistance and inhibits repair of DNA double-strand breaks by targeting the FA/BRCA pathway in multidrug resistant multiple myeloma cell line RPMI8226/R.

    PubMed

    Xiong, Ting; Wei, Heng; Chen, Xiaoqiong; Xiao, Hui

    2015-01-01

    There is still no ideal treatment for multidrug resistant multiple myeloma, looking for drugs which can reverse chemotherapy resistance and enhance curative effects of chemotherapy drugs becomes a problem that needs to be solved urgently. Poly(ADP-ribose) polymerase inhibitors appear to be an important tool for medical therapy of several malignancies. In the present study, we investigated the potential of the PARP-1 inhibitor PJ34, in vitro, to further enhance the ef?cacy of the traditional chemotherapy drug melphalan in the multidrug-resistant multiple myeloma cell line RPMI8226/R. The effects of different concentrations of PJ34 and melphalan on cell proliferation were determined by the CCK-8 assay. The expressions of FA/BRCA pathway-related factors were detected by western blotting and RT-PCR. The percentage of cell apoptosis was measured with flow cytometry. DNA double-strand break (DSB) repair was quanti?ed by ?H2AX immuno?uorescence. In addition, DNA damage repair at the level of the individual cell was determined by comet assay. Co-administration of PJ34 and melphalan had synergistic inhibitory effects on the proliferation of RPMI8226/R cells, suggesting more powerful antitumor activities. The apoptosis percentage also was increased more obviously by the treatment of melphalan plus PJ34. The activation of FA/BRCA pathway was inhibited by downregulation of related factors including FANCD2, BRCA2 and Rad51. PJ34 significantly increased the ratio of ?H2AX-positive cells and the number of foci/cells. The comet tail rate of cells, tail length, tail moment and Olive tail moment all increased after PJ34 treatment in RPMI8226/R cells. These results indicate that PJ34 combined treatment with melphalan produces synergistic effects and reverses multidrug resistance of RPMI8226/R cells effectively. PJ34 cannot induce DNA damage directly, but it may increase the DNA damage induced by melphalan through inhibiting DNA damage repair. The suppression of FA/BRCA pathway may be the mechanism. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of multidrug resistant multiple myeloma. PMID:25351371

  14. Extended Pausing by Humans on Multiple Fixed-Ratio Schedules with Varied Reinforcer Magnitude and Response Requirements

    ERIC Educational Resources Information Center

    Williams, Dean C.; Saunders, Kathryn J.; Perone, Michael

    2011-01-01

    We conducted three experiments to reproduce and extend Perone and Courtney's (1992) study of pausing at the beginning of fixed-ratio schedules. In a multiple schedule with unequal amounts of food across two components, they found that pigeons paused longest in the component associated with the smaller amount of food (the lean component), but only…

  15. Paramutation in Drosophila Requires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and Induces Cis-spreading of piRNA Production.

    PubMed

    Hermant, Catherine; Boivin, Antoine; Teysset, Laure; Delmarre, Valérie; Asif-Laidin, Amna; van den Beek, Marius; Antoniewski, Christophe; Ronsseray, Stéphane

    2015-12-01

    Transposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations. PMID:26482790

  16. Uptake of Shiga-toxigenic Escherichia coli SubAB by HeLa cells requires an actin– and lipid raft -dependent pathway

    PubMed Central

    Nagasawa, Sayaka; Ogura, Kohei; Tsutsuki, Hiroyasu; Saitoh, Hisako; Moss, Joel; Iwase, Hirotaro; Noda, Masatoshi; Yahiro, Kinnosuke

    2014-01-01

    The novel cytotoxic factor Subtilase cytotoxin (SubAB) is produced mainly by non-O157 Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves the molecular chaperone BiP/GRP78 in the endoplasmic reticulum (ER), leading to activation of RNA-dependent protein kinase (PKR)-like ER kinase (PERK), followed by caspase-dependent cell death. However, the SubAB uptake mechanism in HeLa cells is unknown. In this study, a variety of inhibitors and siRNAs were employed to characterize the SubAB uptake process. SubAB-induced BiP cleavage was inhibited by high concentrations of Dynasore, and methyl-?-cyclodextrin (m?CD) and Filipin III, but not suppressed in clathrin-, dynamin I/II-, caveolin1- and caveolin2-knockdown cells. We observed that SubAB treatment led to dramatic actin rearrangements, e.g., formation of plasma membrane blebs, with a significant increase in fluid uptake. Confocal microscopy analysis showed that SubAB uptake required actin cytoskeleton remodeling and lipid raft cholesterol. Furthermore, internalized SubAB in cells was found in the detergent-resistant domain (DRM) structure. Interestingly, IPA-3, an inhibitor of serine/threonine kinase p21-activated kinase (PAK1), an important protein of macropinocytosis, directly inhibited SubAB-mediated BiP cleavage and SubAB internalization. Thus, our findings suggest that SubAB uses lipid raft- and actin-dependent, but not clathrin-, caveolin- and dynamin-dependent pathways as its major endocytic translocation route. PMID:24844382

  17. Observation of multiple ionization pathways for OCS in an intense laser field resolved by three-dimensional covariance mapping and visualized by hierarchical ionization topology

    E-print Network

    W. A. Bryan; W. R. Newell; J. H. Sanderson; A. J. Langley

    2006-10-10

    The two- and three-body Coulomb explosion of carbonyl sulfide (OCS) by 790 nm, 50 fs laser pulses focussed to $\\approx $ 10$^{16}$ Wcm$^{-2}$ has been investigated by three-dimensional covariance mapping technique. For the first time in a triatomic molecule, a single charge state, in this case the trication, has been observed to dissociate into two distinct energy channels. With the aid of a three dimensional visualization technique to reveal the ionization hierarchy, evidence is presented for the existence of two sets of ionization pathways resulting from these two initial states. While one group of ions can be modeled using a Classical enhanced ionization model, the second group, consisting of mainly asymmetric channels, can not. The results provide clear evidence that an enhanced ionization approach must also be accompanied by an appreciation of the effects of excited ionic states and multi-electronic processes.

  18. Tick-borne encephalitis virus triggers inositol-requiring enzyme 1 (IRE1) and transcription factor 6 (ATF6) pathways of unfolded protein response.

    PubMed

    Yu, Chao; Achazi, Katharina; Niedrig, Matthias

    2013-12-26

    Tick-borne encephalitis (TBE) is a serious human neurological disease caused by TBE virus (TBEV). However, the mechanisms of TBEV-caused pathogenesis remain unclear. The endoplasmic reticulum (ER) stress response, also defined as the unfolded protein response (UPR), is an important conserved molecular signaling pathway that modulates many biological functions including innate immunity and viral pathogenesis. Here, we investigated the effects of the two UPR signaling pathways upon TBEV infection in Vero E6 cells. We showed that the amount of heat shock protein 72 (Hsp72) increased in the course of TBEV infection. We then confirmed that TBEV infection activates the IRE1 pathway, leading to RNA and protein expression of the spliced X box binding protein 1 (sXBP1). Furthermore, we observed the translocation of ATF6 during TBEV infection and expression of cleaved transcription factor 6 (ATF6) which suggest activation of ATF6 pathway. Finally, we examined whether inhibition of the IRE1 pathway has an effect on TBEV infection. Cell treatment with 3,5-Dibromosalicylaldehyde (IRE1 inhibitor) and tauroursodeoxycholic acid (TUDCA) showed that TBEV replication was significantly limited. These findings provide the first evidence that TBEV infection activates the two UPR signaling pathways. Moreover, inhibition of TBEV replication by UPR inhibitors may provide a novel therapeutic strategy against TBE. PMID:24177270

  19. Alternative Pathways to Talent Development in Music: The Narrative of an Eminent Filipino Singer-Songwriter

    ERIC Educational Resources Information Center

    Garces-Bacsal, Rhoda Myra

    2014-01-01

    The narrative of an eminent Filipino singer-songwriter, Noel Cabangon, provides a description of an alternative pathway to musical talent development. Most theories on talent development assume that a young artist would have access to the resources required for one to advance in the domain. The results of multiple in-depth interviews suggested…

  20. Pathway Regulation of p63, a Director of Epithelial Cell Fate

    PubMed Central

    Yoh, Kathryn; Prywes, Ron

    2015-01-01

    The p53-related gene p63 is required for epithelial cell establishment and its expression is often altered in tumor cells. Great strides have been made in understanding the pathways and mechanisms that regulate p63 levels, such as the Wnt, Hedgehog, Notch, and EGFR pathways. We discuss here the multiple signaling pathways that control p63 expression as well as transcription factors and post-transcriptional mechanisms that regulate p63 levels. While a unified picture has not emerged, it is clear that the fine-tuning of p63 has evolved to carefully control epithelial cell differentiation and fate. PMID:25972840

  1. A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways.

    PubMed Central

    Kudla, A J; John, M L; Bowen-Pope, D F; Rainish, B; Olwin, B B

    1995-01-01

    The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF beta receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF beta receptor activated the PDGF beta receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF beta receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF beta receptor- and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF beta receptor. Activation of PDGF beta receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation. PMID:7760819

  2. Arabidopsis Phosphoglycerate Dehydrogenase1 of the Phosphoserine Pathway Is Essential for Development and Required for Ammonium Assimilation and Tryptophan Biosynthesis[C][W][OPEN

    PubMed Central

    Benstein, Ruben Maximilian; Ludewig, Katja; Wulfert, Sabine; Wittek, Sebastian; Gigolashvili, Tamara; Frerigmann, Henning; Gierth, Markus; Flügge, Ulf-Ingo; Krueger, Stephan

    2013-01-01

    In plants, two independent serine biosynthetic pathways, the photorespiratory and glycolytic phosphoserine (PS) pathways, have been postulated. Although the photorespiratory pathway is well characterized, little information is available on the function of the PS pathway in plants. Here, we present a detailed characterization of phosphoglycerate dehydrogenases (PGDHs) as components of the PS pathway in Arabidopsis thaliana. All PGDHs localize to plastids and possess similar kinetic properties, but they differ with respect to their sensitivity to serine feedback inhibition. Furthermore, analysis of pgdh1 and phosphoserine phosphatase mutants revealed an embryo-lethal phenotype and PGDH1-silenced lines were inhibited in growth. Metabolic analyses of PGDH1-silenced lines grown under ambient and high CO2 conditions indicate a direct link between PS biosynthesis and ammonium assimilation. In addition, we obtained several lines of evidence for an interconnection between PS and tryptophan biosynthesis, because the expression of PGDH1 and PHOSPHOSERINE AMINOTRANSFERASE1 is regulated by MYB51 and MYB34, two activators of tryptophan biosynthesis. Moreover, the concentration of tryptophan-derived glucosinolates and auxin were reduced in PGDH1-silenced plants. In essence, our results provide evidence for a vital function of PS biosynthesis for plant development and metabolism. PMID:24368794

  3. Health risk assessment of various metal(loid)s via multiple exposure pathways on children living near a typical lead-acid battery plant, China.

    PubMed

    Cao, Suzhen; Duan, Xiaoli; Zhao, Xiuge; Wang, Beibei; Ma, Jin; Fan, Delong; Sun, Chengye; He, Bin; Wei, Fusheng; Jiang, Guibin

    2015-05-01

    Manufacture of lead-acid batteries is of widespread interest because of its emissions of heavy metals and metalloids into environment, harming environmental quality and consequently causing detrimental effects on human health. In this study, exposure pathways and health risks of children to heavy metal(loid)s (Pb, Cd, As, etc) were investigated based on field sampling and questionnaire. Pb was one of the most abundant elements in children's blood, with an elevated blood lead level of 12.45 ?g dL(-1). Soil/dust and food were heavily polluted by targeted metal(loid)s. Food ingestion accounted for more than 80% of the total exposure for most metal(loid)s. The non-cancer risks to children were 3-10 times higher than the acceptable level of 1, while the cancer risks were 5-200 times higher than the maximum acceptable level of 1.0 × 10(-4). The study emphasized the significance of effective environmental management, particularly to ensure food security near battery facilities. PMID:25686884

  4. PPE38 of Mycobacterium marinum triggers the cross-talk of multiple pathways involved in the host response, as revealed by subcellular quantitative proteomics.

    PubMed

    Wang, Hui; Dong, Dandan; Tang, Siwei; Chen, Xian; Gao, Qian

    2013-05-01

    The PE/PPE family of proteins which are in high abundance in pathogenic species such as Mycobacterium tuberculosis and M. marinum , play the critical role in generating antigenic variation and evasion of host immune responses. However, little is known about their functional roles in mycobacterial pathogenesis. Previously, we found that PPE38 is associated with the virulence of mycobacteria, presumably by modulating the host immune response. To clarify the link between PPE38 and host response, we employed a subcellular, amino acid-coded mass tagging (AACT)/SILAC-based quantitative proteomic approach to determine the proteome changes during host response to M. marinum PPE38. As a result, 291 or 290 proteins were found respectively to be up- or down-regulated in the nucleus. Meanwhile, 576 upregulated and 272 downregulated proteins were respectively detected in the cytosol. The data of quantitative proteomic changes and concurrent biological validations revealed that M. marinum PPE38 could trigger extensive inflammatory responses in macrophages, probably through interacting with toll-like receptor 2 (TLR2). We also found that PPE38 may arrest MHC-1 processing and presentation in infected macrophages. Using bioinformatics tools to analyze global changes in the host proteome, we obtained a PPE38-respondor network involved in various transcriptional factors (TFs) and TF-associated proteins. The results of our systems investigation now indicate that there is cross-talk involving a broad range of diverse biological pathways/processes that coordinate the host response to M. marinum PPE38. PMID:23514422

  5. Robust Selection Algorithm (RSA) for Multi-Omic Biomarker Discovery; Integration with Functional Network Analysis to Identify miRNA Regulated Pathways in Multiple Cancers

    PubMed Central

    Sehgal, Vasudha; Seviour, Elena G.; Moss, Tyler J.; Mills, Gordon B.; Azencott, Robert; Ram, Prahlad T.

    2015-01-01

    MicroRNAs (miRNAs) play a crucial role in the maintenance of cellular homeostasis by regulating the expression of their target genes. As such, the dysregulation of miRNA expression has been frequently linked to cancer. With rapidly accumulating molecular data linked to patient outcome, the need for identification of robust multi-omic molecular markers is critical in order to provide clinical impact. While previous bioinformatic tools have been developed to identify potential biomarkers in cancer, these methods do not allow for rapid classification of oncogenes versus tumor suppressors taking into account robust differential expression, cutoffs, p-values and non-normality of the data. Here, we propose a methodology, Robust Selection Algorithm (RSA) that addresses these important problems in big data omics analysis. The robustness of the survival analysis is ensured by identification of optimal cutoff values of omics expression, strengthened by p-value computed through intensive random resampling taking into account any non-normality in the data and integration into multi-omic functional networks. Here we have analyzed pan-cancer miRNA patient data to identify functional pathways involved in cancer progression that are associated with selected miRNA identified by RSA. Our approach demonstrates the way in which existing survival analysis techniques can be integrated with a functional network analysis framework to efficiently identify promising biomarkers and novel therapeutic candidates across diseases. PMID:26505200

  6. Extended LineSets: a visualization technique for the interactive inspection of biological pathways

    PubMed Central

    2015-01-01

    Background Biologists make use of pathway visualization tools for a range of tasks, including investigating inter-pathway connectivity and retrieving details about biological entities and interactions. Some of these tasks require an understanding of the hierarchical nature of elements within the pathway or the ability to make comparisons between multiple pathways. We introduce a technique inspired by LineSets that enables biologists to fulfill these tasks more effectively. Results We introduce a novel technique, Extended LineSets, to facilitate new explorations of biological pathways. Our technique incorporates intuitive graphical representations of different levels of information and includes a well-designed set of user interactions for selecting, filtering, and organizing biological pathway data gathered from multiple databases. Conclusions Based on interviews with domain experts and an analysis of two use cases, we show that our technique provides functionality not currently enabled by current techniques, and moreover that it helps biologists to better understand both inter-pathway connectivity and the hierarchical structure of biological elements within the pathways. PMID:26361500

  7. Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery

    PubMed Central

    Eulitt, Patrick J; Park, Margaret A; Hamed, Hossein A; Cruikshanks, Nichola; Yang, Chen; Dmitriev, Igor P; Yacoub, Adly; Curiel, David T; Fisher, Paul B

    2010-01-01

    We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsakie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected “bystander” RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer. PMID:20948318

  8. Disulfiram/copper-disulfiram Damages Multiple Protein Degradation and Turnover Pathways and Cytotoxicity is Enhanced by Metformin in Oesophageal Squamous Cell Carcinoma Cell Lines.

    PubMed

    Jivan, Rupal; Damelin, Leonard Howard; Birkhead, Monica; Rousseau, Amanda Louise; Veale, Robin Bruce; Mavri-Damelin, Demetra

    2015-10-01

    Disulfiram (DSF), used since the 1950s in the treatment of alcoholism, is reductively activated to diethyldithiocarbamate and both compounds are thiol-reactive and readily complex copper. More recently DSF and copper-DSF (Cu-DSF) have been found to exhibit potent anticancer activity. We have previously shown that the anti-diabetic drug metformin is anti-proliferative and induces an intracellular reducing environment in oesophageal squamous cell carcinoma (OSCC) cell lines. Based on these observations, we investigated the effects of Cu-DSF and DSF, with and without metformin, in this present study. We found that Cu-DSF and DSF caused considerable cytotoxicity across a panel of OSCC cells, and metformin significantly enhanced the effects of DSF. Elevated copper transport contributes to DSF and metformin-DSF-induced cytotoxicity since the cell-impermeable copper chelator, bathocuproinedisulfonic acid, partially reversed the cytotoxic effects of these drugs, and interestingly, metformin-treated OSCC cells contained higher intracellular copper levels. Furthermore, DSF may target cancer cells preferentially due to their high dependence on protein degradation/turnover pathways, and we found that metformin further enhances the role of DSF as a proteasome inhibitor. We hypothesized that the lysosome could be an additional, novel, target of DSF. Indeed, this acid-labile compound decreased lysosomal acidification, and DSF-metformin co-treatment interfered with the progression of autophagy in these cells. In summary, this is the first such report identifying the lysosome as a target of DSF and based on the considerable cytotoxic effects of DSF either alone or in the presence of metformin, in vitro, and we propose these as novel potential chemotherapeutic approaches for OSCC. PMID:25846272

  9. Targeted inhibition of heat shock protein 90 disrupts multiple oncogenic signaling pathways, thus inducing cell cycle arrest and programmed cell death in human urinary bladder cancer cell lines

    PubMed Central

    2013-01-01

    Background Geldanamycin (GA) can be considered a relatively new component with a promising mode of action against human malignancies. It specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone. Methods In this study, we have investigated the effects of geldanamycin on the regulation of Hsp90-dependent oncogenic signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cells. In order to assess the biological outcome of Hsp90 inhibition on RT4 (grade I) and T24 (grade III) human urinary bladder cancer cell lines, we applied MTT assay, FACS analysis, Western blotting, semi-quantitative (sq) RT-PCR, electrophoretic mobility shift assay (EMSA), immunofluorescence and scratch-wound assay. Results We have herein demonstrated that, upon geldanamycin treatment, bladder cancer cells are prominently arrested in the G1 phase of cell cycle and eventually undergo programmed cell death via combined activation of apoptosis and autophagy. Furthermore, geldanamycin administration proved to induce prominent downregulation of several Hsp90 protein clients and downstream effectors, such as membrane receptors (IGF-IR and c-Met), protein kinases (Akt, IKK?, IKK? and Erk1/2) and transcription factors (FOXOs and NF-??), therefore resulting in the impairment of proliferative -oncogenic- signaling and reduction of cell motility. Conclusions In toto, we have evinced the dose-dependent and cell line-specific actions of geldanamycin on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of critical Hsp90 clients and subsequent disruption of signaling -oncogenic- integrity. PMID:23394616

  10. Chilling requirement, cold hardiness and fruiting characteristics of a "Draper" X "Jewel" population planted at multiple sites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A mapping population of 105 individuals has been generated between the southern highbush ‘Jewel’ and the northern highbush ‘Draper’, to produce a genetic linkage map and search for QTL associated with chilling requirement, cold tolerance and fruiting characteristics. ‘Draper’ is adapted to plant h...

  11. PATHWAYS - ELECTRON TUNNELING PATHWAYS IN PROTEINS

    NASA Technical Reports Server (NTRS)

    Beratan, D. N.

    1994-01-01

    The key to understanding the mechanisms of many important biological processes such as photosynthesis and respiration is a better understanding of the electron transfer processes which take place between metal atoms (and other groups) fixed within large protein molecules. Research is currently focused on the rate of electron transfer and the factors that influence it, such as protein composition and the distance between metal atoms. Current models explain the swift transfer of electrons over considerable distances by postulating bridge-mediated tunneling, or physical tunneling pathways, made up of interacting bonds in the medium around and between donor and acceptor sites. The program PATHWAYS is designed to predict the route along which electrons travel in the transfer processes. The basic strategy of PATHWAYS is to begin by recording each possible path element on a connectivity list, including in each entry which two atoms are connected and what contribution the connection would make to the overall rate if it were included in a pathway. The list begins with the bonded molecular structure (including the backbone sequence and side chain connectivity), and then adds probable hydrogen bond links and through-space contacts. Once this list is completed, the program runs a tree search from the donor to the acceptor site to find the dominant pathways. The speed and efficiency of the computer search offers an improvement over manual techniques. PATHWAYS is written in FORTRAN 77 for execution on DEC VAX series computers running VMS. The program inputs data from four data sets and one structure file. The software was written to input BIOGRAF (old format) structure files based on x-ray crystal structures and outputs ASCII files listing the best pathways and BIOGRAF vector files containing the paths. Relatively minor changes could be made in the input format statements for compatibility with other graphics software. The executable and source code are included with the distribution. The main memory requirement for execution is 2.6 Mb. This program is available in DEC VAX BACKUP format on a 9-track 1600 BPI magnetic tape (standard distribution) or on a TK50 tape cartridge. PATHWAYS was developed in 1988. PATHWAYS is a copyrighted work with all copyright vested in NASA. DEC, VAX, VMS, and TK50 are trademarks of Digital Equipment Corporation. BIOGRAF is a trademark of Molecular Simulations, Inc., Sunnyvale, CA.

  12. MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma

    PubMed Central

    He, Jie; Liu, Xi; Qu, Ying; Yan, Wenqing; Fan, Jianling; Li, Rong; Xi, Hao; Fu, Weijun; Zhang, Chunyang; Yang, Jing; Hou, Jian

    2015-01-01

    Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma. PMID:25915427

  13. MicroRNA-451 regulates stemness of side population cells via PI3K/Akt/mTOR signaling pathway in multiple myeloma.

    PubMed

    Du, Juan; Liu, Shuyan; He, Jie; Liu, Xi; Qu, Ying; Yan, Wenqing; Fan, Jianling; Li, Rong; Xi, Hao; Fu, Weijun; Zhang, Chunyang; Yang, Jing; Hou, Jian

    2015-06-20

    Side population (SP) cells are an enriched source of cancer-initiating cells with stemness characteristics, generated by increased ABC transporter activity, which has served as a unique hallmark for multiple myeloma (MM) stem cell studies. Here we isolated and identified MM SP cells via Hoechst 33342 staining. Furthermore, we demonstrate that SP cells possess abnormal cell cycle, clonogenicity, and high drug efflux characteristics-all of which are features commonly seen in stem cells. Interestingly, we found that bortezomib, As2O3, and melphalan all affected apoptosis and clonogenicity in SP cells. We followed by characterizing the miRNA signature of MM SP cells and validated the specific miR-451 target tuberous sclerosis 1 (TSC1) gene to reveal that it activates the PI3K/Akt/mTOR signaling in MM SP cells. Inhibition of miR-451 enhanced anti-myeloma novel agents' effectiveness, through increasing cells apoptosis, decreasing clonogenicity, and reducing MDR1 mRNA expression. Moreover, the novel specific PI3K/Akt/mTOR signaling inhibitor S14161 displayed its prowess as a potential therapeutic agent by targeting MM SP cells. Our findings offer insights into the mechanisms regulating MM SP cells and provide a novel strategy to overcome resistance to existing therapies against myeloma. PMID:25915427

  14. miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells

    PubMed Central

    2012-01-01

    Background Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance between various inhibitory transcription factors and MyoD activity kept rhabdomyosarcomas trapped in a proliferative state. Methods Potential myogenic co-factors were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments. Results Modulation of the transcription factors RUNX1 and ZNF238 can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter. Conclusions These findings support a network model composed of coupled regulatory circuits with miR-206 functioning as a switch regulating the transition from one stable state (growth) to another (differentiation). PMID:22541669

  15. Ursolic Acid Inhibits Growth and Metastasis of Human Colorectal Cancer in an Orthotopic Nude Mouse Model by Targeting Multiple Cell Signaling Pathways: Chemosensitization with Capecitabine

    PubMed Central

    Prasad, Sahdeo; Yadav, Vivek R.; Sung, Bokyung; Reuter, Simone; Kannappan, Ramaswamy; Deorukhkar, Amit; Diagaradjane, Parmeswaran; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Krishnan, Sunil; Guha, Sushovan; Aggarwal, Bharat B.

    2013-01-01

    Purpose Development of chemoresistance, poor prognosis, and metastasis often renders the current treatments for colorectal cancer (CRC) ineffective. Whether ursolic acid (UA), a component of numerous medicinal plants, either alone or in combination with capecitabine, can inhibit the growth and metastasis of human CRC was investigated. Experimental design The effect of UA on proliferation of colorectal cancer cell lines was examined by mitochondrial dye-uptake assay, apoptosis by esterase staining, NF-?B activation by DNA binding assay and protein expression by western blot. The effect of UA on the growth and chemosensitization was also examined in orthotopically-implanted CRC in nude mice. Results We found that UA inhibited the proliferation of different colon cancer cell lines. This is correlated with inhibition of constitutive NF-?B activation and downregulation of cell survival (Bcl-xL, Bcl-2, cFLIP, survivin), proliferative (Cyclin D1), and metastatic (MMP-9, VEGF, ICAM-1) proteins. When examined in an orthotopic nude-mice model, UA significantly inhibited tumor volume, ascites formation and distant organ metastasis, and this effect was enhanced with capecitabine. Immunohistochemistry of tumor tissue indicated that UA downregulated biomarkers of proliferation (Ki-67) and microvessel density (CD31). This effect was accompanied by suppression of NF-?B, STAT3, and ?-catenin. In addition, UA suppressed EGFR, and induced p53, and p21 expression. We also observed bioavailability of UA in the serum and tissue of animals. Conclusion Overall our results demonstrate that UA can inhibit the growth and metastasis of CRC and further enhance the therapeutic effects of capecitabine through suppression of multiple biomarkers linked to inflammation, proliferation, invasion, angiogenesis, and metastasis. PMID:22832932

  16. Multiple-neutral-meson decays of the /tau/ lepton and electromagnetic calorimeter requirements at Tau-Charm Factory

    SciTech Connect

    Gan, K.K.

    1989-08-01

    This is a study of the physics sensitivity to the multiple-neutral-meson decays of the /tau/ lepton at the Tau-Charm Factory. The sensitivity is compared for a moderate and an ultimate electromagnetic calorimeter. With the high luminosity of the Tau- Charm Factory, a very large sample of the decays /tau//sup /minus// /yields/ /pi//sup /minus//2/pi//sup 0//nu//sub /tau// and /tau//sup /minus// /yields/ /pi//sup /minus//3/pi//sup 0//nu//sub /tau// can be collected with both detectors. However, with the ultimate detector, 2/pi//sup 0/ and 3/pi//sup 0/ can be unambiguously reconstructed with very little background. For the suppressed decay /tau//sup /minus// /yields/ /pi//sup /minus///eta//pi//sup 0//nu//sub /tau//, only the ultimate detector has the sensitivity. The ultimate detector is also sensitive to the more suppressed decay /tau//sup /minus// /yields/ K/sup /minus///eta//nu//sub /tau// and the moderate detector may have the sensitivity if the hadronic background is not significantly larger than that predicted by Lund. In the case of the highly suppressed second-class-current decay /tau//sup /minus// /yields/ /pi//sup /minus///eta//nu//sub /tau//, only the ultimate detector has sensitivity. The sensitivity can be greatly enhanced with a small-angle photon veto. 16 refs., 9 figs., 2 tabs.

  17. Hydrogen sulfide induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry plants through modification of reactive species biosynthesis and transcriptional regulation of multiple defence pathways

    PubMed Central

    Christou, Anastasis; Manganaris, George A.; Papadopoulos, Ioannis; Fotopoulos, Vasileios

    2013-01-01

    Hydrogen sulfide (H2S) has been recently found to act as a potent priming agent. This study explored the hypothesis that hydroponic pretreatment of strawberry (Fragaria × ananassa cv. Camarosa) roots with a H2S donor, sodium hydrosulfide (NaHS; 100 ?M for 48h), could induce long-lasting priming effects and tolerance to subsequent exposure to 100mM NaCI or 10% (w/v) PEG-6000 for 7 d. Hydrogen sulfide pretreatment of roots resulted in increased leaf chlorophyll fluorescence, stomatal conductance and leaf relative water content as well as lower lipid peroxidation levels in comparison with plants directly subjected to salt and non-ionic osmotic stress, thus suggesting a systemic mitigating effect of H2S pretreatment to cellular damage derived from abiotic stress factors. In addition, root pretreatment with NaHS resulted in the minimization of oxidative and nitrosative stress in strawberry plants, manifested via lower levels of synthesis of NO and H2O2 in leaves and the maintenance of high ascorbate and glutathione redox states, following subsequent salt and non-ionic osmotic stresses. Quantitative real-time RT-PCR gene expression analysis of key antioxidant (cAPX, CAT, MnSOD, GR), ascorbate and glutathione biosynthesis (GCS, GDH, GS), transcription factor (DREB), and salt overly sensitive (SOS) pathway (SOS2-like, SOS3-like, SOS4) genes suggests that H2S plays a pivotal role in the coordinated regulation of multiple transcriptional pathways. The ameliorative effects of H2S were more pronounced in strawberry plants subjected to both stress conditions immediately after NaHS root pretreatment, rather than in plants subjected to stress conditions 3 d after root pretreatment. Overall, H2S-pretreated plants managed to overcome the deleterious effects of salt and non-ionic osmotic stress by controlling oxidative and nitrosative cellular damage through increased performance of antioxidant mechanisms and the coordinated regulation of the SOS pathway, thus proposing a novel role for H2S in plant priming, and in particular in a fruit crop such as strawberry. PMID:23567865

  18. Status of safety issues at licensed power plants: TMI Action Plan requirements; unresolved safety issues; generic safety issues; other multiplant action issues. Supplement 3

    SciTech Connect

    Not Available

    1993-12-01

    As part of ongoing US Nuclear Regulatory Commission (NRC) efforts to ensure the quality and accountability of safety issue information, the NRC established a program for publishing an annual report on the status of licensee implementation and NRC verification of safety issues in major NRC requirements areas. This information was initially compiled and reported in three NUREG-series volumes. Volume 1, published in March 1991, addressed the status of Three Mile Island (TMI) Action Plan Requirements. Volume 2, published in May 1991, addressed the status of unresolved safety issues (USIs). Volume 3, published in June 1991, addressed the implementation and verification status of generic safety issues (GSIs). The first annual supplement, which combined these volumes into a single report and presented updated information as of September 30, 1991, was published in December 1991. The second annual supplement, which provided updated information as of September 30, 1992, was published in December 1992. Supplement 2 also provided the status of licensee implementation and NRC verification of other multiplant action (MPA) issues not related to TMI Action Plan requirements, USIs, or GSIs. This third annual NUREG report, Supplement 3, presents updated information as of September 30, 1993. This report gives a comprehensive description of the implementation and verification status of TMI Action Plan requirements, safety issues designated as USIs, GSIs, and other MPAs that have been resolved and involve implementation of an action or actions by licensees. This report makes the information available to other interested parties, including the public. Additionally, this report serves as a follow-on to NUREG-0933, ``A Prioritization of Generic Safety Issues,`` which tracks safety issues until requirements are approved for imposition at licensed plants or until the NRC issues a request for action by licensees.

  19. Protective T cell immunity in mice following protein-TLR7/8 agonist-conjugate immunization requires aggregation, type I IFN, and multiple DC subsets

    PubMed Central

    Kastenmüller, Kathrin; Wille-Reece, Ulrike; Lindsay, Ross W.B.; Trager, Lauren R.; Darrah, Patricia A.; Flynn, Barbara J.; Becker, Maria R.; Udey, Mark C.; Clausen, Björn E.; Igyarto, Botond Z.; Kaplan, Daniel H.; Kastenmüller, Wolfgang; Germain, Ronald N.; Seder, Robert A.

    2011-01-01

    The success of a non-live vaccine requires improved formulation and adjuvant selection to generate robust T cell immunity following immunization. Here, using protein linked to a TLR7/8 agonist (conjugate vaccine), we investigated the functional properties of vaccine formulation, the cytokines, and the DC subsets required to induce protective multifunctional T cell immunity in vivo. The conjugate vaccine required aggregation of the protein to elicit potent Th1 CD4+ and CD8+ T cell responses. Remarkably, the conjugate vaccine, through aggregation of the protein and activation of TLR7 in vivo, led to an influx of migratory DCs to the LN and increased antigen uptake by several resident and migratory DC subsets, with the latter effect strongly influenced by vaccine-induced type I IFN. Ex vivo migratory CD8–DEC205+CD103–CD326– langerin-negative dermal DCs were as potent in cross-presenting antigen to naive CD8+ T cells as CD11c+CD8+ DCs. Moreover, these cells also influenced Th1 CD4+ T cell priming. In summary, we propose a model in which broad-based T cell–mediated responses upon vaccination can be maximized by codelivery of aggregated protein and TLR7/8 agonist, which together promote optimal antigen acquisition and presentation by multiple DC subsets in the context of critical proinflammatory cytokines. PMID:21540549

  20. HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana

    PubMed Central

    Soyano, Takashi; Kosetsu, Ken; Sasabe, Michiko; Machida, Yasunori

    2010-01-01

    Cytokinesis is regulated to ensure the precise partitioning of cytoplasm and duplicated chromosomes to daughter cells. The NACK-PQR pathway, which includes NACK1 kinesin-like protein (KLP) and a mitogen-activated protein kinase (MAPK) cascade, plays a key role in cytokinesis in tobacco cells. Although HINKEL/AtNACK1 (HIK) KLP, ANP MAP kinase kinase kinases (MAPKKKs) and MKK6/ ANQ MAP kinase kinase (MAPKK) have been identified independently as regulators of cytokinesis in Arabidopsis thaliana, the involvement of HIK, ANPs and MKK6/ANQ in a regulatory cascade remains to be demonstrated. Here we provide details of the protein kinase pathway that controls cytokinesis in A. thaliana. Analysis of the subcellular distribution of six MAPKKs of A. thaliana that had been fused to green fluorescent protein revealed that only MKK6/ANQ protein was concentrated at the equatorial plane of the phragmoplast, at the site of localization of HIK. Expression of MKK6/ANQ in yeast cells replaced the growth-control function of the MAPKK encoded by yeast PBS2, provided that both ANP1 MAPKKK and HIK [or TETRASPORE/AtNACK2 (TES)] KLP were coexpressed, suggesting that ANP1 activates MKK6/ANQ in the presence of HIK (or TES). Coexpression of HIK and ANP3 (another member of the ANP MAPKKK family) weakly activated MKK6/ANQ but that of TES and ANP3 did not. MKK6/ANQ phosphorylated MPK4 MAPK in vitro to activate the latter kinase. Thus cytokinesis in A. thaliana is controlled by a pathway that consists of ANP MAPKKKs that can be activated by HIK and MKK6/ANQ MAPKK, with MPK4 MAPK being a probable target of MKK6/ANQ. PMID:20802223

  1. Requirement of ER? and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells.

    PubMed

    Song, Xiulong; Wei, Zhengxi; Shaikh, Zahir A

    2015-08-15

    Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ER?, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1-3?M Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ER? with siRNA or use of ER? antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ER?, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. PMID:26006730

  2. Virus interactions with human signal transduction pathways

    PubMed Central

    Zhao, Zhongming; Xia, Junfeng; Tastan, Oznur; Singh, Irtisha; Kshirsagar, Meghana; Carbonell, Jaime; Klein-Seetharaman, Judith

    2011-01-01

    Viruses depend on their hosts at every stage of their life cycles and must therefore communicate with them via Protein-Protein Interactions (PPIs). To investigate the mechanisms of communication by different viruses, we overlay reported pairwise human-virus PPIs on human signalling pathways. Of 671 pathways obtained from NCI and Reactome databases, 355 are potentially targeted by at least one virus. The majority of pathways are linked to more than one virus. We find evidence supporting the hypothesis that viruses often interact with different proteins depending on the targeted pathway. Pathway analysis indicates overrepresentation of some pathways targeted by viruses. The merged network of the most statistically significant pathways shows several centrally located proteins, which are also hub proteins. Generally, hub proteins are targeted more frequently by viruses. Numerous proteins in virus-targeted pathways are known drug targets, suggesting that these might be exploited as potential new approaches to treatments against multiple viruses. PMID:21330695

  3. Sustainable and efficient pathways for bioenergy recovery from low-value process streams via bioelectrochemical systems in biorefineries

    SciTech Connect

    Borole, Abhijeet P.

    2015-01-01

    Conversion of biomass into bioenergy is possible via multiple pathways resulting in production of biofuels, bioproducts and biopower. Efficient and sustainable conversion of biomass, however, requires consideration of many environmental and societal parameters in order to minimize negative impacts. Integration of multiple conversion technologies and inclusion of upcoming alternatives such as bioelectrochemical systems can minimize these impacts and improve conservation of resources such as hydrogen, water and nutrients via recycle and reuse. This report outlines alternate pathways integrating microbial electrolysis in biorefinery schemes to improve energy efficiency while evaluating environmental sustainability parameters.

  4. In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways.

    PubMed

    Dey, Swati; North, Justin A; Sriram, Jaya; Evans, Bradley S; Tabita, F Robert

    2015-12-25

    All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions. PMID:26511314

  5. The activity of RTA2, a downstream effector of the calcineurin pathway, is required during tunicamycin-induced ER stress response in Candida albicans.

    PubMed

    Thomas, Edwina; Sircaik, Shabnam; Roman, Elvira; Brunel, Jean-Michel; Johri, Atul K; Pla, Jesús; Panwar, Sneh L

    2015-12-01

    In this study, we demonstrate a novel function of a downstream effector molecule of the calcineurin pathway, RTA2 (Resistance To Aminocholesterol), in ER stress response. The deletion of RTA2 increases susceptibility to the ER stressor tunicamycin and morpholine-like drug, 7-aminocholesterol. Additionally, the expression of RTA2 is also transcriptionally induced by ergosterol biosynthesis inhibitors and cell-wall-damaging agents. As tunicamycin induces the unfolded protein response pathway (UPR) via the transcription factor, HAC1, we monitored the expression of a subset of HAC1-dependent UPR target genes in rta2?/? cells. Upon tunicamycin exposure, rta2?/? cells displayed a significantly reduced expression of UPR genes, in spite of only a moderate decrease in the HAC1 spliced mRNA levels and no change in Hac1 protein levels. Furthermore, hac1?/?rta2?/? cells display an exacerbated sensitivity to tunicamycin compared to the single mutants. We propose that functional RTA2 is requisite for the regulation of Hac1p-dependent UPR target genes to maximal levels, thereby assisting survival during ER stress. Collectively, this study proposes, for the first time, existence of an interplay between the Hac1p- and calcineurin- controlled networks via a downstream effector molecule of the latter, RTA2, to facilitate survival during ER stress in Candida albicans. PMID:26518191

  6. IL13 Receptor ?2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis.

    PubMed

    Bartolomé, Rubén A; García-Palmero, Irene; Torres, Sofía; López-Lucendo, María; Balyasnikova, Irina V; Casal, J Ignacio

    2015-06-15

    IL13 signaling through its receptor IL13R?2 plays a critical role in colon cancer invasion and liver metastasis, but the mechanistic features of this process are obscure. In this study, we identified a scaffold protein, FAM120A (C9ORF10), as a signaling partner in this process. FAM120A was overexpressed in human colon cancer cell lines and 55% of human colon cancer specimens. IL13R?2-FAM120A coimmunoprecipitation experiments revealed further signaling network associations that could regulate the activity of IL13R?2, including FAK, SRC, PI3K, G-protein-coupled receptors, and TRAIL receptors. In addition, FAM120A associated with kinesins and motor proteins involved in cargo movement along microtubules. IL13R?2-triggered activation of the FAK and PI3K/AKT/mTOR pathways was mediated by FAM120A, which also recruited PI3K and functioned as a scaffold protein to enable phosphorylation and activation of PI3K by Src family kinases. FAM120A silencing abolished IL13-induced cell migration, invasion, and survival. Finally, antibody blockade of IL13R?2 or FAM120A silencing precluded liver colonization in nude mice or metastasis. In conclusion, we identified FAM120A in the IL13/IL13R?2 signaling pathway as a key mediator of invasion and liver metastasis in colon cancer. PMID:25896327

  7. ERManI (Endoplasmic Reticulum Class I ?-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-01

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) ?-mannosidases, ERManI, and ER-degradation enhancing ?-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four ?-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 ?-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process. PMID:26205822

  8. Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing

    PubMed Central

    Naranjo, Silvia; Voesenek, Krysta; de la Calle-Mustienes, Elisa; Robert-Moreno, Alex; Kokotas, Haris; Grigoriadou, Maria; Economides, John; Van Camp, Guy; Hilgert, Nele; Moreno, Felipe; Alsina, Berta; Petersen, Michael B.; Kremer, Hannie

    2010-01-01

    POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development. Electronic supplementary material The online version of this article (doi:10.1007/s00439-010-0864-x) contains supplementary material, which is available to authorized users. PMID:20668882

  9. Pathway Analysis

    Cancer.gov

    Projects such as The Cancer Genome Atlas have gathered enormous quantities of data from human tumor samples. Informaticians at the National Lab are looking within such data for insights about the influence of mutant RAS genes on signaling pathways in cancers. On a smaller scale, the RAS Initiative will use numerous experimental platforms to interrogate cell lines expressing mutant RAS genes.

  10. Cross-talk between the mitogen activated protein kinase and bone morphogenetic protein/hemojuvelin pathways is required for the induction of hepcidin by holotransferrin in primary mouse hepatocytes

    PubMed Central

    Ramey, Guillemette; Deschemin, Jean-Christophe; Vaulont, Sophie

    2009-01-01

    Background The circulating hormone hepcidin plays a central role in iron homeostasis. Our goal was to establish an ex vivo iron-sensing model and to characterize the molecular mechanisms linking iron to hepcidin. Design and Methods Murine hepatocytes were isolated by the collagenase method, either from wild type or HFE knockout mice, and cultured 42 h without serum before treatments. Results After 42 h of serum-free culture, hepcidin gene expression was undetectable in the hepatocytes. Hepcidin gene expression could, however, be re-activated by an additional 24 h of incubation with 10% serum. Interestingly, addition of 30 ?M holotransferrin consistently increased serum-dependent hepcidin levels 3- to 5-fold. The effects of serum and serum+holotransferrin were direct, transcriptional, independent of de novo protein synthesis and required the presence of bone morphogenetic protein. Transferrin receptor-2 activation by its ligand holotransferrin led to extracellular signal regulated kinase (ERK)/mitogen activated protein kinase pathway stimulation and the ERK specific inhibitor U0-126 blunted holotransferrin-mediated induction of hepcidin. ERK activation by holotransferrin provoked increased levels of phospho-Smad1/5/8 highlighting cross-talk between the bone morphogenetic protein/hemojuvelin and ERK1/2 pathways. Finally, we demonstrated, using hepatocytes isolated from Hfe?/? mice, that HFE was not critical for the hepcidin response to holotransferrin but important for basal hepcidin expression. Conclusions We demonstrate that hepatocytes are liver iron-sensor cells and that transferrin receptor-2, by signaling through the ERK1/2 pathway, and bone morphogenetic protein/hemojuvelin, by signaling through the Smad pathways, coordinately regulate the iron-sensing machinery linking holotransferrin to hepcidin. PMID:19454495

  11. The lipoxygenase pathway.

    PubMed

    Feussner, Ivo; Wasternack, Claus

    2002-01-01

    Lipid peroxidation is common to all biological systems, both appearing in developmentally and environmentally regulated processes of plants. The hydroperoxy polyunsaturated fatty acids, synthesized by the action of various highly specialized forms of lipoxygenases, are substrates of at least seven different enzyme families. Signaling compounds such as jasmonates, antimicrobial and antifungal compounds such as leaf aldehydes or divinyl ethers, and a plant-specific blend of volatiles including leaf alcohols are among the numerous products. Cloning of many lipoxygenases and other key enzymes within the lipoxygenase pathway, as well as analyses by reverse genetic and metabolic profiling, revealed new reactions and the first hints of enzyme mechanisms, multiple functions, and regulation. These aspects are reviewed with respect to activation of this pathway as an initial step in the interaction of plants with pathogens, insects, or abiotic stress and at distinct stages of development. PMID:12221977

  12. Systems biology of endothelial mechano-activated pathways

    E-print Network

    Koo, Andrew Jia-An

    2013-01-01

    Multiple signaling pathways are employed by endothelial cells to differentially respond to distinct hemodynamic environments and acquire functional phenotypes, including regulation of inflammation, angiogenesis, blood ...

  13. INVESTIGATION Multiple Pathways of Duplication Formation

    E-print Network

    Roth, John R.

    A) in Salmonella enterica Andrew B. Reams, Eric Kofoid, Elisabeth Kugelberg, and John R. Roth1 Department/division) (Anderson and Roth 1981; Reams et al. 2010). 2. In unselected populations, the frequency of duplications

  14. A Comparison of Continuous Subcutaneous Insulin Infusion vs. Multiple Daily Insulin Injection in Children with Type I Diabetes in Kuwait: Glycemic Control, Insulin Requirement, and BMI

    PubMed Central

    Mousa, Mohammad; Al-Mahdi, Maria; Al-Sanaa, Hala; Al-Kandari, Hessa

    2015-01-01

    Objective Continuous subcutaneous insulin infusion (CSII) and multiple daily insulin injections (MDI) are two methods currently used to manage type I diabetes mellitus (T1DM). Here we compare our experiences with CSII and MDI in a large cohort of pediatric patients in Kuwait. Methods Data on 326 patients with T1DM who were started on CSII between 2007 and 2012 were retrospectively compared with those of 326 patients on MDI. They were matched for sex, age at diagnosis, T1DM duration, glycemic control, insulin requirement, and body mass index (BMI). Data were collected at baseline and every three months and included glycated hemoglobin (HbA1c), insulin dose, and adverse events (severe hypoglycemia, diabetic ketoacidosis, and skin problems). Results The main reason for switching to CSII was to achieve better glycemic control (37%), followed by reducing hypoglycemia, and improving the quality of life (13.3% each). Although HbA1c decrease was most significant in the first year, it continued to be significantly lower in the CSII group compared to the MDI throughout the study period. Total daily insulin requirements were significantly lower in the CSII group. BMI increased in both groups, but the difference was significant only at the end of the fifth year. There was no significant change in the rate of diabetic ketoacidosis in either group. The CSII patients had more severe hypoglycemic episodes at baseline; however, it significantly decreased throughout the study period. Only five patients discontinued CSII therapy and two of these restarted within three months. Conclusion CSII is a safe intensive insulin therapy in youngsters with T1DM and achieved markedly fewer severe hypoglycemic episodes and lower daily insulin requirements PMID:26421114

  15. PPE26 induces TLR2-dependent activation of macrophages and drives Th1-type T-cell immunity by triggering the cross-talk of multiple pathways involved in the host response.

    PubMed

    Su, Haibo; Kong, Cong; Zhu, Lin; Huang, Qi; Luo, Liulin; Wang, Honghai; Xu, Ying

    2015-11-17

    The pathophysiological functions and the underlying molecular basis of PE /PPE proteins of M. tuberculosis remain largely unknown. In this study, we focused on the link between PPE26 and host response. We demonstrated that PPE26 can induce extensive inflammatory responses in macrophages through triggering the cross-talk of multiple pathways involved in the host response, as revealed by iTRAQ-based subcellular quantitative proteomics. We observed that PPE26 is able to specifically bind to TLR2 leading to the subsequent activation of MAPKs and NF-?B signaling. PPE26 functionally stimulates macrophage activation by augmenting pro-inflammatory cytokine production (TNF-?, IL-6 and IL-12 p40) and the expression of cell surface markers (CD80, CD86, MHC class I and II). We observed that PPE26-treated macrophages effectively polarizes naïve CD4+ T cells to up-regulate CXCR3 expression, and to secrete IFN-? and IL-2, indicating PPE26 contributes to the Th1 polarization during the immune response. Importantly, rBCG::PPE26 induces stronger antigen-specific TNF-? and IFN-? activity, and higher levels of the Th1 cytokines TNF-? and IFN-? comparable to BCG. Moreover, PPE26 effectively induces the reciprocal expansion of effector/memory CD4+/CD8+ CD44highCD62Llow T cells in the spleens of mice immunized with this strain. These results suggest that PPE26 may be a TLR2 agonist that stimulates innate immunity and adaptive immunity, indicating that PPE26 is a potential antigen for the rational design of an efficient vaccine against M. tuberculosis. PMID:26439698

  16. An Evaluation of the Implementation of Maternal Obesity Pathways of Care: A Mixed Methods Study with Data Integration

    PubMed Central

    Heslehurst, Nicola; Dinsdale, Sarah; Sedgewick, Gillian; Simpson, Helen; Sen, Seema; Summerbell, Carolyn Dawn; Rankin, Judith

    2015-01-01

    Objectives Maternal obesity has multiple associated risks and requires substantial intervention. This research evaluated the implementation of maternal obesity care pathways from multiple stakeholder perspectives. Study Design A simultaneous mixed methods model with data integration was used. Three component studies were given equal priority. 1: Semi-structured qualitative interviews explored obese pregnant women’s experiences of being on the pathways. 2: A quantitative and qualitative postal survey explored healthcare professionals’ experiences of delivering the pathways. 3: A case note audit quantitatively assessed pathway compliance. Data were integrated using following a thread and convergence coding matrix methods to search for agreement and disagreement between studies. Results Study 1: Four themes were identified: women’s overall (positive and negative) views of the pathways; knowledge and understanding of the pathways; views on clinical and weight management advice and support; and views on the information leaflet. Key results included positive views of receiving additional clinical care, negative experiences of risk communication, and weight management support was considered a priority. Study 2: Healthcare professionals felt the pathways were worthwhile, facilitated good practice, and increased confidence. Training was consistently identified as being required. Healthcare professionals predominantly focussed on women’s response to sensitive obesity communication. Study 3: There was good compliance with antenatal clinical interventions. However, there was poor compliance with public health and postnatal interventions. There were some strong areas of agreement between component studies which can inform future development of the pathways. However, disagreement between studies included a lack of shared priorities between healthcare professionals and women, different perspectives on communication issues, and different perspectives on women’s prioritisation of weight management. Conclusion The differences between healthcare professionals’ and women’s priorities and perspectives are important factors to consider when developing care pathways. Shared perspectives could help facilitate more effective implementation of the pathway interventions that have poor compliance. PMID:26018338

  17. Arabidopsis Raf-Like Mitogen-Activated Protein Kinase Kinase Kinase Gene Raf43 Is Required for Tolerance to Multiple Abiotic Stresses

    PubMed Central

    Virk, Nasar; Li, Dayong; Tian, Limei; Huang, Lei; Hong, Yongbo; Li, Xiaohui; Zhang, Yafen; Liu, Bo; Zhang, Huijuan; Song, Fengming

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are critical signaling modules that mediate the transduction of extracellular stimuli into intracellular response. A relatively large number of MAPKKKs have been identified in a variety of plant genomes but only a few of them have been studied for their biological function. In the present study, we identified an Arabidopsis Raf-like MAPKKK gene Raf43 and studied its function in biotic and abiotic stress response using a T-DNA insertion mutant raf43-1 and two Raf43-overexpressing lines Raf43-OE#1 and Raf43-OE#13. Expression of Raf43 was induced by multiple abiotic and biotic stresses including treatments with drought, mannitol and oxidative stress or defense signaling molecule salicylic acid and infection with necrotrophic fungal pathogen Botrytis cinerea. Seed germination and seedling root growth of raf43-1 were significantly inhibited on MS medium containing mannitol, NaCl, H2O2 or methyl viologen (MV) while seed germination and seedling root growth of the Raf43-OE#1 and Raf43-OE#13 lines was similar to wild type Col-0 under the above stress conditions. Soil-grown raf43-1 plants exhibited reduced tolerance to MV, drought and salt stress. Abscisic acid inhibited significantly seed germination and seedling root growth of the raf43-1 line but had no effect on the two Raf43-overexpressing lines. Expression of stress-responsive RD17 and DREB2A genes was significantly down-regulated in raf43-1 plants. However, the raf43-1 and Raf43-overexpressing plants showed similar disease phenotype to the wild type plants after infection with B. cinerea or Pseudomonas syringae pv. tomato DC3000. Our results demonstrate that Raf43, encoding for a Raf-like MAPKKK, is required for tolerance to multiple abiotic stresses in Arabidopsis. PMID:26222830

  18. Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha.

    PubMed

    Silvestrini, Lucia; Rossi, Beatrice; Gallmetzer, Andreas; Mathieu, Martine; Scazzocchio, Claudio; Berardi, Enrico; Strauss, Joseph

    2015-01-01

    A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems. PMID:26335797

  19. Structural requirements and reaction pathways in condensation reactions of alcohols on Mg{sub y}AlO{sub x} catalysts

    SciTech Connect

    Di Cosimo, J.I.; Apesteguia, C.R.; Gines, M.J.L.; Iglesia, E.

    2000-03-10

    The effect of composition and of surface properties on alcohol-coupling reactions was studied on Mg{sub y}AlO{sub x} catalysts using C{sub 2}H{sub 5}OH or {sup 13}CH{sub 3}OH/1-{sup 12}C{sub 3}H{sub 7}OH mixtures as reactants. Samples with Mg/Al ratios of 0.5--9.0 were obtained by thermal decomposition of precipitated hydrotalcite precursors. The nature, density, and strength of surface basic sites were obtained by temperature-programmed desorption (TPD) of CO{sub 2} and by {sup 13}CO{sub 2}/{sup 12}CO isotopic switch methods, whereas the acid site densities were measured by TPD or NH{sub 3}. The catalyst ability for activating H-H bonds was investigated by performing H{sub 2}-D{sub 2} steady-state equilibration reactions. Isotopic tracer studies were carried out in order to probe chain growth pathways in the synthesis of isobutanol. The rates and product selectivity for C{sub 2}H{sub 5}OH or CH{sub 3}OH/C{sub 3}H{sub 7}OH reactions strongly depended on the chemical composition of Mg{sub y}AlO{sub x} samples. In turn, the chemical composition affected the acid-base properties of Mg{sub y}AlO{sub x} samples by modifying surface acid and base site densities and the distribution of strength for such sites. The rate of alcohol dehydration to ethers and olefins increased with increasing Al content. Al-rich Mg{sub y}AlO{sub x} samples contained a high density of Al{sup 3+}{minus}O{sup 2{minus}} site pairs and of moderate strength basic sites, the combination of which promoted the formation of ethylene or propylene from primary alcohols via E{sub 2} elimination pathways. The competitive dehydration to form ethers involved the adsorption of two alcohol molecules on neighboring active sites offering different acid-base properties.

  20. Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha

    PubMed Central

    Silvestrini, Lucia; Rossi, Beatrice; Gallmetzer, Andreas; Mathieu, Martine; Scazzocchio, Claudio; Berardi, Enrico; Strauss, Joseph

    2015-01-01

    A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems. PMID:26335797

  1. Protein design for pathway engineering

    SciTech Connect

    Eriksen, DT; Lian, JZ; Zhao, HM

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

  2. Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

    PubMed Central

    Yang, Ming; Wang, Shuo; Yue, Xiu-Li

    2014-01-01

    ABSTRACT Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids. IMPORTANCE To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132 deletion on the virus and multiple new findings on ac132. PMID:25142609

  3. Production Pathways and Separation Procedures for High-Diagnostic-Value Activation Species, Fission Products, and Actinides Required for Preparation of Realistic Synthetic Post-Detonation Nuclear Debris

    SciTech Connect

    Faye, S A; Shaughnessy, D A

    2015-08-19

    The objective of this project is to provide a comprehensive study on the production routes and chemical separation requirements for activation products, fission products, and actinides required for the creation of realistic post-detonation surrogate debris. Isotopes that have been prioritized by debris diagnosticians will be examined for their ability to be produced at existing irradiation sources, production rates, and availability of target materials, and chemical separation procedures required to rapidly remove the products from the bulk target matrix for subsequent addition into synthetic debris samples. The characteristics and implications of the irradiation facilities on the isotopes of interest will be addressed in addition to a summary of the isotopes that are already regularly produced.

  4. JWA is required for arsenic trioxide induced apoptosis in HeLa and MCF-7 cells via reactive oxygen species and mitochondria linked signal pathway

    SciTech Connect

    Zhou Jinhong; Ye Jian; Zhao Xiaojia; Li Aiping; Zhou Jianwei

    2008-07-01

    Arsenic trioxide, emerging as a standard therapy for refractory acute promyelocytic leukemia, induces apoptosis in a variety of malignant cell lines. JWA, a novel retinoic acid-inducible gene, is known to be involved in apoptosis induced by various agents, for example, 12-O-tetradecanoylphorbol 13-acetate, N-4-hydroxy-phenyl-retinamide and arsenic trioxide. However, the molecular mechanisms underlying how JWA gene is functionally involved in apoptosis remain largely unknown. Herein, our studies demonstrated that treatment of arsenic trioxide produced apoptosis in HeLa and MCF-7 cells in a dose-dependent manner and paralleled with increased JWA expression. JWA expression was dependent upon generation of intracellular reactive oxygen species induced by arsenic trioxide. Knockdown of JWA attenuated arsenic trioxide induced apoptosis, and was accompanied by significantly reduced activity of caspase-9, enhanced Bad phosphorylation and inhibited MEK1/2, ERK1/2 and JNK phosphorylations. Arsenic trioxide induced loss of mitochondrial transmembrane potential was JWA-dependent. These findings suggest that JWA may serve as a pro-apoptotic molecule to mediate arsenic trioxide triggered apoptosis via a reactive oxygen species and mitochondria-associated signal pathway.

  5. Analysis of Fc?RIIA Cytoplasmic Tail Requirements in Signaling for Serotonin Secretion: Evidence for an ITAM-dependent, PI3K-dependent Pathway

    PubMed Central

    Daniels, Anthony B.; Worth, Randall G.; Dickstein, Rian J.; Dickstein, Joshua S.; Kim-Han, Tae-Hee; Kim, Moo-Kyung; Schreiber, Alan D.

    2010-01-01

    The human Fc receptor, Fc?RIIA, is known to mediate phagocytosis and endocytosis, yet the greatest numbers of these receptors are expressed on the surface of non-phagocytic platelets, where they are involved in serotonin secretion. Fc?RIIA harbors three tyrosine (Y) residues within its cytoplasmic domain. Y1 is upstream of both Y2 and Y3, which are contained within an immunoreceptor tyrosine-based activation motif (ITAM), required for many signaling events. We have demonstrated that the two ITAM tyrosines are required for phagocytic signaling and that mutation of a single ITAM tyrosine decreases but does not abolish phagocytic signaling. Furthermore, we have identified that the YMTL motif is required for endocytosis. These observations suggest that Fc?RIIA utilizes different sequences for various signaling events. Therefore, we investigated the sequence requirements for another important Fc?RIIA-mediated signaling event, serotonin secretion, using Rat Basophilic Leukemia (RBL-2H3) cells transfected with wildtype (WT) Fc?RIIA or mutant Fc?RIIA. Stimulation of cells expressing WT Fc?RIIA induced release of serotonin at a level 7-fold greater than that in nonstimulated WT Fc?RIIA-transfected cells or nontransfected RBL cells. Mutation of either ITAM tyrosine (Y2 or Y3) to phenylalanine was sufficient to abolish serotonin secretion. Further, while inhibition of Syk with piceatannol blocked phagocytosis as expected, it did not inhibit serotonin secretion. Additionally, inhibition of phosphoinositol-3-kinase (PI3K) with wortmannin only had a partial effect on serotonin signaling, despite the fact that the concentrations used completely abolished phagocytic signaling. These data suggest that the requirements for serotonin secretion differ from those for phagocytosis mediated by Fc?RIIA. PMID:20384866

  6. Auditory pathways: anatomy and physiology.

    PubMed

    Pickles, James O

    2015-01-01

    This chapter outlines the anatomy and physiology of the auditory pathways. After a brief analysis of the external, middle ears, and cochlea, the responses of auditory nerve fibers are described. The central nervous system is analyzed in more detail. A scheme is provided to help understand the complex and multiple auditory pathways running through the brainstem. The multiple pathways are based on the need to preserve accurate timing while extracting complex spectral patterns in the auditory input. The auditory nerve fibers branch to give two pathways, a ventral sound-localizing stream, and a dorsal mainly pattern recognition stream, which innervate the different divisions of the cochlear nucleus. The outputs of the two streams, with their two types of analysis, are progressively combined in the inferior colliculus and onwards, to produce the representation of what can be called the "auditory objects" in the external world. The progressive extraction of critical features in the auditory stimulus in the different levels of the central auditory system, from cochlear nucleus to auditory cortex, is described. In addition, the auditory centrifugal system, running from cortex in multiple stages to the organ of Corti of the cochlea, is described. PMID:25726260

  7. Evaluation of microbial triglyceride oil purification requirements for the CelTherm process: an efficient biochemical pathway to renewable fuels and chemicals.

    PubMed

    Linnen, Michael; Seames, Wayne; Kubatova, Alena; Menon, Suresh; Alisala, Kashinatham; Hash, Sara

    2014-10-01

    CelTherm is a biochemical process to produce renewable fuels and chemicals from lignocellulosic biomass. The present study's objective was to determine the level of treatment/purity of the microbial triacylglyceride oil (TAG) necessary to facilitate fuel production. After a unique microbe aerobically synthesizes TAG from biomass-derived sugars, the microbes were harvested and dried then crude TAG was chemically extracted from the residual biomass. Some TAGs were further purified to hydrotreating process requirements. Both grades were then noncatalytically cracked into a petroleum-like intermediate characterized by gas chromatography. Experiments were repeated using refined soybean oil for comparison to previous studies. The products from crude microbial TAG cracking were then further refined into a jet fuel product. Fuel tests indicate that this jet fuel corresponds to specifications for JP-8 military turbine fuel. It was thus concluded that the crude microbial TAG is a suitable feedstock with no further purification required, demonstrating CelTherm's commercial potential. PMID:24781206

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

    PubMed

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

    2015-04-24

    p38 mitogen-activated protein kinases (MAPKs) play important roles in various cellular stress responses, including cell death, which is roughly categorized into apoptosis and necrosis. Although p38 signaling has been extensively studied, the molecular mechanisms of p38-mediated cell death are unclear. ASK1 is a stress-responsive MAP3K that acts as an upstream kinase of p38 and is activated by various stresses, such as oxidative stress. Here, we show that NR4A2, a member of the NR4A nuclear receptor family, acts as a necrosis promoter downstream of ASK1-p38 pathway during oxidative stress. Although NR4A2 is well known as a nucleus-localized transcription factor, we found that it is translocated into the cytosol after phosphorylation by p38. Because the phosphorylation site mutants of NR4A2 cannot rescue the cell death-promoting activity, ASK1-p38 pathway-dependent phosphorylation and subsequent cytoplasmic translocation of NR4A2 may be required for oxidative stress-induced cell death. In addition, NR4A2-mediated cell death does not depend on caspases and receptor-interacting protein 1 (RIP1)-RIP3 complex, suggesting that NR4A2 promotes an RIP kinase-independent necrotic type of cell death. Our findings may enable a more precise understanding of molecular mechanisms that regulate oxidative stress-induced and p38-mediated necrosis. PMID:25752609

  9. Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast.

    PubMed

    Vaga, Stefania; Bernardo-Faura, Marti; Cokelaer, Thomas; Maiolica, Alessio; Barnes, Christopher A; Gillet, Ludovic C; Hegemann, Björn; van Drogen, Frank; Sharifian, Hoda; Klipp, Edda; Peter, Matthias; Saez-Rodriguez, Julio; Aebersold, Ruedi

    2014-01-01

    Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time-resolved phosphorylation site dynamics after pathway co-stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone-induced down-regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways. PMID:25492886

  10. Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast

    PubMed Central

    Vaga, Stefania; Bernardo-Faura, Marti; Cokelaer, Thomas; Maiolica, Alessio; Barnes, Christopher A; Gillet, Ludovic C; Hegemann, Björn; van Drogen, Frank; Sharifian, Hoda; Klipp, Edda; Peter, Matthias; Saez-Rodriguez, Julio; Aebersold, Ruedi

    2014-01-01

    Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time-resolved phosphorylation site dynamics after pathway co-stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone-induced down-regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways. PMID:25492886

  11. The Protease-activated Receptor-2-specific Agonists 2-Aminothiazol-4-yl-LIGRL-NH2 and 6-Aminonicotinyl-LIGRL-NH2 Stimulate Multiple Signaling Pathways to Induce Physiological Responses in Vitro and in Vivo*

    PubMed Central

    Flynn, Andrea N.; Tillu, Dipti V.; Asiedu, Marina N.; Hoffman, Justin; Vagner, Josef; Price, Theodore J.; Boitano, Scott

    2011-01-01

    Protease-activated receptor-2 (PAR2) is one of four protease-activated G-protein-coupled receptors. PAR2 is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR2 reveals a tethered ligand that activates PAR2 and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca2+ signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR2 (e.g. SLIGRL-NH2 and SLIGKV-NH2) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH2 (2-f-LIGRLO-NH2) underscores the use of peptidomimetic PAR2 ligands as a mechanism to enhance pharmacological action at PAR2, 2-f-LIGRLO-NH2 has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH2 and two recently described pentapeptidomimetic PAR2-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at-LIGRL-NH2) and 6-aminonicotinyl-LIGRL-NH2 (6-an-LIGRL-NH2). All peptidomimetic agonists stimulated PAR2-dependent in vitro physiological responses, MAPK signaling, and Ca2+ signaling with an overall rank order of potency of 2-f-LIGRLO-NH2 ? 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2 ? SLIGRL-NH2. Because PAR2 plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca2+ signaling in nociceptors in vitro, and both 2-at-LIGRL-NH2 and 2-f-LIGRLO-NH2 stimulated PAR2-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR2 in a variety of systems and pathologies. PMID:21467041

  12. Characterization of the cis elements in the proximal promoter regions of the anthocyanin pathway genes reveals a common regulatory logic that governs pathway regulation

    PubMed Central

    Zhu, Zhixin; Wang, Hailong; Wang, Yiting; Guan, Shan; Wang, Fang; Tang, Jingyu; Zhang, Ruijuan; Xie, Lulu; Lu, Yingqing

    2015-01-01

    Cellular activities such as compound synthesis often require the transcriptional activation of an entire pathway; however, the molecular mechanisms underlying pathway activation have rarely been explained. Here, the cis regulatory architecture of the anthocyanin pathway genes targeted by the transcription factor (TF) complex including MYB, bHLH, and WDR was systematically analysed in one species and the findings extended to others. In Ipomoea purpurea, the IpMYB1-IpbHLH2-IpWDR1 (IpMBW) complex was found to be orthologous to the PAP1-GL3-TTG1 (AtPGT) complex of Arabidopsis thaliana, and interacted with a 7-bp MYB-recognizing element (MRE) and a 6-bp bHLH-recognizing element (BRE) at the proximal promoter region of the pathway genes. There was little transcription of the gene in the absence of the MRE or BRE. The cis elements identified experimentally converged on two syntaxes, ANCNNCC for MREs and CACN(A/C/T)(G/T) for BREs, and our bioinformatic analysis showed that these were present within anthocyanin gene promoters in at least 35 species, including both gymnosperms and angiosperms. For the anthocyanin pathway, IpMBW and AtPGT recognized the interspecific promoters of both early and later genes. In A. thaliana, the seed-specific TF complex (TT2, TT8, and TTG1) may regulate all the anthocyanin pathway genes, in addition to the proanthocyanidin-specific BAN. When multiple TF complexes in the anthocyanin pathway were compared, the cis architecture played a role larger than the TF complex in determining the variation in promoter activity. Collectively, a cis logic common to the pathway gene promoters was found, and this logic is essential for the trans factors to regulate the pathway. PMID:25911741

  13. Multiple Sclerosis

    MedlinePLUS

    MENU Return to Web version Multiple Sclerosis Overview What is multiple sclerosis (MS)? Multiple sclerosis is an autoimmune disease that affects the nervous system. Normally, antibodies produced by ...

  14. A genetic screen for mutations affecting gonad formation in Drosophila reveals a role for the slit/robo pathway

    PubMed Central

    Weyers, Jill J.; Milutinovich, Allison B.; Takeda, Yasuko; Jemc, Jennifer C.; Doren, Mark Van

    2013-01-01

    Organogenesis is a complex process requiring multiple cell types to associate with one another through correct cell contacts and in the correct location to achieve proper organ morphology and function. To better understand the mechanisms underlying gonad formation, we performed a mutagenesis screen in Drosophila and identified twenty-four genes required for gonadogenesis. These genes affect all different aspects of gonad formation and provide a framework for understanding the molecular mechanisms that control these processes. We find that gonad formation is regulated by multiple, independent pathways; some of these regulate the key cell adhesion molecule DE-cadherin, while others act through distinct mechanisms. In addition, we discover that the Slit/Roundabout pathway, best known for its role in regulating axonal guidance, is essential for proper gonad formation. Our findings shed light on the complexities of gonadogenesis and the genetic regulation required for proper organ formation. PMID:21377458

  15. The Exosome Component Rrp6 Is Required for RNA Polymerase II Termination at Specific Targets of the Nrd1-Nab3 Pathway

    PubMed Central

    Fox, Melanie J.; Gao, Hongyu; Smith-Kinnaman, Whitney R.; Liu, Yunlong; Mosley, Amber L.

    2015-01-01

    The exosome and its nuclear specific subunit Rrp6 form a 3’-5’ exonuclease complex that regulates diverse aspects of RNA biology including 3’ end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3’ end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3’ end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3’ RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3’ end RNA processing and/or degradation at specific targets. PMID:25680078

  16. Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

    PubMed Central

    Babayeva, Sima; Rocque, Brittany; Aoudjit, Lamine; Zilber, Yulia; Li, Jane; Baldwin, Cindy; Kawachi, Hiroshi; Takano, Tomoko; Torban, Elena

    2013-01-01

    The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/?-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate. PMID:23824190

  17. Improving carbon fixation pathways

    SciTech Connect

    Ducat, DC; Silver, PA

    2012-08-01

    A recent resurgence in basic and applied research on photosynthesis has been driven in part by recognition that fulfilling future food and energy requirements will necessitate improvements in crop carbon-fixation efficiencies. Photosynthesis in traditional terrestrial crops is being reexamined in light of molecular strategies employed by photosynthetic microbes to enhance the activity of the Calvin cycle. Synthetic biology is well-situated to provide original approaches for compartmentalizing and enhancing photosynthetic reactions in a species independent manner. Furthermore, the elucidation of alternative carbon-fixation routes distinct from the Calvin cycle raises possibilities that novel pathways and organisms can be utilized to fix atmospheric carbon dioxide into useful materials.

  18. Multiple sclerosis.

    PubMed

    Files, Daniel Kane; Jausurawong, Tani; Katrajian, Ruba; Danoff, Robert

    2015-06-01

    Multiple sclerosis (MS) is a chronic, debilitating disease that can have devastating effects. Presentation varies widely in symptoms, pace, and progression. In addition to a thorough history and physical examination, diagnostic tools required to diagnose MS and exclude other diagnoses include MRI, evoked potential testing, and cerebrospinal fluid analysis. Although the disease is not curable presently, quality of life can be improved by minimizing the frequency and severity of disease burden. Disease modification, symptom management, preservation of function, and treatment of psychosocial issues are paramount to enhance the quality of life for the patient affected with MS. PMID:25979578

  19. Too Many Mutants with Multiple Mutations

    PubMed Central

    Drake, John W.

    2007-01-01

    It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These “multiples” appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts. PMID:17687667

  20. Methotrexate Is a JAK/STAT Pathway Inhibitor

    PubMed Central

    Thomas, Sally; Fisher, Katherine H.; Snowden, John A.; Danson, Sarah J.; Brown, Stephen; Zeidler, Martin P.

    2015-01-01

    Background The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. Methods & Findings We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin. Conclusions Aminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, we suggest that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential. PMID:26131691

  1. SUSTAINABLE TRANSPORTATION ENERGY PATHWAYS

    E-print Network

    California at Davis, University of

    SUSTAINABLE TRANSPORTATION ENERGY PATHWAYS A Research Summary for Decision Makers Edited by Joan Ogden and Lorraine Anderson #12;SUSTAINABLE TRANSPORTATION ENERGY PATHWAYS #12;SUSTAINABLE TRANSPORTATION ENERGY PATHWAYS A Research Summary for Decision Makers Edited by Joan Ogden and Lorraine Anderson

  2. TARGETING 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE AND THE SHIKIMATE PATHWAY

    E-print Network

    Funke, Todd Arnold

    2008-08-21

    Bacteria, plants, fungi, and apicomplexan parasites require the functionality of the shikimate pathway for biosynthesis of essential aromatic compounds. Animals lack the enzymes that constitute the shikimate pathway, making these attractive...

  3. Minimal metabolic pathway structure is consistent with associated biomolecular interactions

    PubMed Central

    Bordbar, Aarash; Nagarajan, Harish; Lewis, Nathan E; Latif, Haythem; Ebrahim, Ali; Federowicz, Stephen; Schellenberger, Jan; Palsson, Bernhard O

    2014-01-01

    Pathways are a universal paradigm for functionally describing cellular processes. Even though advances in high-throughput data generation have transformed biology, the core of our biological understanding, and hence data interpretation, is still predicated on human-defined pathways. Here, we introduce an unbiased, pathway structure for genome-scale metabolic networks defined based on principles of parsimony that do not mimic canonical human-defined textbook pathways. Instead, these minimal pathways better describe multiple independent pathway-associated biomolecular interaction datasets suggesting a functional organization for metabolism based on parsimonious use of cellular components. We use the inherent predictive capability of these pathways to experimentally discover novel transcriptional regulatory interactions in Escherichia coli metabolism for three transcription factors, effectively doubling the known regulatory roles for Nac and MntR. This study suggests an underlying and fundamental principle in the evolutionary selection of pathway structures; namely, that pathways may be minimal, independent, and segregated. PMID:24987116

  4. The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

    PubMed

    de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

    2015-11-01

    The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa. PMID:26338184

  5. TEGS-CN: A Statistical Method for Pathway Analysis of Genome-wide Copy Number Profile

    PubMed Central

    Huang, Yen-Tsung; Hsu, Thomas; Christiani, David C

    2014-01-01

    The effects of copy number alterations make up a significant part of the tumor genome profile, but pathway analyses of these alterations are still not well established. We proposed a novel method to analyze multiple copy numbers of genes within a pathway, termed Test for the Effect of a Gene Set with Copy Number data (TEGS-CN). TEGS-CN was adapted from TEGS, a method that we previously developed for gene expression data using a variance component score test. With additional development, we extend the method to analyze DNA copy number data, accounting for different sizes and thus various numbers of copy number probes in genes. The test statistic follows a mixture of X2 distributions that can be obtained using permutation with scaled X2 approximation. We conducted simulation studies to evaluate the size and the power of TEGS-CN and to compare its performance with TEGS. We analyzed a genome-wide copy number data from 264 patients of non-small-cell lung cancer. With the Molecular Signatures Database (MSigDB) pathway database, the genome-wide copy number data can be classified into 1814 biological pathways or gene sets. We investigated associations of the copy number profile of the 1814 gene sets with pack-years of cigarette smoking. Our analysis revealed five pathways with significant P values after Bonferroni adjustment (<2.8 × 10?5), including the PTEN pathway (7.8 × 10?7), the gene set up-regulated under heat shock (3.6 × 10?6), the gene sets involved in the immune profile for rejection of kidney transplantation (9.2 × 10?6) and for transcriptional control of leukocytes (2.2 × 10?5), and the ganglioside biosynthesis pathway (2.7 × 10?5). In conclusion, we present a new method for pathway analyses of copy number data, and causal mechanisms of the five pathways require further study. PMID:25452685

  6. The autographa californica multiple nucleopolyhedrovirus ODV-E56 envelope protein is required for oral infectivity and can be functionally substituted by rachiplusia ou multiple nucleopolyhedrovirus ODV-E56

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of viral ho...

  7. SRNL ALL-PATHWAYS APPLICATION

    SciTech Connect

    Koffman, L; Elmer Wilhite, E; Leonard Collard, L

    2007-05-29

    The Environmental Analysis and Performance Modeling group of Savannah River National Laboratory (SRNL) performs performance assessments of the Savannah River Site (SRS) low-level waste facilities to meet the requirements of DOE Order 435.1. One of the performance objectives in the DOE Order is that the radiological dose to representative members of the public shall not exceed 25 mrem in a year total effective dose equivalent from all exposure pathways, excluding radon. Analysis to meet this performance objective is generally referred to as all-pathways analysis. SRNL performs detailed transient groundwater transport analysis for the waste disposal units, which has been used as input for the groundwater part of all-pathways analysis. The desire to better integrate all-pathways analysis with the groundwater transport analysis lead to the development of a software application named the SRNL All-Pathways Application. Another requirement of DOE Order 435.1 is to assess the impact of nuclear waste disposal on water resources, which SRS has interpreted for groundwater protection as meeting the EPA regulations for radionuclides in drinking water. EPA specifies four separate criteria as part of their implementation guidance for radionuclides, which are specified as maximum contaminant levels (MCL). (1) Beta/gamma emitters have a combined dose limit of 4 mrem/year. (2) Alpha emitters have a combined concentration limit of 15 pCi/L (called gross alpha), excluding uranium and radon, but including radium-226. (3) Combined radium-226 and radium-228 have a concentration limit of 5 pCi/L. (4) Isotopes of uranium have a combined concentration limit of 30 {micro}g/L. The All-Pathways Application was designed to be an easy-to-use software application that utilizes transient concentration results from groundwater transport analysis to (1) calculate the groundwater part of all-pathways dose and to (2) evaluate the four EPA criteria for groundwater protection.

  8. Major Academic Pathway (MAP) Spanish (concentration required)

    E-print Network

    Alexandrova, Ivana

    Elective Spring Semester 2 ASpn 301Z UL WI ASpn 223 HU Minor course Global and Cross Cultural Gen Ed GC (min. 3 crs.) GC Global and Cross-Cultural Studies (min 3 crs) DP U.S. Diversity and Pluralism (min. 3 Disciplinary Perspectives AR Arts (min. 3 crs.) Communication and Reasoning Competencies HU Humanities (min. 3

  9. A Distal Enhancer in Il12b Is the Target of Transcriptional Repression by the STAT3 Pathway and Requires the Basic Leucine Zipper (B-ZIP) Protein NFIL3*

    PubMed Central

    Smith, Amber M.; Qualls, Joseph E.; O'Brien, Kevin; Balouzian, Liza; Johnson, Peter F.; Schultz-Cherry, Stacey; Smale, Stephen T.; Murray, Peter J.

    2011-01-01

    Deregulated IL-12 and IL-23 production from activated myeloid lineage cells is a key driver of numerous T cell-dependent autoimmune and inflammatory diseases. IL-12 and IL-23 share a common p40 subunit encoded by Il12b, which is negatively regulated at the transcriptional level by the STAT3 (signal transducer and activator of transcription 3)-activating anti-inflammatory cytokine IL-10. We found that IL-10 targets an enhancer 10 kb upstream of the Il12b transcriptional start site. Within the enhancer, a single 10-bp site is required for the inhibitory effects of IL-10 and is bound by NFIL3 (nuclear factor, interleukin 3-regulated), a B-ZIP transcription factor. Myeloid cells lacking NFIL3 produce excessive IL-12p40 and increased IL-12p70. Thus, the STAT3-dependent expression of NFIL3 is a key component of a negative feedback pathway in myeloid cells that suppresses proinflammatory responses. PMID:21566115

  10. Multiple Sclerosis

    MedlinePLUS

    ... Awards Enhancing Diversity Find People About NINDS NINDS Multiple Sclerosis Information Page Condensed from Multiple Sclerosis: Hope Through ... en Español Additional resources from MedlinePlus What is Multiple Sclerosis? An unpredictable disease of the central nervous system, ...

  11. Experimental Approaches to Systematic Discovery and Development of Reproductive Adverse Outcome Pathways in Fish

    EPA Science Inventory

    Adverse outcome pathways (AOPs) are conceptual frameworks that portray causal and predictive linkages between key events at multiple scales of biological organization that connect molecular initiating events and early cellular perturbations (e.g., initiation of toxicity pathways)...

  12. Molecular pathways of angiogenesis inhibition

    SciTech Connect

    Tabruyn, Sebastien P.; Griffioen, Arjan W. . E-mail: aw.griffioen@path.unimaas.nl

    2007-03-30

    A large body of evidence now demonstrates that angiostatic therapy represents a promising way to fight cancer. This research recently resulted in the approval of First angiostatic agent for clinical treatment of cancer. Progress has been achieved in decrypting the cellular signaling in endothelial cells induced by angiostatic agents. These agents predominantly interfere with the molecular pathways involved in migration, proliferation and endothelial cell survival. In the current review, these pathways are discussed. A thorough understanding of the mechanism of action of angiostatic agents is required to develop efficient anti-tumor therapies.

  13. SRNL All-Pathways Application

    SciTech Connect

    Koffman, Larry D.; Wilhite, Elmer L.; Collard, Leonard B.

    2008-01-15

    The Environmental Analysis and Performance Modeling group of Savannah River National Laboratory (SRNL) performs performance assessments of the Savannah River Site (SRS) low-level waste facilities to meet the requirements of DOE Order 435.1. One of the performance objectives in the DOE Order is that the radiological dose to representative members of the public shall not exceed 25 mrem in a year total effective dose equivalent from all exposure pathways, excluding radon. Analysis to meet this performance objective is generally referred to as all-pathways analysis. SRNL performs detailed transient groundwater transport analysis for the waste disposal units, which has been used as input for the groundwater part of all-pathways analysis. The desire to better integrate all-pathways analysis with the groundwater transport analysis lead to the development of a software application named the SRNL All-Pathways Application. Another requirement of DOE Order 435.1 is to assess the impact of nuclear waste disposal on water resources, which SRS has interpreted for groundwater protection as meeting the EPA regulations for radionuclides in drinking water. EPA specifies four separate criteria as part of their implementation guidance for radionuclides, which are specified as maximum contaminant levels (MCL). 1. Beta/gamma emitters have a combined dose limit of 4 mrem/year. 2. Alpha emitters have a combined concentration limit of 15 pCi/L (called gross alpha), excluding uranium and radon, but including radium-226. 3. Combined radium-226 and radium-228 have a concentration limit of 5 pCi/L. 4. Isotopes of uranium have a combined concentration limit of 30 {mu}g/L. The All-Pathways Application was designed to be an easy-to-use software application that utilizes transient concentration results from groundwater transport analysis to (1) calculate the groundwater part of all-pathways dose and to (2) evaluate the four EPA criteria for groundwater protection. An application has been developed to streamline the assessment of performance objective criteria for groundwater using concentration results from groundwater transport analyses. The SRNL All-Pathways Application evaluates the groundwater portion of all-pathways analysis and also evaluates EPA groundwater protection criteria. The application can perform a calculation using a specified inventory to determine if that inventory meets performance objectives, or it can perform a sum of fractions calculation to provide results for each parent chain that are subsequently used to establish limits on the amount of parent radionuclides that can be placed in the waste disposal unit.

  14. The Timing of Multiple Retrieval Events Can Alter GluR1 Phosphorylation and the Requirement for Protein Synthesis in Fear Memory Reconsolidation

    ERIC Educational Resources Information Center

    Jarome, Timothy J.; Kwapis, Janine L.; Werner, Craig T.; Parsons, Ryan G.; Gafford, Georgette M.; Helmstetter, Fred J.

    2012-01-01

    Numerous studies have indicated that maintaining a fear memory after retrieval requires de novo protein synthesis. However, no study to date has examined how the temporal dynamics of repeated retrieval events affect this protein synthesis requirement. The present study varied the timing of a second retrieval of an established auditory fear memory…

  15. Logic models of pathway biology.

    PubMed

    Watterson, Steven; Marshall, Stephen; Ghazal, Peter

    2008-05-01

    Living systems seamlessly perform complex information processing and control tasks using combinatorially complex sets of biochemical reactions. Drugs that therapeutically modulate the biological processes of disease are developed using single protein target strategies, often with limited knowledge of the complex underlying role of the targets. Approaches that attempt to consider the combinatorial complexity from the outset might help identify any causal relationships that could lead to undesirable or adverse side effects earlier in the development pipeline. Such approaches, in particular logic methodologies, might also aid pathway selection and multiple target strategies during the drug discovery phase. Here, we describe the use of logic as a tractable and informative approach to modelling biological pathways that can allow us to improve our understanding of the dependencies in complex biological processes. PMID:18468563

  16. Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

    USGS Publications Warehouse

    Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

    2002-01-01

    Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.