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Sample records for copper-dioxygen complex mediated

  1. Copper-Dioxygen Complex Mediated C-H Bond Oxygenation: Relevance for Particulate Methane Monooxygenase (pMMO)

    OpenAIRE

    Himes, Richard A.; Karlin, Kenneth D.

    2009-01-01

    Particulate methane monooxygenase (pMMO), an integral membrane protein found in methanotrophic bacteria, catalyzes the oxidation of methane to methanol. Expression and greater activity of the enzyme in the presence of copper ion suggest that pMMO is a cuprous metalloenzyme. Recent advances – especially the first crystal structures of pMMO – have energized the field, but the nature of the active site(s) and the mechanism of methane oxidation remain poorly understood – yet hotly contested. Here...

  2. Complex Mediation

    DEFF Research Database (Denmark)

    Bødker, Susanne; Andersen, Peter Bøgh

    2005-01-01

    This article has its starting point in a large number of empirical findings regarding computer-mediated work. These empirical findings have challenged our understanding of the role of mediation in such work; on the one hand as an aspect of communication and cooperation at work and on the other hand...... as an aspect of human engagement with instruments of work. On the basis of previous work in activity-theoretical and semiotic human—computer interaction, we propose a model to encompass both of these aspects. In a dialogue with our empirical findings we move on to propose a number of types of mediation...... that have helped to enrich our understanding of mediated work and the design of computer mediation for such work....

  3. Copper dioxygen (bio)inorganic chemistry.

    Science.gov (United States)

    Solomon, Edward I; Ginsbach, Jake W; Heppner, David E; Kieber-Emmons, Matthew T; Kjaergaard, Christian H; Smeets, Pieter J; Tian, Li; Woertink, Julia S

    2011-01-01

    Cu/O2 intermediates in biological, homogeneous, and heterogeneous catalysts exhibit unique spectral features that reflect novel geometric and electronic structures that make significant contributions to reactivity. This review considers how the respective intermediate electronic structures overcome the spin-forbidden nature of O2 binding, activate O2 for electrophilic aromatic attack and H-atom abstraction, catalyze the 4 e- reduction of O2 to H2O, and discusses the role of exchange coupling between Cu ions in determining reactivity.

  4. The Mediator complex and transcription regulation

    Science.gov (United States)

    Poss, Zachary C.; Ebmeier, Christopher C.

    2013-01-01

    The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

  5. Catalytic Organic Transformations Mediated by Actinide Complexes

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    Isabell S. R. Karmel

    2015-10-01

    Full Text Available This review article presents the development of organoactinides and actinide coordination complexes as catalysts for homogeneous organic transformations. This chapter introduces the basic principles of actinide catalysis and deals with the historic development of actinide complexes in catalytic processes. The application of organoactinides in homogeneous catalysis is exemplified in the hydroelementation reactions, such as the hydroamination, hydrosilylation, hydroalkoxylation and hydrothiolation of alkynes. Additionally, the use of actinide coordination complexes for the catalytic polymerization of α-olefins and the ring opening polymerization of cyclic esters is presented. The last part of this review article highlights novel catalytic transformations mediated by actinide compounds and gives an outlook to the further potential of this field.

  6. Plant Mediator complex and its critical functions in transcription regulation.

    Science.gov (United States)

    Yang, Yan; Li, Ling; Qu, Li-Jia

    2016-02-01

    The Mediator complex is an important component of the eukaryotic transcriptional machinery. As an essential link between transcription factors and RNA polymerase II, the Mediator complex transduces diverse signals to genes involved in different pathways. The plant Mediator complex was recently purified and comprises conserved and specific subunits. It functions in concert with transcription factors to modulate various responses. In this review, we summarize the recent advances in understanding the plant Mediator complex and its diverse roles in plant growth, development, defense, non-coding RNA production, response to abiotic stresses, flowering, genomic stability and metabolic homeostasis. In addition, the transcription factors interacting with the Mediator complex are also highlighted.

  7. Proteomic Analysis of the Mediator Complex Interactome in Saccharomyces cerevisiae

    Science.gov (United States)

    Uthe, Henriette; Vanselow, Jens T.; Schlosser, Andreas

    2017-01-01

    Here we present the most comprehensive analysis of the yeast Mediator complex interactome to date. Particularly gentle cell lysis and co-immunopurification conditions allowed us to preserve even transient protein-protein interactions and to comprehensively probe the molecular environment of the Mediator complex in the cell. Metabolic 15N-labeling thereby enabled stringent discrimination between bona fide interaction partners and nonspecifically captured proteins. Our data indicates a functional role for Mediator beyond transcription initiation. We identified a large number of Mediator-interacting proteins and protein complexes, such as RNA polymerase II, general transcription factors, a large number of transcriptional activators, the SAGA complex, chromatin remodeling complexes, histone chaperones, highly acetylated histones, as well as proteins playing a role in co-transcriptional processes, such as splicing, mRNA decapping and mRNA decay. Moreover, our data provides clear evidence, that the Mediator complex interacts not only with RNA polymerase II, but also with RNA polymerases I and III, and indicates a functional role of the Mediator complex in rRNA processing and ribosome biogenesis. PMID:28240253

  8. Power Analysis for Complex Mediational Designs Using Monte Carlo Methods

    Science.gov (United States)

    Thoemmes, Felix; MacKinnon, David P.; Reiser, Mark R.

    2010-01-01

    Applied researchers often include mediation effects in applications of advanced methods such as latent variable models and linear growth curve models. Guidance on how to estimate statistical power to detect mediation for these models has not yet been addressed in the literature. We describe a general framework for power analyses for complex…

  9. Complement-mediated solubilization of immune complexes and their interaction with complement C3 receptors

    DEFF Research Database (Denmark)

    Petersen, Ivan; Baatrup, Gunnar; Jepsen, H H;

    1985-01-01

    Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components of the me......Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components...

  10. Mediated attachment as a mechanism for growth of complex networks

    CERN Document Server

    Shekatkar, Snehal M

    2014-01-01

    Connection topologies of many networked systems like human brain, biological cell, world wide web, power grids, human society and ecological food webs markedly deviate from that of completely random networks indicating the presence of organizing principles behind their evolution. The five important features that characterize such networks are scale-free topology, small average path length, high clustering, hierarchical community structure and assortative mixing. Till now the generic mechanisms underlying the existence of these properties are not well understood. Here we show that potentially a single mechanism, which we call "mediated attachment", where two nodes get connected through a mediator or common neighbor, could be responsible for the emergence of all important properties of real networks. The mediated attachment naturally unifies scale-free topology, high clustering, small world nature, hierarchical community structure and dissortative nature of networks. Further, with additional mixing by age, this...

  11. Emerging functions of multi-protein complex Mediator with special emphasis on plants.

    Science.gov (United States)

    Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh

    2017-10-01

    Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.

  12. Complement-mediated solubilization of immune complexes. Solubilization inhibition and complement factor levels in SLE patients

    DEFF Research Database (Denmark)

    Baatrup, Gunnar; Petersen, Ivan; Kappelgaard, E;

    1984-01-01

    Thirty-two of 36 serum samples from 19 SLE patients showed reduced capacity to mediate complement-dependent solubilization of immune complexes (IC). SLE patients with nephritis exerted the lowest complement-mediated solubilization capacity (CMSC) whereas sera from patients with inactive disease g...

  13. C-NH2 bond formation mediated by iridium complexes

    OpenAIRE

    Mena, Inmaculada; Casado, Miguel A.; Polo, Víctor; García-Orduña, P.; Lahoz, Fernando J.; Oro, Luis A.

    2014-01-01

    In the presence of phosphanes (PR3), the amido-bridged trinuclear complex [{Ir(μ-NH2)(tfbb)}3] (tfbb=tetrafluorobenzobarrelene) transforms into mononuclear discrete compounds [Ir(1,2-η2-4-κ-C12H8F 4N)(PR3)3], which are the products of the C-N coupling between the amido moiety and a vinylic carbon of the diolefin. An alternative synthetic approach to these species involves the reaction of the 18-e- complex [Ir(Cl)(tfbb)(PMePh2)2] with gaseous ammonia and additional phosphane. DFT studies show ...

  14. Cdk11-CyclinL Controls the Assembly of the RNA Polymerase II Mediator Complex

    Directory of Open Access Journals (Sweden)

    Julie Drogat

    2012-11-01

    Full Text Available The large Mediator (L-Mediator is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1 complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.

  15. Cdk11-cyclinL controls the assembly of the RNA polymerase II mediator complex.

    Science.gov (United States)

    Drogat, Julie; Migeot, Valérie; Mommaerts, Elise; Mullier, Caroline; Dieu, Marc; van Bakel, Harm; Hermand, Damien

    2012-11-29

    The large Mediator (L-Mediator) is a general coactivator of RNA polymerase II transcription and is formed by the reversible association of the small Mediator (S-Mediator) and the kinase-module-harboring Cdk8. It is not known how the kinase module association/dissociation is regulated. We describe the fission yeast Cdk11-L-type cyclin pombe (Lcp1) complex and show that its inactivation alters the global expression profile in a manner very similar to that of mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylates the Med27 and Med4 Mediator subunits on conserved residues. The association of the kinase module and the S-Mediator is strongly decreased by the inactivation of either Cdk11 or the mutation of its target residues on the Mediator. These results show that Cdk11-Lcp1 regulates the association of the kinase module and the S-Mediator to form the L-Mediator complex.

  16. Reduced complement-mediated immune complex solubilizing capacity and the presence of incompletely solubilized immune complexes in SLE sera

    DEFF Research Database (Denmark)

    Baatrup, Gunnar; Petersen, I; Jensenius, J C

    1983-01-01

    Reduced complement-mediated solubilization (CMS) of pre-formed immune complexes (IC) was demonstrated in sera from 11 out of 12 SLE patients. The presence of incompletely solubilized endogeneous IC in SLE sera was indicated by the following findings: (1) When IC positive SLE sera with reduced CMS...

  17. Complex-mediated microwave-assisted synthesis of polyacrylonitrile nanoparticles

    Directory of Open Access Journals (Sweden)

    Trinath Biswal

    2010-10-01

    Full Text Available Trinath Biswal, Ramakanta Samal, Prafulla K SahooDepartment of Chemistry, Utkal University, Vani Vihar, Bhubaneswar 751004, IndiaAbstract: The polymerization of acrylonitrile (AN is efficiently, easily, and quickly achieved in the presence of trans-[Co(IIIen2Cl2]Cl complex in a domestic microwave (MW oven. MW irradiation notably promoted the polymerization reaction; this phenomenon is ascribed to the acceleration of the initiator, ammonium persulfate (APS, decomposition by microwave irradiation in the presence of [Co(IIIen2Cl2]Cl. The conversion of monomer to the polymer was mostly excellent in gram scale. Irradiation at low power and time produced more homogeneous polymers with high molecular weight and low polydispersity when compared with the polymer formed by a conventional heating method. The interaction of reacting components was monitored by UV-visible spectrometer. The average molecular weight was derived by gel permeation chromatography (GPC, viscosity methods, and sound velocity by ultrasonic interferometer. The uniform and reduced molecular size was characterized by transmission electron microscopy, the diameter of polyacrylonitrile nanoparticles (PAN being in the range 50–115 nm and 40–230 nm in microwave and conventional heating methods respectively. The surface morphology of PAN prepared by MW irradiation was characterized by scanning electron microscope (SEM. From the kinetic results, the rate of polymerization (Rp was expressed as Rp = [AN]0.63 [APS]0.57 [complex (I].0.88Keywords: microwave, complex catalyst, nanoparticle, kinetics

  18. Comprehensive analysis of the transcriptional profile of the Mediator complex across human cancer types.

    Science.gov (United States)

    Syring, Isabella; Klümper, Niklas; Offermann, Anne; Braun, Martin; Deng, Mario; Boehm, Diana; Queisser, Angela; von Mässenhausen, Anne; Brägelmann, Johannes; Vogel, Wenzel; Schmidt, Doris; Majores, Michael; Schindler, Anne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg; Shaikhibrahim, Zaki; Perner, Sven

    2016-04-26

    The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking.We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

  19. C-NH2 bond formation mediated by iridium complexes.

    Science.gov (United States)

    Mena, Inmaculada; Casado, Miguel A; Polo, Víctor; García-Orduña, Pilar; Lahoz, Fernando J; Oro, Luis A

    2014-09-01

    In the presence of phosphanes (PR3 ), the amido-bridged trinuclear complex [{Ir(μ-NH2 )(tfbb)}3 ] (tfbb=tetrafluorobenzobarrelene) transforms into mononuclear discrete compounds [Ir(1,2-η(2) -4-κ-C12 H8 F4 N)(PR3 )3 ], which are the products of the CN coupling between the amido moiety and a vinylic carbon of the diolefin. An alternative synthetic approach to these species involves the reaction of the 18 e(-) complex [Ir(Cl)(tfbb)(PMePh2 )2 ] with gaseous ammonia and additional phosphane. DFT studies show that both transformations occur through nucleophilic attack. In the first case the amido moiety attacks a diolefin coordinated to a neighboring molecule following a bimolecular mechanism induced by the highly basic NH2 moiety; the second pathway involves a direct nucleophilic attack of ammonia to a coordinated tfbb molecule. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Transition metal complexes as mediator-titrants in protein redox potentiometry.

    Science.gov (United States)

    Bernhardt, Paul V; Chen, Kuan-I; Sharpe, Philip C

    2006-10-01

    A selection of nine macrocyclic Fe(III/II) and Co(III/II) transition metal complexes has been chosen to serve as a universal set of mediator-titrants in redox potentiometry of protein samples. The potential range spanned by these mediators is approximately from +300 to -700 mV vs the normal hydrogen electrode, which covers the range of most protein redox potentials accessible in aqueous solution. The complexes employed exhibit stability in both their oxidized and their reduced forms as well as pH-independent redox potentials within the range 6 < pH < 9. The mediators were also chosen on the basis of their very weak visible absorption maxima in both oxidation states, which will enable (for the first time) optical redox potentiometric titrations of proteins with relatively low extinction coefficients. This has previously been impractical with organic mediators, such as indoles, viologens and quinones, whose optical spectra interfere strongly with those of the protein.

  1. A standardized method for quantitating the complement-mediated immune complex solubilizing capacity of human serum

    DEFF Research Database (Denmark)

    Baatrup, G; Peterson, I; Svehag, S E;

    1983-01-01

    A standardized radioassay for measuring the complement-mediated immune complex solubilizing capacity (CMSC) and the initial kinetics of the solubilization (IKS) reaction is described. The total complement (C)-mediated solubilizing capacity was determined after incubation of diluted serum and 125I......-BSA-anti-BSA. Percentage C-mediated solubilization (CMS) was measured after centrifugation by determining the distribution of radioactivity. The dependency of CMSC upon factors such as serum dilution and buffer system used, amount of IC added to serum, serum storage conditions and centrifugation conditions...

  2. Generalization mediates sensitivity to complex odor features in the honeybee.

    Directory of Open Access Journals (Sweden)

    Geraldine A Wright

    Full Text Available Animals use odors as signals for mate, kin, and food recognition, a strategy which appears ubiquitous and successful despite the high intrinsic variability of naturally-occurring odor quantities. Stimulus generalization, or the ability to decide that two objects, though readily distinguishable, are similar enough to afford the same consequence, could help animals adjust to variation in odor signals without losing sensitivity to key inter-stimulus differences. The present study was designed to investigate whether an animal's ability to generalize learned associations to novel odors can be influenced by the nature of the associated outcome. We use a classical conditioning paradigm for studying olfactory learning in honeybees to show that honeybees conditioned on either a fixed- or variable-proportion binary odor mixture generalize learned responses to novel proportions of the same mixture even when inter-odor differences are substantial. We also show that the resulting olfactory generalization gradients depend critically on both the nature of the stimulus-reward paradigm and the intrinsic variability of the conditioned stimulus. The reward dependency we observe must be cognitive rather than perceptual in nature, and we argue that outcome-dependent generalization is necessary for maintaining sensitivity to inter-odor differences in complex olfactory scenes.

  3. NMNAT2:HSP90 Complex Mediates Proteostasis in Proteinopathies.

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    Yousuf O Ali

    2016-06-01

    Full Text Available Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2 is neuroprotective in numerous preclinical models of neurodegeneration. Here, we show that brain nmnat2 mRNA levels correlate positively with global cognitive function and negatively with AD pathology. In AD brains, NMNAT2 mRNA and protein levels are reduced. NMNAT2 shifts its solubility and colocalizes with aggregated Tau in AD brains, similar to chaperones, which aid in the clearance or refolding of misfolded proteins. Investigating the mechanism of this observation, we discover a novel chaperone function of NMNAT2, independent from its enzymatic activity. NMNAT2 complexes with heat shock protein 90 (HSP90 to refold aggregated protein substrates. NMNAT2's refoldase activity requires a unique C-terminal ATP site, activated in the presence of HSP90. Furthermore, deleting NMNAT2 function increases the vulnerability of cortical neurons to proteotoxic stress and excitotoxicity. Interestingly, NMNAT2 acts as a chaperone to reduce proteotoxic stress, while its enzymatic activity protects neurons from excitotoxicity. Taken together, our data indicate that NMNAT2 exerts its chaperone or enzymatic function in a context-dependent manner to maintain neuronal health.

  4. Evidence for copper-dioxygen reactivity during alpha-synuclein fibril formation.

    Science.gov (United States)

    Lucas, Heather R; Debeer, Serena; Hong, Myoung-Soon; Lee, Jennifer C

    2010-05-19

    Alpha-synuclein (alpha-syn), a presynaptic protein implicated in Parkinson's disease, binds copper(II) ion (1:1) with submicromolar affinity in vitro. Insights on the molecular details of soluble- and fibrillar-Cu-alpha-syn are gained through X-ray absorption spectroscopy. Our results indicate that the copper coordination environment (3-to-4 N/O ligands, average Cu-ligand distance approximately 1.96 A) exhibits little structural rearrangement upon amyloid formation in spite of the overall polypeptide conformational change from disordered-to-beta-sheet. Interestingly, we find that some population of Cu(II)-alpha-syn reduces to Cu(I)-alpha-syn in the absence of O(2). This autoreduction event appears diminished in the presence of O(2) suggestive of preceding Cu(I)/O(2) chemistry. Evidence for generation of reactive oxygen species is obtained by the observation of new emission features attributed to dityrosine cross-links in fibrillar samples.

  5. Core Self-Evaluations as Causes of Satisfaction: The Mediating Role of Seeking Task Complexity

    Science.gov (United States)

    Srivastava, Abhishek; Locke, Edwin A.; Judge, Timothy A.; Adams, John W.

    2010-01-01

    This study examined the mediating role of task complexity in the relationship between core self-evaluations (CSE) and satisfaction. In Study 1, eighty three undergraduate business students worked on a strategic decision-making simulation. The simulated environment enabled us to verify the temporal sequence of variables, use an objective measure of…

  6. Core Self-Evaluations as Causes of Satisfaction: The Mediating Role of Seeking Task Complexity

    Science.gov (United States)

    Srivastava, Abhishek; Locke, Edwin A.; Judge, Timothy A.; Adams, John W.

    2010-01-01

    This study examined the mediating role of task complexity in the relationship between core self-evaluations (CSE) and satisfaction. In Study 1, eighty three undergraduate business students worked on a strategic decision-making simulation. The simulated environment enabled us to verify the temporal sequence of variables, use an objective measure of…

  7. Peer-Mediated vs. Individual Writing: Measuring Fluency, Complexity, and Accuracy in Writing

    Science.gov (United States)

    Soleimani, Maryam; Modirkhamene, Sima; Sadeghi, Karim

    2017-01-01

    Drawing upon Vygotsky's Sociocultural Theory (SCT), this study aimed at investigating the effect of two writing modes, namely, peer-mediated/collaborative vs. individual writing on measures of fluency, accuracy, and complexity of female EFL learners' writing. Based on an in-house placement test and the First Certificate in English writing paper, a…

  8. Electron Transfer Mediators for Photoelectrochemical Cells Based on Cu(I Metal Complexes

    Directory of Open Access Journals (Sweden)

    Michele Brugnati

    2007-01-01

    Full Text Available The preparation and the photoelectrochemical characterization of a series of bipyridine and pyridyl-quinoline Cu(I complexes, used as electron transfer mediators in regenerative photoelectrochemical cells, are reported. The best performing mediators produced maximum IPCEs of the order of 35–40%. The J-V curves recorded under monochromatic light showed that the selected Cu(I/(II couples generated higher Vocs and fill factors compared to an equivalent I-/I3- cell, due to a decreased dark current.

  9. Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

    Science.gov (United States)

    Jin, Shunying; Merchant, Michael L; Ritzenthaler, Jeffrey D; McLeish, Kenneth R; Lederer, Eleanor D; Torres-Gonzalez, Edilson; Fraig, Mostafa; Barati, Michelle T; Lentsch, Alex B; Roman, Jesse; Klein, Jon B; Rane, Madhavi J

    2015-01-01

    Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a

  10. Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

    Directory of Open Access Journals (Sweden)

    Shunying Jin

    Full Text Available Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC deposition-induced acute lung injury (ALI. Components of gamma amino butyric acid (GABA signaling, including GABA B receptor 2 (GABABR2, GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP, in the bronchoalveolar lavage fluid (BALF. Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting

  11. Polymorphism of DNA-anionic liposome complexes reveals hierarchy of ion-mediated interactions.

    Science.gov (United States)

    Liang, Hongjun; Harries, Daniel; Wong, Gerard C L

    2005-08-09

    Self-assembled DNA delivery systems based on anionic lipids (ALs) complexed with DNA mediated by divalent cations have been recently introduced as an alternative to cationic lipid-DNA complexes because of their low cytotoxicity. We investigate AL-DNA complexes induced by different cations by using synchrotron small angle x-ray scattering and confocal microscopy to show how different ion-mediated interactions are expressed in the self-assembled structures and phase behavior of AL-DNA complexes. The governing interactions in AL-DNA systems are complex: divalent ions can mediate strong attractions between different combinations of the components (such as DNA-DNA and membrane-membrane). Moreover, divalent cations can coordinate non-electrostatically with lipids and modify the resultant membrane structure. We find that at low membrane charge densities AL-DNA complexes organize into a lamellar structure of alternating DNA and membrane layers crosslinked by ions. At high membrane charge densities, a new phase with no analog in cationic lipid-DNA systems is observed: DNA is expelled from the complex, and a lamellar stack of membranes and intercalated ions is formed. For a subset of the ionic species, high ion concentrations generate an inverted hexagonal phase comprised of DNA strands wrapped by ion-coated lipid tubes. A simple theoretical model that takes into account the electrostatic and membrane elastic contributions to the free energy shows that this transition is consistent with an ion-induced change in the membrane spontaneous curvature, c0. Moreover, the crossover between the lamellar and inverted hexagonal phases occurs at a critical c0 that agrees well with experimental values.

  12. Structural basis for Spt5-mediated recruitment of the Paf1 complex to chromatin.

    Science.gov (United States)

    Wier, Adam D; Mayekar, Manasi K; Héroux, Annie; Arndt, Karen M; VanDemark, Andrew P

    2013-10-22

    Polymerase associated factor 1 complex (Paf1C) broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription elongation. The Plus3 domain of the Rtf1 subunit mediates Paf1C recruitment to genes by binding a repeating domain within the elongation factor Spt5 (suppressor of Ty). Here we provide a molecular description of this interaction by reporting the structure of human Rtf1 Plus3 in complex with a phosphorylated Spt5 repeat. We find that Spt5 binding is mediated by an extended surface containing phosphothreonine recognition and hydrophobic interfaces that interact with residues outside the Spt5 motif. Changes within these interfaces diminish binding of Spt5 in vitro and chromatin localization of Rtf1 in vivo. The structure reveals the basis for recognition of the repeat motif of Spt5, a key player in the recruitment of gene regulatory factors to RNA polymerase II.

  13. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of); Shin, Ki Soon [Department of Biology, Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kang, Shin Jung, E-mail: sjkang@sejong.ac.kr [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of)

    2013-08-09

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

  14. Transferrin serves as a mediator to deliver organometallic ruthenium(II) anticancer complexes into cells.

    Science.gov (United States)

    Guo, Wei; Zheng, Wei; Luo, Qun; Li, Xianchan; Zhao, Yao; Xiong, Shaoxiang; Wang, Fuyi

    2013-05-06

    We report herein a systematic study on interactions of organometallic ruthenium(II) anticancer complex [(η(6)-arene)Ru(en)Cl](+) (arene = p-cymene (1) or biphenyl (2), en = ethylenediamine) with human transferrin (hTf) and the effects of the hTf-ligation on the bioavailability of these complexes with cisplatin as a reference. Incubated with a 5-fold excess of complex 1, 2, or cisplatin, 1 mol of diferric hTf (holo-hTf) attached 0.62 mol of 1, 1.01 mol of 2, or 2.14 mol of cisplatin. Mass spectrometry revealed that both ruthenium complexes coordinated to N-donors His242, His273, His578, and His606, whereas cisplatin bound to O donors Tyr136 and Tyr317 and S-donor Met256 in addition to His273 and His578 on the surface of both apo- and holo-hTf. Moreover, cisplatin could bind to Thr457 within the C-lobe iron binding cleft of apo-hTf. Neither ruthenium nor platinum binding interfered with the recognition of holo-hTf by the transferrin receptor (TfR). The ruthenated/platinated holo-hTf complexes could be internalized via TfR-mediated endocytosis at a similar rate to that of holo-hTf itself. Moreover, the binding to holo-hTf well preserved the bioavailability of the ruthenium complexes, and the hTf-bound 1 and 2 showed a similar cytotoxicity toward the human breast cancer cell line MCF-7 to those of the complexes themselves. However, the conjugation with holo-hTf significantly reduced the cellular uptake of cisplatin and the amount of platinated DNA adducts formed intracellularly, leading to dramatic reduction of cisplatin cytotoxicity toward MCF-7. These findings suggest that hTf can serve as a mediator for the targeting delivery of Ru(arene) anticancer complexes while deactivating cisplatin.

  15. Dia2 controls transcription by mediating assembly of the RSC complex.

    Directory of Open Access Journals (Sweden)

    Edward J Andress

    Full Text Available BACKGROUND: Dia2 is an F-box protein found in the budding yeast, S. cerevisiae. Together with Skp1 and Cul1, Dia2 forms the substrate-determining part of an E3 ubiquitin ligase complex, otherwise known as the SCF. Dia2 has previously been implicated in the control of replication and genome stability via its interaction with the replisome progression complex. PRINCIPAL FINDINGS: We identified components of the RSC chromatin remodelling complex as genetic interactors with Dia2, suggesting an additional role for Dia2 in the regulation of transcription. We show that Dia2 is involved in controlling assembly of the RSC complex. RSC belongs to a group of ATP-dependent nucleosome-remodelling complexes that controls the repositioning of nucleosomes. The RSC complex is expressed abundantly and its 17 subunits are recruited to chromatin in response to both transcription activation and repression. In the absence of Dia2, RSC-mediated transcription regulation was impaired, with concomitant abnormalities in nucleosome positioning. CONCLUSIONS: Our findings imply that Dia2 is required for the correct assembly and function of the RSC complex. Dia2, by controlling the RSC chromatin remodeller, fine-tunes transcription by controlling nucleosome positioning during transcriptional activation and repression.

  16. Identifying causal gateways and mediators in complex spatio-temporal systems

    Science.gov (United States)

    Runge, Jakob; Petoukhov, Vladimir; Donges, Jonathan; Hlinka, Jaroslav; Jajcay, Nikola; Vejmelka, Martin; Hartman, David; Marwan, Norbert; Palus, Milan; Kurths, Jürgen

    2016-04-01

    Identifying regions important for spreading and mediating perturbations is crucial to assess the susceptibilities of spatio-temporal complex systems such as the Earth's climate to volcanic eruptions, extreme events or geoengineering. Here a data-driven approach is introduced based on a dimension reduction, causal reconstruction, and novel network measures based on causal effect theory that go beyond standard complex network tools by distinguishing direct from indirect pathways. Applied to a data set of atmospheric dynamics, the method identifies several strongly uplifting regions acting as major gateways of perturbations spreading in the atmosphere. Additionally, the method provides a stricter statistical approach to pathways of atmospheric teleconnections, yielding insights into the Pacific-Indian Ocean interaction relevant for monsoonal dynamics. The novel causal interaction perspective provides a complementary approach to simulations or experiments for understanding the functioning of complex spatio-temporal systems with potential applications in increasing their resilience to shocks or extreme events. Reference: Runge, J., Petoukhov, V., Donges, J. F., Hlinka, J., Jajcay, N., Vejmelka, M., Hartman, D., Marwan, M., Paluš, M., Kurths, J. (2015). Identifying causal gateways and mediators in complex spatio-temporal systems. Nature Communications, 6, 8502. doi:10.1038/ncomms9502

  17. Differential role of lipocalin-2 during immune-complex mediated acute and chronic inflammation

    Science.gov (United States)

    Shashidharamurthy, Rangaiah; Machiah, Deepa; Aitken, Jesse D; Putty, Kalyani; Srinivasan, Gayathri; Chassaing, Benoit; Parkos, Charles A; Selvaraj, Periasamy; Vijay-Kumar, Matam

    2013-01-01

    Objectives Lipocalin-2 (Lcn2) is an innate immune protein expressed by a variety of cells and is highly upregulated during several pathological conditions including immune-complex (IC) mediated inflammatory/autoimmune disorders. However, the function of Lcn2 during IC-mediated inflammation is largely unknown. Therefore our objective was to investigate the role of Lcn2 in IC-mediated diseases. Methods The upregulation of Lcn2 was determined by ELISA in three different mouse models of IC-mediated autoimmune disease: systemic lupus erythematosus, collagen-induced arthritis and serum-induced arthritis. The in vivo role of Lcn2 during IC-mediated inflammation was investigated using Lcn2 knockout (Lcn2KO) mice and their wild type (WT) littermates. Results Lcn2 levels were significantly elevated in all the three autoimmune disease models. Further, in an acute skin inflammation model, Lcn2KO mice demonstrated a 50% reduction in inflammation with histopathological analysis revealing strikingly reduced immune cell infiltration compared to WT mice. Administration of recombinant Lcn2 to Lcn2KO mice restored inflammation to levels observed in WT mice. Neutralization of Lcn2 using a monoclonal antibody significantly reduced inflammation in WT mice. In contrast, Lcn2KO mice developed more severe serum-induced arthritis compared to WT mice. Histological analysis revealed extensive tissue and bone destruction with significantly reduced neutrophil infiltration but considerably more macrophage migration in Lcn2KO mice when compared to WT. Conclusion These results demonstrate that Lcn2 may regulate immune cell recruitment to the site of inflammation, a process essential for the controlled initiation, perpetuation and resolution of inflammatory processes. Thus, Lcn2 may present a promising target in the treatment of IC-mediated inflammatory/autoimmune diseases. PMID:23280250

  18. A study of the Fenton-mediated oxidation of methylene blue-cucurbit[n]uril complexes.

    Science.gov (United States)

    Fuenzalida, Tomás; Fuentealba, Denis

    2015-04-01

    Cucurbit[n]urils efficiently decreased the Fenton-mediated oxidation of encapsulated dyes, providing a mechanism for some control and selectivity over the degradation. The encapsulation of methylene blue into cucurbit[7]uril made it highly refractory against Fenton oxidation in the dark or under UVA light irradiation. However, the oxidation of the encapsulated dye was significantly enhanced under visible light irradiation. This behavior was selective for the cucurbit[7]uril complex and not for the cucurbit[8]uril complex, which achieved the same degree of protection irrespective of the irradiation conditions. This different reactivity of the complexes was further discussed in terms of their excited state properties. The main mechanism for protection was the seclusion of the dye into cucurbit[n]urils as shown by the fact that the non-encapsulated dye safranin was protected much less than methylene blue. Additionally, cucurbit[n]urils efficiently trapped hydroxyl radicals, which contributed significantly to the protection of the dyes from Fenton-mediated oxidation.

  19. The talin head domain reinforces integrin-mediated adhesion by promoting adhesion complex stability and clustering.

    Directory of Open Access Journals (Sweden)

    Stephanie J Ellis

    2014-11-01

    Full Text Available Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.

  20. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Somanath, Sangeeta [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Partridge, Christopher J. [Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Churchill Hospital, University of Oxford, Oxford, OX3 7LJ (United Kingdom); Marshall, Catriona [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Rowe, Tony [CSL Limited, 45 Poplar Road, Parkville, Victoria 3052 (Australia); Turner, Mark D., E-mail: mark.turner@ntu.ac.uk [Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS (United Kingdom)

    2016-04-29

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.

  1. Ammonia-Borane and Amine-Borane Dehydrogenation Mediated by Complex Metal Hydrides.

    Science.gov (United States)

    Rossin, Andrea; Peruzzini, Maurizio

    2016-08-10

    This review is a comprehensive survey of the last 10 years of research on ammonia-borane and amine-borane dehydrogenation mediated by complex metal hydrides (CMHs), within the broader context of chemical hydrogen storage. The review also collects those cases where CMHs are the catalyst spent form or its resting state. Highlights on the reaction mechanism (strictly dependent on the CMH of choice) and the catalysts efficiency (in terms of equivalents of H2 produced and relative reaction rates) are provided throughout the discussion.

  2. Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma.

    Science.gov (United States)

    Verstraete, Kenneth; Peelman, Frank; Braun, Harald; Lopez, Juan; Van Rompaey, Dries; Dansercoer, Ann; Vandenberghe, Isabel; Pauwels, Kris; Tavernier, Jan; Lambrecht, Bart N; Hammad, Hamida; De Winter, Hans; Beyaert, Rudi; Lippens, Guy; Savvides, Savvas N

    2017-04-03

    The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.

  3. Transition Metal Polypyridine Complexes: Studies of Mediation in Dye-Sensitized Solar Cells and Charge Separation

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, C. Michael [Colorado State Univ., Fort Collins, CO (United States). Dept. of Chemistry; Prieto, Amy L. [Colorado State Univ., Fort Collins, CO (United States). Dept. of Chemistry

    2017-02-08

    The Elliott group has long been supported by DOE for studies of cobalt(II/III) trisbypiridine (DTB) mediator complexes in dye sensitized solar cells. Previous work demonstrated that Co(II/III) chemistry is sensitive to the environment, showing unprecedented electrode-surface and electrolyte dependant voltammetry. In electrolytes that have large lipophilic cations, voltammetry of the [Co(DTB)3]2+/3+ couple is nearly Nernstian in appearance on nominally oxide-free metal surfaces. In contrast, on semiconductor electrodes in electrolytes with small, hard cations such as Li+, the electron transfer rates are so slow that it is difficult to measure any Faradaic current even at overpotentials of ±1 V. These studies are of direct relevance to the operation of cobalt-based mediators in solar cells. The research has also shown that these mediators are compatible with copper phenantroline based dyes, in contrast to I- due to the insolubility of CuI.

  4. The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis.

    Science.gov (United States)

    Weimer, Annika K; Biedermann, Sascha; Harashima, Hirofumi; Roodbarkelari, Farshad; Takahashi, Naoki; Foreman, Julia; Guan, Yonsheng; Pochon, Gaëtan; Heese, Maren; Van Damme, Daniël; Sugimoto, Keiko; Koncz, Csaba; Doerner, Peter; Umeda, Masaaki; Schnittger, Arp

    2016-10-04

    Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  5. A functional portrait of Med7 and the mediator complex in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Faiza Tebbji

    2014-11-01

    Full Text Available Mediator is a multi-subunit protein complex that regulates gene expression in eukaryotes by integrating physiological and developmental signals and transmitting them to the general RNA polymerase II machinery. We examined, in the fungal pathogen Candida albicans, a set of conditional alleles of genes encoding Mediator subunits of the head, middle, and tail modules that were found to be essential in the related ascomycete Saccharomyces cerevisiae. Intriguingly, while the Med4, 8, 10, 11, 14, 17, 21 and 22 subunits were essential in both fungi, the structurally highly conserved Med7 subunit was apparently non-essential in C. albicans. While loss of CaMed7 did not lead to loss of viability under normal growth conditions, it dramatically influenced the pathogen's ability to grow in different carbon sources, to form hyphae and biofilms, and to colonize the gastrointestinal tracts of mice. We used epitope tagging and location profiling of the Med7 subunit to examine the distribution of the DNA sites bound by Mediator during growth in either the yeast or the hyphal form, two distinct morphologies characterized by different transcription profiles. We observed a core set of 200 genes bound by Med7 under both conditions; this core set is expanded moderately during yeast growth, but is expanded considerably during hyphal growth, supporting the idea that Mediator binding correlates with changes in transcriptional activity and that this binding is condition specific. Med7 bound not only in the promoter regions of active genes but also within coding regions and at the 3' ends of genes. By combining genome-wide location profiling, expression analyses and phenotyping, we have identified different Med7p-influenced regulons including genes related to glycolysis and the Filamentous Growth Regulator family. In the absence of Med7, the ribosomal regulon is de-repressed, suggesting Med7 is involved in central aspects of growth control.

  6. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  7. Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ruochan [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Fu, Sha; Fan, Xue-Gong [Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Lotze, Michael T.; Zeh, Herbert J. [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Tang, Daolin, E-mail: tangd2@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Kang, Rui, E-mail: kangr@upmc.edu [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-03-13

    High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis and necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.

  8. Mediator complex subunit 12 exon 2 mutation analysis in different subtypes of smooth muscle tumors confirms genetic heterogeneity.

    Science.gov (United States)

    de Graaff, Marieke A; Cleton-Jansen, Anne-Marie; Szuhai, Károly; Bovée, Judith V M G

    2013-08-01

    Recently, heterozygous mutations in exon 2 of the mediator complex subunit 12 gene have been described in 50% to 70% of uterine leiomyomas; the recurrent nature of these mutations suggests an important role in their pathogenesis. Mediator complex subunit 12 is involved in regulation of transcription and Wnt signaling. So far, little is known about the pathogenesis of the different subtypes of extrauterine leiomyomas and leiomyosarcomas. We performed mutation analysis of mediator complex subunit 12 and immunohistochemistry for β-catenin, using 69 tumors of 64 patients including 19 uterine leiomyomas, 6 abdominal leiomyomas, 9 angioleiomyomas, 5 piloleiomyomas, and 7 uterine and 23 soft tissue leiomyosarcomas. In line with previous observations, 58% of uterine leiomyomas carried a mediator complex subunit 12 mutation. However, all other extrauterine leiomyomas were negative with the exception of 1 abdominal leiomyoma with a likely primary uterine origin. Of the 30 leiomyosarcomas, only 1 uterine tumor harbored a mutation. A new observation is the identification of 3 tumors with a homozygous mutation; a monosomy X or interstitial deletion was excluded. β-Catenin immunohistochemistry showed nuclear positivity in only 55% of the mediator complex subunit 12-mutated uterine leiomyomas, suggesting the involvement of pathways other than canonical Wnt signaling in tumorigenesis. Interestingly, 80% of mediator complex subunit 12 wild-type sporadic piloleiomyomas displayed nuclear β-catenin positivity, indicating its involvement in this leiomyoma subtype. The lack of mediator complex subunit 12 mutations in extrauterine leiomyomas and leiomyosarcomas indicates that these tumors arise through a different pathway, emphasizing the genetic heterogeneity of smooth muscle tumors.

  9. Efficient Wnt mediated intestinal hyperproliferation requires the cyclin D2-CDK4/6 complex

    Directory of Open Access Journals (Sweden)

    Sansom Owen

    2011-02-01

    Full Text Available Abstract Inactivation of the gene encoding the adenomatous polyposis coli (APC tumour suppressor protein is recognized as the key early event in the development of colorectal cancers (CRC. Apc loss leads to nuclear localization of beta-catenin and constitutive activity of the beta-catenin-Tcf4 transcription complex. This complex drives the expression of genes involved in cell cycle progression such as c-Myc and cyclin D2. Acute loss of Apc in the small intestine leads to hyperproliferation within the intestinal crypt, increased levels of apoptosis, and perturbed differentiation and migration. It has been demonstrated that c-Myc is a critical mediator of the phenotypic abnormalities that follow Apc loss in the intestine. As it may be difficult to pharmacologically inhibit transcription factors such as c-Myc, investigating more druggable targets of the Wnt-c-Myc pathway within the intestine may reveal potential therapeutic targets for CRC. Recent work in our laboratory has shown that the cyclin D2-cyclin-dependent kinase 4/6 (CDK4/6 complex promotes hyperproliferation in Apc deficient intestinal tissue and ApcMin/+ adenomas. We showed that the hyperproliferative phenotype associated with Apc loss in vivo was partially dependent on the expression of cyclin D2. Most importantly, tumour growth and development in ApcMin/+ mice was strongly perturbed in mice lacking cyclin D2. Furthermore, pharmacological inhibition of CDK4/6 suppressed the proliferation of adenomatous cells. This commentary discusses the significance of this work in providing evidence for the importance of the cyclin D2-CDK4/6 complex in colorectal adenoma formation. It also argues that inhibition of this complex may be an effective chemopreventative strategy in CRC.

  10. Force-Mediated Kinetics of Single P-Selectin/Ligand Complexes Observed by Atomic Force Microscopy

    Science.gov (United States)

    Fritz, Jurgen; Katopodis, Andreas G.; Kolbinger, Frank; Anselmetti, Dario

    1998-10-01

    Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm-1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s-1 under zero force up to 15 s-1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

  11. The transcriptional coactivator DRIP/mediator complex is involved in vitamin D receptor function and regulates keratinocyte proliferation and differentiation.

    Science.gov (United States)

    Oda, Yuko; Chalkley, Robert J; Burlingame, Alma L; Bikle, Daniel D

    2010-10-01

    Mediator is a multisubunit coactivator complex that facilitates transcription of nuclear receptors. We investigated the role of the mediator complex as a coactivator for vitamin D receptor (VDR) in keratinocytes. Using VDR affinity beads, the vitamin D receptor interacting protein (DRIP)/mediator complex was purified from primary keratinocytes, and its subunit composition was determined by mass spectrometry. The complex included core subunits, such as DRIP205/MED1 (MED1), that directly binds to VDR. Additional subunits were identified that are components of the RNA polymerase II complex. The functions of different mediator components were investigated by silencing its subunits. The core subunit MED1 facilitates VDR activity and regulating keratinocyte proliferation and differentiation. A newly described subunit MED21 also has a role in promoting keratinocyte proliferation and differentiation, whereas MED10 has an inhibitory role. Blocking MED1/MED21 expression caused hyperproliferation of keratinocytes, accompanied by increases in mRNA expression of the cell cycle regulator cyclin D1 and/or glioma-associated oncogene homolog. Blocking MED1 or MED21 expression also resulted in defects in calcium-induced keratinocyte differentiation, as indicated by decreased expression of differentiation markers and decreased translocation of E-cadherin to the membrane. These results show that keratinocytes use the transcriptional coactivator mediator to regulate VDR functions and control keratinocyte proliferation and differentiation.

  12. Measurement of the formation of complexes in tyrosine kinase-mediated signal transduction

    Energy Technology Data Exchange (ETDEWEB)

    Ladbury, John E., E-mail: j.ladbury@biochem.ucl.ac.uk [Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT (United Kingdom)

    2007-01-01

    The use of isothermal titration calorimetry (ITC) provides a full thermodynamic characterization of an interaction in one experiment. The determination of the affinity is an important value; however, the additional layer of information provided by the change in enthalpy and entropy can help in understanding the biology. This is demonstrated with respect to tyrosine kinase-mediated signal transduction. Isothermal titration calorimetry (ITC) provides highly complementary data to high-resolution structural detail. An overview of the methodology of the technique is provided. Ultimately, the correlation of the thermodynamic parameters determined by ITC with structural perturbation observed on going from the free to the bound state should be possible at an atomic level. Currently, thermodynamic data provide some insight as to potential changes occurring on complex formation. Here, this is demonstrated in the context of in vitro quantification of intracellular tyrosine kinase-mediated signal transduction and the issue of specificity of the important interactions. The apparent lack of specificity in the interactions of domains of proteins involved in early signalling from membrane-bound receptors is demonstrated using data from ITC.

  13. Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

    Science.gov (United States)

    Cahill, Thomas J.; Thomsen, Alex R. B.; Tarrasch, Jeffrey T.; Plouffe, Bianca; Nguyen, Anthony H.; Yang, Fan; Huang, Li-Yin; Kahsai, Alem W.; Bassoni, Daniel L.; Gavino, Bryant J.; Lamerdin, Jane E.; Triest, Sarah; Shukla, Arun K.; Berger, Benjamin; Little, John; Antar, Albert; Blanc, Adi; Qu, Chang-Xiu; Chen, Xin; Kawakami, Kouki; Inoue, Asuka; Aoki, Junken; Steyaert, Jan; Sun, Jin-Peng; Bouvier, Michel; Skiniotis, Georgios; Lefkowitz, Robert J.

    2017-01-01

    β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR–βarr complexes: the “tail” conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the “core” conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown. Here, we created a mutant form of βarr lacking the “finger-loop” region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and βarr signaling but not desensitization of G protein signaling. Thus, the two GPCR–βarr conformations can carry out distinct functions. PMID:28223524

  14. Dab2, megalin, cubilin and amnionless receptor complex might mediate intestinal endocytosis in the suckling rat.

    Science.gov (United States)

    Vázquez-Carretero, María D; Palomo, Marta; García-Miranda, Pablo; Sánchez-Aguayo, Inmaculada; Peral, María J; Calonge, María L; Ilundain, Anunciación A

    2014-03-01

    We previously proposed that Dab2 participates in the endocytosis of milk macromolecules in rat small intestine. Here we investigate the receptors that may mediate this endocytosis by studying the effects of age and diet on megalin, VLDLR, and ApoER2 expression, and that of age on the expression of cubilin and amnionless. Of megalin, VLDLR and ApoER2, only the megalin expression pattern resembles that of Dab2 previously reported. Thus the mRNA and protein levels of megalin and Dab2 are high in the intestine of the suckling rat, down-regulated by age and up-regulated by milk diet, mainly in the ileum. Neither age nor diet affect ApoER2 mRNA levels. The effect of age on VLDLR mRNA levels depends on the epithelial cell tested but they are down-regulated by milk diet. In the suckling rat, the intestinal expressions of both cubilin and amnionless are similar to that of megalin and megalin, cubilin, amnionless and Dab2 co-localize at the microvilli and in the apical endocytic apparatus. Co-localization of Dab2 with ApoER2 and VLDLR at the microvilli and in the apical endocytic apparatus is also observed. This is the first report showing intestinal co-localization of: megalin/cubilin/amnionless/Dab2, VLDLR/Dab2 and ApoER2/Dab2. We conclude that the megalin/cubilin/amnionless/Dab2 complex/es participate in intestinal processes, mainly during the lactation period and that Dab2 may act as an adaptor in intestinal processes mediated by ApoER2 and VLDLR.

  15. Defluoridation of water using dicarboxylic acids mediated chitosan-polyaniline/zirconium biopolymeric complex.

    Science.gov (United States)

    Muthu Prabhu, Subbaiah; Meenakshi, Sankaran

    2016-04-01

    The present investigation describes the preparation of hydrogen bonded chitosan-polyaniline/zirconium biopolymeric matrix by grafting method under dicarboxylic acid medium for the removal of fluoride, first time. Herein, the dicarboxylic acids, oxalic acid, malonic acid, succinic acid were used as medium. The synthesized complex was characterized by usual analytical techniques like FTIR, XRD, SEM and EDAX analysis. From the batch equilibrium experiments, the maximum defluoridation capacity (DC) was found to be 8.713 mg/g at room temperature with the minimum contact time of 24 min at 100mg of the sorbent dosage. The temperature study results of adsorption kinetics showed the adsorption behavior could be better described by the pseudo-second-order equation than pseudo-first-order kinetic model. The adsorption isotherm was well fitted by the Freundlich equation rather than Langmuir and D-R isotherms. The mechanism of fluoride removal was ligand exchange at neutral pH and electrostatic attraction at acidic pH of the medium. Regeneration studies were carried out to identify the best regenerant which makes the process cost-effective. Conclusions of this work demonstrate the potential applicability of the dicarboxylic acid mediated chitosan-polyaniline/zirconium complex as an effective adsorbent for fluoride removal from water.

  16. An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity.

    Science.gov (United States)

    Albert, Isabell; Böhm, Hannah; Albert, Markus; Feiler, Christina E; Imkampe, Julia; Wallmeroth, Niklas; Brancato, Caterina; Raaymakers, Tom M; Oome, Stan; Zhang, Heqiao; Krol, Elzbieta; Grefen, Christopher; Gust, Andrea A; Chai, Jijie; Hedrich, Rainer; Van den Ackerveken, Guido; Nürnberger, Thorsten

    2015-10-05

    Plants and animals employ innate immune systems to cope with microbial infection. Pattern-triggered immunity relies on the recognition of microbe-derived patterns by pattern recognition receptors (PRRs). Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) constitute plant immunogenic patterns that are unique, as these proteins are produced by multiple prokaryotic (bacterial) and eukaryotic (fungal, oomycete) species. Here we show that the leucine-rich repeat receptor protein (LRR-RP) RLP23 binds in vivo to a conserved 20-amino-acid fragment found in most NLPs (nlp20), thereby mediating immune activation in Arabidopsis thaliana. RLP23 forms a constitutive, ligand-independent complex with the LRR receptor kinase (LRR-RK) SOBIR1 (Suppressor of Brassinosteroid insensitive 1 (BRI1)-associated kinase (BAK1)-interacting receptor kinase 1), and recruits a second LRR-RK, BAK1, into a tripartite complex upon ligand binding. Stable, ectopic expression of RLP23 in potato (Solanum tuberosum) confers nlp20 pattern recognition and enhanced immunity to destructive oomycete and fungal plant pathogens, such as Phytophthora infestans and Sclerotinia sclerotiorum. PRRs that recognize widespread microbial patterns might be particularly suited for engineering immunity in crop plants.

  17. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

    Science.gov (United States)

    Decker, Logan M.; Xiao, Hua; Boone, Erin C.; Vierling, Michael M.; Shanker, Benjamin S.; Kingston, Shanika L.; Boone, Shannon F.; Haynes, Jackson B.; Shiu, Patrick K.T.

    2017-01-01

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD), which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi) proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC). Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors. PMID:28179391

  18. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

    Directory of Open Access Journals (Sweden)

    Logan M. Decker

    2017-04-01

    Full Text Available In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD, which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC. Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute of the perinuclear meiotic silencing complex (MSC, directly linking the two cellular factors.

  19. The effects of childhood abuse on symptom complexity in a clinical sample: mediating effects of emotion regulation difficulties.

    Science.gov (United States)

    Choi, Ji Young; Choi, Young Min; Gim, Min Sook; Park, Jun Hyun; Park, Soo Hyun

    2014-08-01

    The purpose of the present study was to first examine whether childhood abuse predicts symptom complexity, as indicated by the number of clinically elevated scales on the MMPI-2 in an adult clinical sample. Secondly, we investigated whether emotion regulation difficulties mediated the relationship between childhood abuse and symptom complexity. A total of 162 adult outpatients not presenting with psychotic symptoms completed the Korean Childhood Trauma Questionnaire (K-CTQ), Life Events Checklist (LEC), Difficulties in Emotion Regulation Scale (DERS), and Korean Minnesota Multiphasic Personality Inventory-2 (MMPI-2). Partial correlation analysis results indicated that after controlling for the presence of adulthood trauma, childhood abuse was associated with more symptom complexity, or more clinically elevated scales on the MMPI-2. Furthermore, structural equation modeling results showed that emotion regulation difficulties partially mediated the relationship between childhood abuse and symptom complexity. These findings indicate that individuals who had experienced childhood abuse evidence simultaneous presentation of diverse clinical symptoms.

  20. Understanding the role of ETS-mediated gene regulation in complex biological processes.

    Science.gov (United States)

    Findlay, Victoria J; LaRue, Amanda C; Turner, David P; Watson, Patricia M; Watson, Dennis K

    2013-01-01

    Ets factors are members of one of the largest families of evolutionarily conserved transcription factors, regulating critical functions in normal cell homeostasis, which when perturbed contribute to tumor progression. The well-documented alterations in ETS factor expression and function during cancer progression result in pleiotropic effects manifested by the downstream effect on their target genes. Multiple ETS factors bind to the same regulatory sites present on target genes, suggesting redundant or competitive functions. The anti- and prometastatic signatures obtained by examining specific ETS regulatory networks will significantly improve our ability to accurately predict tumor progression and advance our understanding of gene regulation in cancer. Coordination of multiple ETS gene functions also mediates interactions between tumor and stromal cells and thus contributes to the cancer phenotype. As such, these new insights may provide a novel view of the ETS gene family as well as a focal point for studying the complex biological control involved in tumor progression. One of the goals of molecular biology is to elucidate the mechanisms that contribute to the development and progression of cancer. Such an understanding of the molecular basis of cancer will provide new possibilities for: (1) earlier detection, as well as better diagnosis and staging of disease; (2) detection of minimal residual disease recurrences and evaluation of response to therapy; (3) prevention; and (4) novel treatment strategies. Increased understanding of ETS-regulated biological pathways will directly impact these areas. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Biocombinatorial Synthesis of Novel Lipopeptides by COM Domain-Mediated Reprogramming of the Plipastatin NRPS Complex

    Directory of Open Access Journals (Sweden)

    Hongxia Liu

    2016-11-01

    Full Text Available Both donors and acceptors of communication-mediating (COM domains are essential for coordinating intermolecular communication within nonribosomal peptides synthetases (NRPSs complexes. Different sets of COM domains provide selectivity, allowing NRPSs to utilise different natural biosynthetic templates. In this study, novel lipopeptides were synthesised by reprogramming the plipastatin biosynthetic machinery. A Thr-to-Asp point mutation was sufficient to shift the selectivity of the donor COM domain of ppsB toward that of ppsD. Deletion and/or interchangeability established donor and acceptor function. Variations in acceptor COM domain did not result in novel product formation in the presence of its partner donor, whereas plipastatin formation was completely abrogated by altering donor modules. Five novel lipopeptides (cyclic pentapeptide, linear hexapeptide, nonapeptide, heptapeptide and cyclic octapeptide were identified and verified by high-resolution LC-ESI-MS/MS. In addition, we demonstrated the potential to generate novel strains with the antimicrobial activity by selecting compatible COM domains, and the novel lipopeptides exhibited antimicrobial activity against five of the fungal species at a contention of 31.25-125 μg/ml.

  2. Therapeutic Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton's Tyrosine Kinase.

    Science.gov (United States)

    Chalmers, Samantha A; Doerner, Jessica; Bosanac, Todd; Khalil, Sara; Smith, Dustin; Harcken, Christian; Dimock, Janice; Der, Evan; Herlitz, Leal; Webb, Deborah; Seccareccia, Elise; Feng, Di; Fine, Jay S; Ramanujam, Meera; Klein, Elliott; Putterman, Chaim

    2016-05-19

    Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton's tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1, either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease, which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells, as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN.

  3. Plant root exudates mediate neighbour recognition and trigger complex behavioural changes.

    Science.gov (United States)

    Semchenko, Marina; Saar, Sirgi; Lepik, Anu

    2014-11-01

    Some plant species are able to distinguish between neighbours of different genetic identity and attempt to pre-empt resources through root proliferation in the presence of unrelated competitors, but avoid competition with kin. However, studies on neighbour recognition have met with some scepticism because the mechanisms by which plants identify their neighbours have remained unclear. In order to test whether root exudates could mediate neighbour recognition in plants, we performed a glasshouse experiment in which plants of Deschampsia caespitosa were subjected to root exudates collected from potential neighbours of different genetic identities, including siblings and individuals belonging to the same or a different population or species. Our results show that root exudates can carry specific information about the genetic relatedness, population origin and species identity of neighbours, and trigger different responses at the whole root system level and at the level of individual roots in direct contact with locally applied exudates. Increased root density was mainly achieved through changes in morphology rather than biomass allocation, suggesting that plants are able to limit the energetic cost of selfish behaviour. This study reveals a new level of complexity in the ability of plants to interpret and react to their surroundings.

  4. Hepatitis C virus genomic RNA dimerization is mediated via a kissing complex intermediate

    Science.gov (United States)

    Shetty, Sumangala; Kim, Seungtaek; Shimakami, Tetsuro; Lemon, Stanley M.; Mihailescu, Mihaela-Rita

    2010-01-01

    With over 200 million people infected with hepatitis C virus (HCV) worldwide, there is a need for more effective and better-tolerated therapeutic strategies. The HCV genome is a positive-sense; single-stranded RNA encoding a large polyprotein cleaved at multiple sites to produce at least ten proteins, among them an error-prone RNA polymerase that confers a high mutation rate. Despite considerable overall sequence diversity, in the 3′-untranslated region of the HCV genomic RNA there is a 98-nucleotide (nt) sequence named X RNA, the first 55 nt of which (X55 RNA) are 100% conserved among all HCV strains. The X55 region has been suggested to be responsible for in vitro dimerization of the genomic RNA in the presence of the viral core protein, although the mechanism by which this occurs is unknown. In this study, we analyzed the X55 region and characterized the mechanism by which it mediates HCV genomic RNA dimerization. Similar to a mechanism proposed previously for the human immunodeficiency 1 virus (HIV-1) genome, we show that dimerization of the HCV genome involves formation of a kissing complex intermediate, which is converted to a more stable extended duplex conformation in the presence of the core protein. Mutations in the dimer linkage sequence loop sequence that prevent RNA dimerization in vitro significantly reduced but did not completely ablate the ability of HCV RNA to replicate or produce infectious virus in transfected cells. PMID:20360391

  5. Copper-obatoclax derivative complexes mediate DNA cleavage and exhibit anti-cancer effects in hepatocellular carcinoma.

    Science.gov (United States)

    Su, Jung-Chen; Chang, Jung-Hua; Huang, Jui-Wen; Chen, Peter P-Y; Chen, Kuen-Feng; Tseng, Ping-Hui; Shiau, Chung-Wai

    2015-02-25

    Obatoclax is an indole-pyrrole compound that induces cancer cell apoptosis through targeting the anti-apoptotic Bcl-2 protein family. Previously, we developed a series of obatoclax derivatives and studied their STAT3 inhibition-dependent activity against cancer cell lines. The obatoclax analog, prodigiosin, has been reported to mediate DNA cleavage in cancer cells by coordinating with copper complexes. To gain an understanding of copper-obatoclax complex activity, we applied obatoclax derivatives to examine their copper-mediated nuclease activity as a means to establish a basis for structure activity relationship. Replacement of the indole ring of obatoclax with furanyl, thiophenyl or Boc-indolyl rings reduced the DNA cleavage ability. The same effect was achieved through the replacement of the obatoclax pyrrolyl ring with thiazolidinedione and thioacetal. Among the compounds tested, we demonstrated that the complex of obatoclax or compound 7 with copper exhibited potent DNA strand scission which correlated with HCC cell growth inhibition.

  6. Optimization of ultrasound parameters for microbubble-nanoliposome complex-mediated delivery

    Directory of Open Access Journals (Sweden)

    Young Il Yoon

    2015-10-01

    Full Text Available Purpose: The aim of this study was to identify the optimal ultrasound (US parameters for gene and drug delivery. Methods: In order to target SkBr3, which is a breast cancer cell overexpressing the Her2 receptor, trastuzumab (Herceptin was used. Micobubble-nanoliposome complex (MLC was mixed with trastuzumab and stored overnight. Finally, MLC was combined with Her2Ab. A US device equipped with a 1-MHz probe was used for delivery to the cell. Several parameters, including intensity (w/cm2, time (minutes, and duty cycle (%, were varied within a range from 1 w/cm2, 1 minute, and 20% to 2 w/cm2, 2 minutes, and 60%, respectively. A confocal laser scanning microscope (CLSM was used to confirm the delivery of MLC to the cells after US treatment. Results: MLC with fluorescent dyes and trastuzumab was synthesized successfully. By delivering MLC with Her2Ab to cells, the targeting effect of trastuzumab with MLC was confirmed by CLSM. The cell membranes showed green (fluorescein isothiocyanate and red (Texas red fluorescence but treatments with MLC without Her2Ab did not show any fluorescence. Optimal conditions for US-mediated delivery were 1 or 2 w/cm2, 2 minutes, and 60% (uptake ratio, 95.9% for 1 w/cm2 and 95.7% for 2 w/cm2 for hydrophobic materials and 2 w/cm2, 2 minutes, and 60% (uptake ratio, 95.0% for hydrophilic materials. Conclusion: The greater the strength, duty cycle, and period of US application within the tested range, the more efficiently the fluorescent contents were conveyed.

  7. The TPLATE Adaptor Complex Drives Clathrin-Mediated Endocytosis in Plants

    NARCIS (Netherlands)

    Gadeyne, A.; Sanchez-Rodriguez, C.; Rubbo, Di S.; Ketelaar, T.

    2014-01-01

    Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure.

  8. Feedback regulation on PTEN/AKT pathway by the ER stress kinase PERK mediated by interaction with the Vault complex

    DEFF Research Database (Denmark)

    Zhang, Wei; Neo, Suat Peng; Gunaratne, Jayantha

    2015-01-01

    The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological...... inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress...... results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications...

  9. Proteomic analysis reveals GIT1 as a novel mTOR complex component critical for mediating astrocyte survival.

    Science.gov (United States)

    Smithson, Laura J; Gutmann, David H

    2016-06-15

    As a critical regulator of cell growth, the mechanistic target of rapamycin (mTOR) protein operates as part of two molecularly and functionally distinct complexes. Herein, we demonstrate that mTOR complex molecular composition varies in different somatic tissues. In astrocytes and neural stem cells, we identified G-protein-coupled receptor kinase-interacting protein 1 (GIT1) as a novel mTOR-binding protein, creating a unique mTOR complex lacking Raptor and Rictor. Moreover, GIT1 binding to mTOR is regulated by AKT activation and is essential for mTOR-mediated astrocyte survival. Together, these data reveal that mTOR complex function is partly dictated by its molecuflar composition in different cell types.

  10. Electrocatalytic Oxidation of NADH Based on Self-assembled Colloidal Gold and Nafion Matrixes and Co Complex Mediator

    Institute of Scientific and Technical Information of China (English)

    Na WANG; Ruo YUAN; Ya Qin CHAI; Dian Ping TANG; Qiang ZHU; Xue Lian LI

    2006-01-01

    A novel approach based on self-assembled colloidal gold and Nafion matrixes and Co complex mediator to construct Co(bpy)33+/nano-Au/Co(bpy)33+/nafion/GC electrode, on which formed stable redox-active films. This electrode can decrease the overpotential about 330 mV for the oxidation of NADH with high stability, wide linear range and low detection limit.

  11. Noncanonical role of Arabidopsis COP1/SPA complex in repressing BIN2-mediated PIF3 phosphorylation and degradation in darkness.

    Science.gov (United States)

    Ling, Jun-Jie; Li, Jian; Zhu, Danmeng; Deng, Xing Wang

    2017-03-28

    The E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) has been known to mediate key signaling factors for degradation via the ubiquitin/26S proteasome pathway in both plants and animals. Here, we report a noncanonical function of Arabidopsis COP1, the central repressor of photomorphogenesis, in the form of a COP1/ SUPPRESSOR of phyA-105 (SPA) complex. We show that the COP1/SPA complex associates with and stabilizes PHYTOCHROME INTERACTING FACTOR 3 (PIF3) to repress photomorphogenesis in the dark. We identify the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) as a kinase of PIF3, which induces PIF3 degradation via 26S proteasome during skotomorphogenesis. Mutations on two typical BIN2 phosphorylation motifs of PIF3 lead to a strong stabilization of the protein in the dark. We further show that the COP1/SPA complex promotes PIF3 stability by repressing BIN2 activity. Intriguingly, without affecting BIN2 expression, the COP1/SPA complex modulates BIN2 activity through interfering with BIN2-PIF3 interaction, thereby inhibiting BIN2-mediated PIF3 phosphorylation and degradation. Taken together, our results suggest another paradigm for COP1/SPA complex action in the precise control of skotomorphogenesis.

  12. Single platelets seal neutrophil-induced vascular breaches via GPVI during immune-complex-mediated inflammation in mice.

    Science.gov (United States)

    Gros, Angèle; Syvannarath, Varouna; Lamrani, Lamia; Ollivier, Véronique; Loyau, Stéphane; Goerge, Tobias; Nieswandt, Bernhard; Jandrot-Perrus, Martine; Ho-Tin-Noé, Benoît

    2015-08-20

    Platelets protect vascular integrity during inflammation. Recent evidence suggests that this action is independent of thrombus formation and requires the engagement of glycoprotein VI (GPVI), but it remains unclear how platelets prevent inflammatory bleeding. We investigated whether platelets and GPVI act primarily by preventing detrimental effects of neutrophils using models of immune complex (IC)-mediated inflammation in mice immunodepleted in platelets and/or neutrophils or deficient in GPVI. Depletion of neutrophils prevented bleeding in thrombocytopenic and GPVI(-/-) mice during IC-mediated dermatitis. GPVI deficiency did not modify neutrophil recruitment, which was reduced by thrombocytopenia. Neutrophil cytotoxic activities were reduced in thrombocytopenic and GPVI(-/-) mice during IC-mediated inflammation. Intravital microscopy revealed that in this setting, intravascular binding sites for platelets were exposed by neutrophils, and GPVI supported the recruitment of individual platelets to these spots. Furthermore, the platelet secretory response accompanying IC-mediated inflammation was partly mediated by GPVI, and blocking of GPVI signaling impaired the vasculoprotective action of platelets. Together, our results show that GPVI plays a dual role in inflammation by enhancing neutrophil-damaging activities while supporting the activation and hemostatic adhesion of single platelets to neutrophil-induced vascular breaches.

  13. Discrimination between platelet-mediated and coagulation-mediated mechanisms in a model of complex thrombus formation in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Cadroy, Y.; Horbett, T.A.; Hanson, S.R.

    1989-04-01

    To study mechanisms of complex thrombus formation in vivo, and to compare the relative antithrombotic effects of anticoagulants and antiplatelet agents, a model was developed in baboons. Segments of collagen-coated tubing followed by two sequentially placed expansion chambers exhibiting disturbed flow patterns were exposed to native blood under laminar flow conditions. The device was incorporated for 1 hour into an exteriorized arteriovenous shunt in baboons under controlled blood flow (20 ml/min). Morphologic evaluation by scanning electron microscopy showed that thrombi associated with collagen were relatively rich in platelets but thrombi in the chambers were rich in fibrin and red cells. Deposition of indium 111-labeled platelets was continuously measured with a scintillation camera. Platelet deposition increased in a linear (collagen-coated segment) or exponential (chambers 1 and 2) fashion over time, with values after 40 minutes averaging 24.1 +/- 3.3 x 10(8) platelets (collagen segment), 16.7 +/- 3.4 x 10(8) platelets (chamber 1), and 8.4 +/- 2.4 x 10(8) platelets (chamber 2). Total fibrinogen deposition after 40 minutes was determined by using iodine 125-labeled baboon fibrinogen and averaged 0.58 +/- 0.14 mg in the collagen segment, 1.51 +/- 0.27 mg in chamber 1, and 0.95 +/- 0.25 mg in chamber 2. Plasma levels of beta-thromboglobulin (beta TG), platelet-factor 4 (PF4), and fibrinopeptide A (FPA) increased fourfold to fivefold after 60 minutes of blood exposure to the thrombotic device. Platelet deposition onto the collagen segment, chamber 1, and chamber 2 was linearly dependent on the circulating platelet count. Platelet accumulation in chamber 1 and chamber 2 was also dependent on the presence of the proximal collagen segment.

  14. Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells.

    Science.gov (United States)

    Li, Bo-Chao; Chang, Hao; Ren, Ke-Feng; Ji, Jian

    2016-11-01

    Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants.

  15. Reptin and Pontin function antagonistically with PcG and TrxG complexes to mediate Hox gene control

    Science.gov (United States)

    Diop, Soda Balla; Bertaux, Karine; Vasanthi, Dasari; Sarkeshik, Ali; Goirand, Benjamin; Aragnol, Denise; Tolwinski, Nicholas S; Cole, Michael D; Pradel, Jacques; Yates, John R; Mishra, Rakesh K; Graba, Yacine; Saurin, Andrew J

    2008-01-01

    Pontin (Pont) and Reptin (Rept) are paralogous ATPases that are evolutionarily conserved from yeast to human. They are recruited in multiprotein complexes that function in various aspects of DNA metabolism. They are essential for viability and have antagonistic roles in tissue growth, cell signalling and regulation of the tumour metastasis suppressor gene, KAI1, indicating that the balance of Pont and Rept regulates epigenetic programmes critical for development and cancer progression. Here, we describe Pont and Rept as antagonistic mediators of Drosophila Hox gene transcription, functioning with Polycomb group (PcG) and Trithorax group proteins to maintain correct patterns of expression. We show that Rept is a component of the PRC1 PcG complex, whereas Pont purifies with the Brahma complex. Furthermore, the enzymatic functions of Rept and Pont are indispensable for maintaining Hox gene expression states, highlighting the importance of these two antagonistic factors in transcriptional output. PMID:18259215

  16. Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

    Science.gov (United States)

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

    At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in “ventral F-actin waves” that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These “adhesive F-actin waves” require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization. PMID:22069459

  17. Adhesive F-actin waves: a novel integrin-mediated adhesion complex coupled to ventral actin polymerization.

    Directory of Open Access Journals (Sweden)

    Lindsay B Case

    Full Text Available At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These "adhesive F-actin waves" require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization.

  18. Focus on opportunities as a mediator of the relationships between age, job complexity, and work performance

    NARCIS (Netherlands)

    Zacher, Hannes; Heusner, Sandra; Schmitz, Michael; Zwierzanska, Monika M.; Frese, Michael

    2010-01-01

    Focus on opportunities is a cognitive-motivational facet of occupational future time perspective that describes how many new goals, options, and possibilities individuals expect to have in their personal work-related futures. This study examined focus on opportunities as a mediator of the relationsh

  19. Rsp5-Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast.

    Science.gov (United States)

    Kaida, Daisuke; Toh-e, Akio; Kikuchi, Yoshiko

    2003-07-11

    Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

  20. The Arabidopsis mediator complex subunit16 positively regulates salicylate-mediated systemic acquired resistance and jasmonate/ethylene-induced defense pathways.

    Science.gov (United States)

    Zhang, Xudong; Wang, Chenggang; Zhang, Yanping; Sun, Yijun; Mou, Zhonglin

    2012-10-01

    Systemic acquired resistance (SAR) is a long-lasting plant immunity against a broad spectrum of pathogens. Biological induction of SAR requires the signal molecule salicylic acid (SA) and involves profound transcriptional changes that are largely controlled by the transcription coactivator nonexpressor of pathogenesis-related genes1 (NPR1). However, it is unclear how SAR signals are transduced from the NPR1 signaling node to the general transcription machinery. Here, we report that the Arabidopsis thaliana Mediator subunit16 (MED16) is an essential positive regulator of SAR. Mutations in MED16 reduced NPR1 protein levels and completely compromised biological induction of SAR. These mutations also significantly suppressed SA-induced defense responses, altered the transcriptional changes induced by the avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000/avrRpt2, and rendered plants susceptible to both Pst DC3000/avrRpt2 and Pst DC3000. In addition, mutations in MED16 blocked the induction of several jasmonic acid (JA)/ethylene (ET)-responsive genes and compromised resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. The Mediator complex acts as a bridge between specific transcriptional activators and the RNA polymerase II transcription machinery; therefore, our data suggest that MED16 may be a signaling component in the gap between the NPR1 signaling node and the general transcription machinery and may relay signals from both the SA and the JA/ET pathways.

  1. The Non-canonical Tetratricopeptide Repeat (TPR) Domain of Fluorescent (FLU) Mediates Complex Formation with Glutamyl-tRNA Reductase.

    Science.gov (United States)

    Zhang, Min; Zhang, Feilong; Fang, Ying; Chen, Xuemin; Chen, Yuhong; Zhang, Wenxia; Dai, Huai-En; Lin, Rongcheng; Liu, Lin

    2015-07-10

    The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLU(TPR)) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLU(TPR) at 2.4-Å resolution. Three non-canonical TPR motifs of each FLU(TPR) form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLU(TPR)-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein.

  2. The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

    Directory of Open Access Journals (Sweden)

    Stefan Pils

    Full Text Available BACKGROUND: CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. PRINCIPAL FINDINGS: In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. CONCLUSIONS: Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

  3. Survivin mediates targeting of the chromosomal passenger complex to the centromere and midbody

    NARCIS (Netherlands)

    Vader, G; Kauw, JJW; Medema, RH; Lens, SMA

    2006-01-01

    The chromosomal passenger complex (CPC) coordinates chromosomal and cytoskeletal events of mitosis. The enzymatic core of this complex (Aurora-B) is guided through the mitotic cell by its companion chromosomal passenger proteins, inner centromere protein (INCENP), Survivin and Borealin/Dasra-B, ther

  4. Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans.

    Science.gov (United States)

    Etheridge, Timothy; Oczypok, Elizabeth A; Lehmann, Susann; Fields, Brandon D; Shephard, Freya; Jacobson, Lewis A; Szewczyk, Nathaniel J

    2012-01-01

    Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line- or M-line-specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks

  5. Calpains mediate integrin attachment complex maintenance of adult muscle in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Timothy Etheridge

    2012-01-01

    Full Text Available Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line- or M-line-specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C

  6. Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation.

    NARCIS (Netherlands)

    Nabbe, K.C.A.M.; Lent, P.L.E.M. van; Holthuysen, A.E.M.; Sloetjes, A.W.; Koch, A.E.; Radstake, T.R.D.J.; Berg, W.B. van den

    2005-01-01

    During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcgamma receptors (FcgammaRs) (mainly FcgammaRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with

  7. Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3.

    Science.gov (United States)

    Gu, Shen; Yuan, Bo; Campbell, Ian M; Beck, Christine R; Carvalho, Claudia M B; Nagamani, Sandesh C S; Erez, Ayelet; Patel, Ankita; Bacino, Carlos A; Shaw, Chad A; Stankiewicz, Paweł; Cheung, Sau Wai; Bi, Weimin; Lupski, James R

    2015-07-15

    Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.

  8. Genome-Wide Transcriptional Regulation Mediated by Biochemically Distinct SWI/SNF Complexes.

    Directory of Open Access Journals (Sweden)

    Jesse R Raab

    2015-12-01

    Full Text Available Multiple positions within the SWI/SNF chromatin remodeling complex can be filled by mutually exclusive subunits. Inclusion or exclusion of these proteins defines many unique forms of SWI/SNF and has profound functional consequences. Often this complex is studied as a single entity within a particular cell type and we understand little about the functional relationship between these biochemically distinct forms of the remodeling complex. Here we examine the functional relationships among three complex-specific ARID (AT-Rich Interacting Domain subunits using genome-wide chromatin immunoprecipitation, transcriptome analysis, and transcription factor binding maps. We find widespread overlap in transcriptional regulation and the genomic binding of distinct SWI/SNF complexes. ARID1B and ARID2 participate in wide-spread cooperation to repress hundreds of genes. Additionally, we find numerous examples of competition between ARID1A and another ARID, and validate that gene expression changes following loss of one ARID are dependent on the function of an alternative ARID. These distinct regulatory modalities are correlated with differential occupancy by transcription factors. Together, these data suggest that distinct SWI/SNF complexes dictate gene-specific transcription through functional interactions between the different forms of the SWI/SNF complex and associated co-factors. Most genes regulated by SWI/SNF are controlled by multiple biochemically distinct forms of the complex, and the overall expression of a gene is the product of the interaction between these different SWI/SNF complexes. The three mutually exclusive ARID family members are among the most frequently mutated chromatin regulators in cancer, and understanding the functional interactions and their role in transcriptional regulation provides an important foundation to understand their role in cancer.

  9. Genome-Wide Transcriptional Regulation Mediated by Biochemically Distinct SWI/SNF Complexes

    Science.gov (United States)

    Raab, Jesse R.; Resnick, Samuel; Magnuson, Terry

    2015-01-01

    Multiple positions within the SWI/SNF chromatin remodeling complex can be filled by mutually exclusive subunits. Inclusion or exclusion of these proteins defines many unique forms of SWI/SNF and has profound functional consequences. Often this complex is studied as a single entity within a particular cell type and we understand little about the functional relationship between these biochemically distinct forms of the remodeling complex. Here we examine the functional relationships among three complex-specific ARID (AT-Rich Interacting Domain) subunits using genome-wide chromatin immunoprecipitation, transcriptome analysis, and transcription factor binding maps. We find widespread overlap in transcriptional regulation and the genomic binding of distinct SWI/SNF complexes. ARID1B and ARID2 participate in wide-spread cooperation to repress hundreds of genes. Additionally, we find numerous examples of competition between ARID1A and another ARID, and validate that gene expression changes following loss of one ARID are dependent on the function of an alternative ARID. These distinct regulatory modalities are correlated with differential occupancy by transcription factors. Together, these data suggest that distinct SWI/SNF complexes dictate gene-specific transcription through functional interactions between the different forms of the SWI/SNF complex and associated co-factors. Most genes regulated by SWI/SNF are controlled by multiple biochemically distinct forms of the complex, and the overall expression of a gene is the product of the interaction between these different SWI/SNF complexes. The three mutually exclusive ARID family members are among the most frequently mutated chromatin regulators in cancer, and understanding the functional interactions and their role in transcriptional regulation provides an important foundation to understand their role in cancer. PMID:26716708

  10. Asymmetric catalysis mediated by the ligand sphere of octahedral chiral-at-metal complexes.

    Science.gov (United States)

    Gong, Lei; Chen, Liang-An; Meggers, Eric

    2014-10-06

    Due to the relationship between structure and function in chemistry, access to novel chemical structures ultimately drives the discovery of novel chemical function. In this light, the formidable utility of the octahedral geometry of six-coordinate metal complexes is founded in its stereochemical complexity combined with the ability to access chemical space that might be unavailable for purely organic compounds. In this Minireview we wish to draw attention to inert octahedral chiral-at-metal complexes as an emerging class of metal-templated asymmetric "organocatalysts" which exploit the globular, rigid nature and stereochemical options of octahedral compounds and promise to provide new opportunities in the field of catalysis.

  11. Low temperature syntheses and reactivity of Cu2O2 active-site models.

    Science.gov (United States)

    Citek, Cooper; Herres-Pawlis, Sonja; Stack, T Daniel P

    2015-08-18

    Nature's facility with dioxygen outmatches modern chemistry in the oxidation and oxygenation of materials and substrates for biosynthesis and cellular metabolism. The Earth's most abundant naturally occurring oxidant is-frankly-poorly understood and controlled, and thus underused. Copper-based enzyme metallocofactors are ubiquitous to the efficient consumption of dioxygen by all domains of life. Over the last several decades, we have joined many research groups in the study of copper- and dioxygen-dependent enzymes through close investigation of synthetically derived, small-molecule active-site analogs. Simple copper-dioxygen clusters bearing structural and spectroscopic similarity to dioxygen-activating enzymes can be probed for their fundamental geometrical, electronic, and reactive properties using the tools available to inorganic and synthetic chemistry. Our exploration of the copper-dioxygen arena has sustained product evaluation of the key dynamics and reactivity of binuclear Cu2O2 compounds. Almost exclusively operating at low temperatures, from -78 °C to solution characterization even at -125 °C, we have identified numerous compounds supported by simple and easily accessed, low molecular weight ligands-chiefly families of bidentate diamine chelates. We have found that by stripping away complexity in comparison to extended protein tertiary structures or sophisticated, multinucleating architectures, we can experimentally manipulate activated compounds and open pathways of reactivity toward exogenous substrates that both inform on and extend fundamental mechanisms of oxygenase enzymes. Our recent successes have advanced understanding of the tyrosinase enzyme, and related hemocyanin and NspF, and the copper membrane monooxygenases, specifically particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO). Tyrosinase, ubiquitously distributed throughout life, is fundamental to the copper-based oxidation of phenols and the production of chromophores

  12. Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex.

    Science.gov (United States)

    Vukotic, Milena; Oeljeklaus, Silke; Wiese, Sebastian; Vögtle, F Nora; Meisinger, Chris; Meyer, Helmut E; Zieseniss, Anke; Katschinski, Doerthe M; Jans, Daniel C; Jakobs, Stefan; Warscheid, Bettina; Rehling, Peter; Deckers, Markus

    2012-03-01

    The terminal enzyme of the mitochondrial respiratory chain, cytochrome oxidase, transfers electrons to molecular oxygen, generating water. Within the inner mitochondrial membrane, cytochrome oxidase assembles into supercomplexes, together with other respiratory chain complexes, forming so-called respirasomes. Little is known about how these higher oligomeric structures are attained. Here we report on Rcf1 and Rcf2 as cytochrome oxidase subunits in S. cerevisiae. While Rcf2 is specific to yeast, Rcf1 is a conserved subunit with two human orthologs, RCF1a and RCF1b. Rcf1 is required for growth in hypoxia and complex assembly of subunits Cox13 and Rcf2, as well as for the oligomerization of a subclass of cytochrome oxidase complexes into respirasomes. Our analyses reveal that the cytochrome oxidase of mitochondria displays intrinsic heterogeneity with regard to its subunit composition and that distinct forms of respirasomes can be formed by complex variants.

  13. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells

    National Research Council Canada - National Science Library

    Tchasovnikarova, I. A; Timms, R. T; Matheson, N. J; Wals, K; Antrobus, R; Gottgens, B; Dougan, G; Dawson, M. A; Lehner, P. J

    2015-01-01

    ... (see the Perspective by Brummelkamp). They identified a complex of proteins in human cells they called HUSH that kept particular parts of the genome silent by changing associated histone methylation marks...

  14. Cation-mediated conversion of the state of charge in uranium arene inverted-sandwich complexes

    Energy Technology Data Exchange (ETDEWEB)

    Camp, Clement; Mougel, Victor; Pecaut, Jacques; Mazzanti, Marinella [Laboratoire de Reconnaissance Ionique et Chimie de Coordination, SCIB, UMR-E3 CEA-UJF, INAC, CEA-Grenoble (France); Maron, Laurent [LCPNO, CNRS and INSA, UPS, Universite de Toulouse (France)

    2013-12-16

    Two new arene inverted-sandwich complexes of uranium supported by siloxide ancillary ligands [K{U(OSi(OtBu)_3)_3}{sub 2}(μ-η{sup 6}:η{sup 6}-C{sub 7}H{sub 8})] (3) and [K{sub 2}{U(OSi(OtBu)_3)_3}{sub 2}(μ-η{sup 6}:η{sup 6}-C{sub 7}H{sub 8})] (4) were synthesized by the reduction of the parent arene-bridged complex [{U(OSi(OtBu)_3)_3}{sub 2}(μ-η{sup 6}:η{sup 6}-C{sub 7}H{sub 8})] (2) with stoichiometric amounts of KC{sub 8} yielding a rare family of inverted-sandwich complexes in three states of charge. The structural data and computational studies of the electronic structure are in agreement with the presence of high-valent uranium centers bridged by a reduced tetra-anionic toluene with the best formulation being U{sup V}-(arene{sup 4-})-U{sup V}, KU{sup IV}-(arene{sup 4-})-U{sup V}, and K{sub 2}U{sup IV}-(arene{sup 4-})-U{sup IV} for complexes 2, 3, and 4 respectively. The potassium cations in complexes 3 and 4 are coordinated to the siloxide ligands both in the solid state and in solution. The addition of KOTf (OTf=triflate) to the neutral compound 2 promotes its disproportionation to yield complexes 3 and 4 (depending on the stoichiometry) and the U{sup IV} mononuclear complex [U(OSi(OtBu){sub 3}){sub 3}(OTf)(thf){sub 2}] (5). This unprecedented reactivity demonstrates the key role of potassium for the stability of these complexes. (Copyright copyright 2013 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  15. LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina.

    Directory of Open Access Journals (Sweden)

    David Razafsky

    Full Text Available It has long been observed that many neuronal types position their nuclei within restricted cytoplasmic boundaries. A striking example is the apical localization of cone photoreceptors nuclei at the outer edge of the outer nuclear layer of mammalian retinas. Yet, little is known about how such nuclear spatial confinement is achieved and further maintained. Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes consist of evolutionary-conserved macromolecular assemblies that span the nuclear envelope to connect the nucleus with the peripheral cytoskeleton. Here, we applied a new transgenic strategy to disrupt LINC complexes either in cones or rods. In adult cones, we observed a drastic nuclear mislocalization on the basal side of the ONL that affected cone terminals overall architecture. We further provide evidence that this phenotype may stem from the inability of cone precursor nuclei to migrate towards the apical side of the outer nuclear layer during early postnatal retinal development. By contrast, disruption of LINC complexes within rod photoreceptors, whose nuclei are scattered across the outer nuclear layer, had no effect on the positioning of their nuclei thereby emphasizing differential requirements for LINC complexes by different neuronal types. We further show that Sun1, a component of LINC complexes, but not A-type lamins, which interact with LINC complexes at the nuclear envelope, participate in cone nuclei positioning. This study provides key mechanistic aspects underlying the well-known spatial confinement of cone nuclei as well as a new mouse model to evaluate the pathological relevance of nuclear mispositioning.

  16. Structural characterization of the ternary complex that mediates termination of NF-κB signaling by IκBα.

    Science.gov (United States)

    Mukherjee, Sulakshana P; Quintas, Pedro O; McNulty, Reginald; Komives, Elizabeth A; Dyson, H Jane

    2016-05-31

    The transcription factor NF-κB is used in many systems for the transduction of extracellular signals into the expression of signal-responsive genes. Published structural data explain the activation of NF-κB through degradation of its dedicated inhibitor IκBα, but the mechanism by which NF-κB-mediated signaling is turned off by its removal from the DNA in the presence of newly synthesized IκBα (termed stripping) is unknown. Previous kinetic studies showed that IκBα accelerates NF-κB dissociation from DNA, and a transient ternary complex between NF-κB, its cognate DNA sequence, and IκBα was observed. Here we structurally characterize the >100-kDa ternary complex by NMR and negative stain EM and show a modeled structure that is consistent with the measurements. These data provide a structural basis for previously unidentified insights into the molecular mechanism of stripping.

  17. Synthesis, characterization, and photoactivated DNA cleavage by copper (II)/cobalt (II) mediated macrocyclic complexes.

    Science.gov (United States)

    Naik, H R Prakash; Naik, H S Bhojya; Aravinda, T; Lamani, D S

    2010-01-01

    We report the synthesis of new photonuclease consisting of two Co(II)/Cu(II) complexes of macrocyclic fused quinoline. Metal complexes are [MLX(2)], type where M = Co(II) (5), Cu(II) (6), and X = Cl, and are well characterized by elemental analysis, Fourier transform infrared spectroscopy, (1)H-NMR and electronic spectra. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of Cu(II), and more so than that of cobalt. The chemistry of ternary and binary Co(II) complexes showing efficient light induced (360 nm) DNA cleavage activity is summarized. The role of the metal in photoinduced DNA cleavage reactions is explored by designing complex molecules having macrocyclic structure. The mechanistic pathways are found to be concentration dependent on Co(II)/Cu(II) complexes and the photoexcitation energy photoredox chemistry. Highly effective DNA cleavage ability of 6 is attributed to the effective cooperation of the metal moiety.

  18. A modular phosphate tether-mediated divergent strategy to complex polyols

    Directory of Open Access Journals (Sweden)

    Paul R. Hanson

    2014-10-01

    Full Text Available An efficient and divergent synthesis of polyol subunits utilizing a phosphate tether-mediated, one-pot, sequential RCM/CM/reduction process is reported. A modular, 3-component coupling strategy has been developed, in which, simple “order of addition” of a pair of olefinic-alcohol components to a pseudo-C2-symmetric phosphoryl chloride, coupled with the RCM/CM/reduction protocol, yields five polyol fragments. Each of the product polyols bears a central 1,3-anti-diol subunit with differential olefinic geometries at the periphery.

  19. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

    Science.gov (United States)

    Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

    2014-02-15

    Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

  20. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

    Science.gov (United States)

    Hiraga, Shin-ichiro; Alvino, Gina M.; Chang, FuJung; Lian, Hui-yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J.; Weinreich, Michael; Raghuraman, M.K.; Donaldson, Anne D.

    2014-01-01

    Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism. PMID:24532715

  1. Infrared spectrum of the Ag(+)-(pyridine)2 ionic complex: probing interactions in artificial metal-mediated base pairing.

    Science.gov (United States)

    Chakraborty, Shamik; Dopfer, Otto

    2011-07-11

    The isolated pyridine-Ag(+)-pyridine unit (Py-Ag(+)-Py) is employed as a model system to characterize the recently observed Ag(+)-mediated base pairing in DNA oligonucleotides at the molecular level. The structure and infrared (IR) spectrum of the Ag(+)-Py(2) cationic complex are investigated in the gas phase by IR multiple-photon dissociation (IRMPD) spectroscopy and quantum chemical calculations to determine the preferred metal-ion binding site and other salient properties of the potential-energy surface. The IRMPD spectrum has been obtained in the 840-1720 cm(-1) fingerprint region by coupling the IR free electron laser at the Centre Laser Infrarouge d'Orsay (CLIO) with a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an electrospray ionization source. The spectroscopic results are interpreted with quantum chemical calculations conducted at the B3LYP/aug-cc-pVDZ level. The analysis of the IRMPD spectrum is consistent with a σ complex, in which the Ag(+) ion binds to the nitrogen lone pairs of the two Py ligands in a linear configuration. The binding motif of Py-Ag(+)-Py in the gas phase is the same as that observed in Ag(+)-mediated base pairing in solution. Ag(+) bonding to the π-electron system of the aromatic ring is predicted to be a substantially less-favorable binding motif.

  2. Ikaros mediates gene silencing in T cells through Polycomb repressive complex 2

    Science.gov (United States)

    Oravecz, Attila; Apostolov, Apostol; Polak, Katarzyna; Jost, Bernard; Le Gras, Stéphanie; Chan, Susan; Kastner, Philippe

    2015-01-01

    T-cell development is accompanied by epigenetic changes that ensure the silencing of stem cell-related genes and the activation of lymphocyte-specific programmes. How transcription factors influence these changes remains unclear. We show that the Ikaros transcription factor forms a complex with Polycomb repressive complex 2 (PRC2) in CD4−CD8− thymocytes and allows its binding to more than 500 developmentally regulated loci, including those normally activated in haematopoietic stem cells and others induced by the Notch pathway. Loss of Ikaros in CD4−CD8− cells leads to reduced histone H3 lysine 27 trimethylation and ectopic gene expression. Furthermore, Ikaros binding triggers PRC2 recruitment and Ikaros interacts with PRC2 independently of the nucleosome remodelling and deacetylation complex. Our results identify Ikaros as a fundamental regulator of PRC2 function in developing T cells. PMID:26549758

  3. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

    Science.gov (United States)

    Tchasovnikarova, Iva A; Timms, Richard T; Matheson, Nicholas J; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A; Lehner, Paul J

    2015-06-26

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

  4. Plant structural complexity and mechanical defenses mediate predator-prey interactions in an odonate-bird system.

    Science.gov (United States)

    Grof-Tisza, Patrick; LoPresti, Eric; Heath, Sacha K; Karban, Richard

    2017-03-01

    Habitat-forming species provide refuges for a variety of associating species; these refuges may mediate interactions between species differently depending on the functional traits of the habitat-forming species. We investigated refuge provisioning by plants with different functional traits for dragonfly and damselfly (Odonata: Anisoptera and Zygoptera) nymphs emerging from water bodies to molt into their adult stage. During this period, nymphs experience high levels of predation by birds. On the shores of a small pond, plants with mechanical defenses (e.g., thorns and prickles) and high structural complexity had higher abundances of odonate exuviae than nearby plants which lacked mechanical defenses and exhibited low structural complexity. To disentangle the relative effects of these two potentially important functional traits on nymph emergence-site preference and survival, we conducted two fully crossed factorial field experiments using artificial plants. Nymphs showed a strong preference for artificial plants with high structural complexity and to a lesser extent, mechanical defenses. Both functional traits increased nymph survival but through different mechanisms. We suggest that future investigations attempt to experimentally separate the elements contributing to structural complexity to elucidate the mechanistic underpinnings of refuge provisioning.

  5. Hydrolytic protein cleavage mediated by unusual mononuclear copper(II) complexes: X-ray structures and solution studies.

    Science.gov (United States)

    de Oliveira, Mauricio C B; Scarpellini, Marciela; Neves, Ademir; Terenzi, Hernán; Bortoluzzi, Adailton J; Szpoganics, Bruno; Greatti, Alessandra; Mangrich, Antônio S; de Souza, Emanuel M; Fernandez, Pablo M; Soares, Marcia R

    2005-02-21

    The crystal structures and redox and UV-vis/EPR spectroscopic properties of two new mononuclear copper(II) complexes, [Cu(HL1)Cl2] (1) and [Cu(L1)Cl] (2), prepared through the reaction between copper(II) chloride and the ligand 2-[(bis(pyridylmethyl)amino)methyl]-4-methyl-6-formylphenol (HL1) under distinct base conditions, are reported along with solution studies. Also, we demonstrate that these CuII complexes are able to cleave unactivated peptide bonds from bovine serum albumin (BSA) and the thermostable enzyme Taq DNA polymerase at micromolar concentration, under mild pH and temperature conditions. The cleavage activity seems to be specific with defined proteolytic fragments appearing after protein treatment. The location of the specific cleavage sites was tentatively assigned to solvent-accessible portions of the protein. These are two of the most active Cu(II) complexes described to date, since their cleavage activity is detected in minutes and evidence is here presented for a hydrolytic mechanism mediating protein cleavage by these complexes.

  6. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    Science.gov (United States)

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  7. The Arabidopsis Mediator Complex Subunit16 Is a Key Component of Basal Resistance against the Necrotrophic Fungal Pathogen Sclerotinia sclerotiorum.

    Science.gov (United States)

    Wang, Chenggang; Yao, Jin; Du, Xuezhu; Zhang, Yanping; Sun, Yijun; Rollins, Jeffrey A; Mou, Zhonglin

    2015-09-01

    Although Sclerotinia sclerotiorum is a devastating necrotrophic fungal plant pathogen in agriculture, the virulence mechanisms utilized by S. sclerotiorum and the host defense mechanisms against this pathogen have not been fully understood. Here, we report that the Arabidopsis (Arabidopsis thaliana) Mediator complex subunit MED16 is a key component of basal resistance against S. sclerotiorum. Mutants of MED16 are markedly more susceptible to S. sclerotiorum than mutants of 13 other Mediator subunits, and med16 has a much stronger effect on S. sclerotiorum-induced transcriptome changes compared with med8, a mutation not altering susceptibility to S. sclerotiorum. Interestingly, med16 is also more susceptible to S. sclerotiorum than coronatine-insensitive1-1 (coi1-1), which is the most susceptible mutant reported so far. Although the jasmonic acid (JA)/ethylene (ET) defense pathway marker gene PLANT DEFENSIN1.2 (PDF1.2) cannot be induced in either med16 or coi1-1, basal transcript levels of PDF1.2 in med16 are significantly lower than in coi1-1. Furthermore, ET-induced suppression of JA-activated wound responses is compromised in med16, suggesting a role for MED16 in JA-ET cross talk. Additionally, MED16 is required for the recruitment of RNA polymerase II to PDF1.2 and OCTADECANOID-RESPONSIVE ARABIDOPSIS ETHYLENE/ETHYLENE-RESPONSIVE FACTOR59 (ORA59), two target genes of both JA/ET-mediated and the transcription factor WRKY33-activated defense pathways. Finally, MED16 is physically associated with WRKY33 in yeast and in planta, and WRKY33-activated transcription of PDF1.2 and ORA59 as well as resistance to S. sclerotiorum depends on MED16. Taken together, these results indicate that MED16 regulates resistance to S. sclerotiorum by governing both JA/ET-mediated and WRKY33-activated defense signaling in Arabidopsis.

  8. Ligand-Controlled CO2 Activation Mediated by Cationic Titanium Hydride Complexes, [LTiH](+) (L=Cp2 , O).

    Science.gov (United States)

    Tang, Shi-Ya; Rijs, Nicole J; Li, Jilai; Schlangen, Maria; Schwarz, Helmut

    2015-06-01

    CO2 activation mediated by [LTiH](+) (L=Cp2 , O) is observed in the gas phase at room temperature using electrospray-ionization mass spectrometry, and reaction details are derived from traveling wave ion-mobility mass spectrometry. Wheresas oxygen-atom transfer prevails in the reaction of the oxide complex [OTiH](+) with CO2 , generating [OTi(OH)](+) under the elimination of CO, insertion of CO2 into the metal-hydrogen bond of the cyclopentadienyl complex, [Cp2 TiH](+) , gives rise to the formate complex [Cp2 Ti(O2 CH)](+) . DFT-based methods were employed to understand how the ligand controls the observed variation in reactivity toward CO2 . Insertion of CO2 into the Ti-H bond constitutes the initial step for the reaction of both [Cp2 TiH](+) and [OTiH](+) , thus generating formate complexes as intermediates. In contrast to [Cp2 Ti(O2 CH)](+) which is kinetically stable, facile decarbonylation of [OTi(O2 CH)](+) results in the hydroxo complex [OTi(OH)](+) . The longer lifetime of [Cp2 Ti(O2 CH)](+) allows for secondary reactions with background water, as a result of which, [Cp2 Ti(OH)](+) is formed. Further, computational studies reveal a good linear correlation between the hydride affinity of [LTi](2+) and the barrier for CO2 insertion into various [LTiH](+) complexes. Understanding the intrinsic ligand effects may provide insight into the selective activation of CO2 .

  9. Zeolite-mediated photochemical charge separation using a surface-entrapped ruthenium-polypyridyl complex.

    Science.gov (United States)

    Kim, Yanghee; Lee, Hyunjung; Dutta, Prabir K; Das, Amitava

    2003-06-30

    Employing the strategy of quaternization of the 2,2' N atoms of the conjugated bipyridine ligand 1,4-bis[2-(4'-methyl-2,2'-bipyrid-4-yl)ethenyl]benzene (L), a polypyridyl complex of ruthenium(II) was tethered on the surface of zeolite Y. Electrochemical and spectroscopic properties of the complex suggest that, upon visible photoexcitation of the MLCT band, the electron is localized on the conjugated ligand rather than the bipyridines. Electron transfer from the surface complex to bipyridinium ions (methyl viologen) within the zeolite was observed. Visible light photolysis of the ruthenium-zeolite solid ion-exchanged with diquat and suspended in a propyl viologen sulfonate solution led to permanent formation of the blue propyl viologen sulfonate radical ion in solution. The model that is proposed involves intrazeolitic charge transfer to ion-exchanged diquat followed by interfacial (zeolite to solution) electron transfer to propyl viologen sulfonate in solution. Because of the slow intramolecular back-electron-transfer reaction and the forward electron propagation via the ion-exchanged diquat, Ru(III) is formed. This Ru(III) complex formed on the zeolite is proposed to react rapidly with water in the presence of light, followed by reaction with the propyl viologen sulfonate, to form pyridones and regeneration of Ru(II), which then continues the photochemical process.

  10. Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity

    Science.gov (United States)

    Zhang, Jing; Rouillon, Christophe; Kerou, Melina; Reeks, Judith; Brugger, Kim; Graham, Shirley; Reimann, Julia; Cannone, Giuseppe; Liu, Huanting; Albers, Sonja-Verena; Naismith, James H; Spagnolo, Laura; White, Malcolm F

    2012-01-01

    Summary The prokaryotic Clusters of Regularly Interspaced Palindromic Repeats (CRISPR) system utilizes genomically-encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse “payload” of targeting crRNA. The crystal structure of Cmr7 and low resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8-nucleotide repeat-derived 5′ sequence in the crRNA, but not on the presence of a proto-spacer associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets. PMID:22227115

  11. Mediating Complex Texts in the Upper Grades: Considering Motivation, Instructional Intensity, and Cognitive Challenge

    Science.gov (United States)

    Ford-Connors, Evelyn; Dougherty, Susan; Robertson, Dana A.; Paratore, Jeanne R.

    2015-01-01

    Rising expectations for middle grade students to independently read and comprehend complex, discipline-specific texts have also raised expectations for the ways teachers will teach. Helping all students, despite assessed reading levels, to access grade level texts calls for instructional approaches that not only meet readers where they are, but…

  12. Tuning interaction in dinuclear ruthenium complexes : HOMO versus LUMO mediated superexchange through azole and azine bridges

    NARCIS (Netherlands)

    Browne, Wesley; Hage, R; Vos, Johannes G.

    2006-01-01

    In this review the interaction between metal centers in dinuclear complexes based on azole and azine containing bridging ligands is reviewed. The focus of the review is on the manner in which the interaction pathway can be manipulated by variations in the nature of both the direct bridging unit and

  13. Trigeminal star-like platinum complexes induce cancer cell senescence through quadruplex-mediated telomere dysfunction.

    Science.gov (United States)

    Zheng, Xiao-Hui; Mu, Ge; Zhong, Yi-Fang; Zhang, Tian-Peng; Cao, Qian; Ji, Liang-Nian; Zhao, Yong; Mao, Zong-Wan

    2016-12-01

    Two trigeminal star-like platinum complexes were synthesized to induce the formation of human telomere G-quadruplex (hTel G4) with extremely high selectivity and affinity. The induced hTel G4 activates strong telomeric DNA damage response (TDDR), resulting in telomere dysfunction and cell senescence.

  14. Organometallic mediated radical polymerization of vinyl acetate using bis(imino)pyridine vanadium trichloride complexes.

    Science.gov (United States)

    Perry, Mitchell R; Allan, Laura E N; Decken, Andreas; Shaver, Michael P

    2013-07-07

    The synthesis and characterization of one novel proligand and six novel vanadium(III) trichloride complexes is described. The controlled radical polymerization activity towards vinyl acetate of these, and eight other bis(imino)pyridine vanadium trichloride complexes previously reported, is investigated. Those complexes possessing variation at the N-aryl para-position with no steric protection offered by ortho-substituents (4 examples) result in poor control over poly(vinyl acetate) polymerization. Control is improved with increasing steric bulk at the ortho-position of the N-aryl substituent (4 examples) although attempts to increase steric bulk past isopropyl were unsuccessful. Synthesizing bis(imino)pyridine vanadium trichloride complexes with substituted imine backbones restores polymerization control when aliphatic substituents are used (4 examples) but ceases to make any drastic improvements on catalyst lifetime. Modification of the polymerization conditions is also investigated, in an attempt to improve the catalyst lifetime. Expansion of the monomer scope to include other vinyl esters, particularly those derived from renewable resources, shows promising results.

  15. Recombination-Mediated Telomere Maintenance in Saccharomyces cerevisiae Is Not Dependent on the Shu Complex.

    Directory of Open Access Journals (Sweden)

    Paula M van Mourik

    Full Text Available In cells lacking telomerase, telomeres shorten progressively during each cell division due to incomplete end-replication. When the telomeres become very short, cells enter a state that blocks cell division, termed senescence. A subset of these cells can overcome senescence and maintain their telomeres using telomerase-independent mechanisms. In Saccharomyces cerevisiae, these cells are called 'survivors' and are dependent on Rad52-dependent homologous recombination and Pol32-dependent break-induced replication. There are two main types of survivors: type I and type II. The type I survivors require Rad51 and maintain telomeres by amplification of subtelomeric elements, while the type II survivors are Rad51-independent, but require the MRX complex and Sgs1 to amplify the C1-3A/TG1-3 telomeric sequences. Rad52, Pol32, Rad51, and Sgs1 are also important to prevent accelerated senescence, indicating that recombination processes are important at telomeres even before the formation of survivors. The Shu complex, which consists of Shu1, Shu2, Psy3, and Csm2, promotes Rad51-dependent homologous recombination and has been suggested to be important for break-induced replication. It also promotes the formation of recombination intermediates that are processed by the Sgs1-Top3-Rmi1 complex, as mutations in the SHU genes can suppress various sgs1, top3, and rmi1 mutant phenotypes. Given the importance of recombination processes during senescence and survivor formation, and the involvement of the Shu complex in many of the same processes during DNA repair, we hypothesized that the Shu complex may also have functions at telomeres. Surprisingly, we find that this is not the case: the Shu complex does not affect the rate of senescence, does not influence survivor formation, and deletion of SHU1 does not suppress the rapid senescence and type II survivor formation defect of a telomerase-negative sgs1 mutant. Altogether, our data suggest that the Shu complex is not

  16. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.

    Science.gov (United States)

    McGarvey, Jennifer C; Xiao, Kunhong; Bowman, Shanna L; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W Bruce; Ardura, Juan A; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A; Friedman, Peter A

    2016-05-20

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.

  17. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

    Science.gov (United States)

    McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

    2016-01-01

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

  18. Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.

    Science.gov (United States)

    Gong, Peng; Kortus, Matthew G; Nix, Jay C; Davis, Ralph E; Peersen, Olve B

    2013-01-01

    RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle intermediates.

  19. Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.

    Directory of Open Access Journals (Sweden)

    Peng Gong

    Full Text Available RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle

  20. Bridging function mediated intermetallic coupling in diruthenium-bis(bipyridine) complexes

    Indian Academy of Sciences (India)

    Soma Chakraborty; Biplab Mondal; Biprajit Sarkar; Goutam Kumar Lahiri

    2002-08-01

    The interactions of potentially dinucleating bridging functionalities (I-VI) with the ruthenium-bis(bypyridine) precursor [RuII(bpy)2(EtOH)2]2+ have been explored. The bridging functions I, II and VI directly result in the expected dinuclear complexes of the type [(bpy)2RuII{L}RuII(bpy)2]+ (1, 2, 7 and 8) ( = 0, = 4 and = -2, = 2). The bridging ligand III undergoes N-N or N-C bond cleavage reaction on coordination to the RuII(bpy)2 core which eventually yields a mononuclear complex of the type [(bpy)2RuII(L)]+, 3, where L = -OC6H3(R)C(R′)=N-H. However, the electrogenerated mononuclear ruthenium(III) congener, 3+ in acetonitrile dimerises to [(bpy)2RuIII {-OC6H3(R)C(R′)=N-N=(R′)C(R)C6H3O-}RuIII(bpy)2]4+ (4). In the presence of a slight amount of water content in the acetonitrile solvent the dimeric species (4) reduces back to the starting ruthenium(II) monomer (3). The preformed bridging ligand IV undergoes multiple transformations on coordination to the Ru(bpy)2 core, such as hydrolysis of the imine groups of IV followed by intermolecular head-to-tail oxidative coupling of the resultant amino phenol moieties, which in turn results in a new class of dimeric complex of the type [(bpy)2RuII {-OC6H4-N=C6H3(=NH)O-}RuII(bpy)2]2+ (5). In 5, the bridging ligand comprises of two , chelating binding sites each formally in the semiquinone level and there is a -benzoquinonediimine bridge between the metal centres. In complex 6, the preformed bridging ligand, 3,6-bis(3,5-dimethylpyrazol-1-yl)-1,2-dihydro-1,2,4,5- tetrazine, H2L (V) undergoes oxidative dehydrogenation to aromatic tetrazine based bridging unit, 3,6-bis(3,5-dimethylpyrazol-1-yl)-1,2,4,5-tetrazine, L. The detailed spectroelectrochemical aspects of the complexes have been studied in order to understand the role of the bridging units towards the intermetallic electronic coupling in the dinuclear complexes.

  1. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells*

    Science.gov (United States)

    Matheson, Nicholas J.; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A.; Lehner, Paul J.

    2015-01-01

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A non-lethal forward genetic screen in near-haploid KBM7 cells identified the Human Silencing Hub (HUSH), a complex of three poorly-characterised proteins, TASOR, MPP8, and periphilin, which is absent from Drosophila but conserved from fish to humans. Loss of HUSH subunits resulted in decreased H3K9me3 at both endogenous genomic loci and retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing. PMID:26022416

  2. TWEAK inhibits TRAF2-mediated CD40 signaling by destabilization of CD40 signaling complexes.

    Science.gov (United States)

    Salzmann, Steffen; Lang, Isabell; Rosenthal, Alevtina; Schäfer, Viktoria; Weisenberger, Daniela; Carmona Arana, José Antonio; Trebing, Johannes; Siegmund, Daniela; Neumann, Manfred; Wajant, Harald

    2013-09-01

    We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)-induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L-CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L-CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2-cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L-CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.

  3. Importance of iron complexation for Fenton-mediated hydroxyl radical production at circumneutral pH

    Directory of Open Access Journals (Sweden)

    Christopher J. Miller

    2016-08-01

    Full Text Available The reaction between Fe(II and H2O2 to yield hydroxyl radicals (HO•, the Fenton reaction, is of interest due to its role in trace metal and natural organic matter biogeochemistry, its utility in water treatment and its role in oxidative cell degradation and associated human disease. There is significant dispute over whether HO•, the most reactive of the so-called reactive oxygen species, is formed in this reaction, particularly under circumneutral conditions relevant to natural systems. In this work we have studied the oxidation kinetics of Fe(II complexed by L = citrate, ethylenediaminetetraacetic acid (EDTA and diethylenetriaminepentaacetic acid (DTPA and also measured HO• production using phthalhydrazide as a probe compound at pH 8.2. It is shown that HO• is the sole product of the Fe(IIL-H2O2 reaction for L = EDTA and DTPA, with kinetic modelling of the full reaction pathway utilized to confirm this finding. Quantitative HO• production also appears likely for L = citrate, although uncertainties with the speciation of Fe(II-citrate complexes as well as difficulties in modelling the oxidation kinetics of these complexes has prevented a definitive conclusion. In the absence of ligands at circumneutral pH, inorganic Fe(II reacts with H2O2 to yield a species other than HO•, contrary to the well-established production of HO• from inorganic Fe(II at low pH. Our results suggest that at high pH Fe(II must be complexed for HO• production to occur.

  4. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate.

    Science.gov (United States)

    Vorhagen, Susanne; Niessen, Carien M

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Reductive Cleavage of CO2 by Metal-Ligand-Cooperation Mediated by an Iridium Pincer Complex.

    Science.gov (United States)

    Feller, Moran; Gellrich, Urs; Anaby, Aviel; Diskin-Posner, Yael; Milstein, David

    2016-05-25

    A unique mode of stoichiometric CO2 activation and reductive splitting based on metal-ligand-cooperation is described. The novel Ir hydride complexes [((t)Bu-PNP*)Ir(H)2] (2) ((t)Bu-PNP*, deprotonated (t)Bu-PNP ligand) and [((t)Bu-PNP)Ir(H)] (3) react with CO2 to give the dearomatized complex [((t)Bu-PNP*)Ir(CO)] (4) and water. Mechanistic studies have identified an adduct in which CO2 is bound to the ligand and metal, [((t)Bu-PNP-COO)Ir(H)2] (5), and a di-CO2 iridacycle [((t)Bu-PNP)Ir(H)(C2O4-κC,O)] (6). DFT calculations confirm the formation of 5 and 6 as reversibly formed side products, and suggest an η(1)-CO2 intermediate leading to the thermodynamic product 4. The calculations support a metal-ligand-cooperation pathway in which an internal deprotonation of the benzylic position by the η(1)-CO2 ligand leads to a carboxylate intermediate, which further reacts with the hydride ligand to give complex 4 and water.

  6. The IDOL–UBE2D complex mediates sterol-dependent degradation of the LDL receptor

    Science.gov (United States)

    Zhang, Li; Fairall, Louise; Goult, Benjamin T.; Calkin, Anna C.; Hong, Cynthia; Millard, Christopher J.; Tontonoz, Peter; Schwabe, John W.R.

    2011-01-01

    We previously identified the E3 ubiquitin ligase IDOL as a sterol-dependent regulator of the LDL receptor (LDLR). The molecular pathway underlying IDOL action, however, remains to be determined. Here we report the identification and biochemical and structural characterization of an E2–E3 ubiquitin ligase complex for LDLR degradation. We identified the UBE2D family (UBE2D1–4) as E2 partners for IDOL that support both autoubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1 Å crystal structure of the IDOL RING domain–UBE2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL–UBE2D1 interface also defined the stereochemical basis for the selectivity of IDOL for UBE2Ds over other E2 ligases. Structure-based mutations that inhibit IDOL dimerization or IDOL–UBE2D interaction block IDOL-dependent LDLR ubiquitination and degradation. Furthermore, expression of a dominant-negative UBE2D enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOL–UBE2D complex as an important determinant of LDLR activity, and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake. PMID:21685362

  7. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    Energy Technology Data Exchange (ETDEWEB)

    Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  8. The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex

    Directory of Open Access Journals (Sweden)

    Gribble Kristin E

    2012-08-01

    Full Text Available Abstract Background Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP, a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. Results Isolates of the B. plicatilis species complex have 1–4 copies of mmr-b, each composed of 2–9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. Conclusions Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving

  9. SLAC, a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis.

    Science.gov (United States)

    Feliciano, Daniel; Di Pietro, Santiago M

    2012-11-01

    During clathrin-mediated endocytosis, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (yeast WASp) is the strongest activator of the Arp2/3 complex in yeast cells; it is not autoinhibited and arrives to endocytic sites 20 s before actin polymerization begins. It is unclear how Las17 is kept inactive for 20 s at endocytic sites, thus restricting actin polymerization to late stages of endocytosis. In this paper, we demonstrate that Las17 is part of a large and biochemically stable complex with Sla1, a clathrin adaptor that inhibits Las17 activity. The interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I and class II. In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on the class I/II Las17 polyproline motifs and is based on competition between Sla1 and monomeric actin for binding to Las17. Furthermore, live-cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, inhibition during the initial 20 s, and efficient endocytosis. These results advance our understanding of the regulation of actin polymerization in endocytosis.

  10. A TDG/CBP/RARα Ternary Complex Mediates the Retinoic Acid-dependent Expression of DNA Methylation-sensitive Genes

    Directory of Open Access Journals (Sweden)

    Hélène Léger

    2014-02-01

    Full Text Available The thymine DNA glycosylase (TDG is a multifunctional enzyme, which is essential for embryonic development. It mediates the base excision repair (BER of G:T and G:U DNA mismatches arising from the deamination of 5-methyl cytosine (5-MeC and cytosine, respectively. Recent studies have pointed at a role of TDG during the active demethylation of 5-MeC within CpG islands. TDG interacts with the histone acetylase CREB-binding protein (CBP to activate CBP-dependent transcription. In addition, TDG also interacts with the retinoic acid receptor α (RARα, resulting in the activation of RARα target genes. Here we provide evidence for the existence of a functional ternary complex containing TDG, CBP and activated RARα. Using global transcriptome profiling, we uncover a coupling of de novo methylation-sensitive and RA-dependent transcription, which coincides with a significant subset of CBP target genes. The introduction of a point mutation in TDG, which neither affects overall protein structure nor BER activity, leads to a significant loss in ternary complex stability, resulting in the deregulation of RA targets involved in cellular networks associated with DNA replication, recombination and repair. We thus demonstrate for the first time a direct coupling of TDG’s epigenomic and transcription regulatory function through ternary complexes with CBP and RARα.

  11. Preparation and characterization of nanocrystalline CuO powders with the different surfactants and complexing agent mediated precipitation method

    Energy Technology Data Exchange (ETDEWEB)

    Rajendran, V.; Gajendiran, J., E-mail: gaja.nanotech@gmail.com

    2014-08-15

    Highlights: • CuO nanostructures by surfactants mediated method. • Structural and optical properties of CuO nanostructures changes under the effect of surface modifier. • Citric acid assisted is the best, in terms of size, morphology and optical properties than that of CTAB, SDS and PEG-400. - Abstract: Nanostructures of copper oxide (CuO) was synthesized into crystallite sized ranging from 20 to 50 nm in the presence of different surfactants, and complex agent such as cityl tri methyl ammonium bromide (CTAB), sodium do decyl sulfate (SDS), poly ethylene glycol (PEG-400) and citric acid via a precipitation route. Variations in several parameters and their effects on the structural and optical properties of CuO nanostructures (crystallite size, morphology and band gap) were investigated by XRD, FTIR, SEM and UV analysis. The UV–visible absorption spectra of the different surfactants and complexing agent assisted CuO nanostructures indicates that the estimated optical band gap energy value (1.94–1.98 eV) is higher than that of the bulk CuO value (1.4 eV), which is attributed to the quantum confinement effect. The formation mechanism of different surfactants and complexing agent assisted CuO nanostructures is also proposed.

  12. The evolution of the dystroglycan complex, a major mediator of muscle integrity

    Directory of Open Access Journals (Sweden)

    Josephine C. Adams

    2015-09-01

    Full Text Available Basement membrane (BM extracellular matrices are crucial for the coordination of different tissue layers. A matrix adhesion receptor that is important for BM function and stability in many mammalian tissues is the dystroglycan (DG complex. This comprises the non-covalently-associated extracellular α-DG, that interacts with laminin in the BM, and the transmembrane β-DG, that interacts principally with dystrophin to connect to the actin cytoskeleton. Mutations in dystrophin, DG, or several enzymes that glycosylate α-DG underlie severe forms of human muscular dystrophy. Nonwithstanding the pathophysiological importance of the DG complex and its fundamental interest as a non-integrin system of cell-ECM adhesion, the evolution of DG and its interacting proteins is not understood. We analysed the phylogenetic distribution of DG, its proximal binding partners and key processing enzymes in extant metazoan and relevant outgroups. We identify that DG originated after the divergence of ctenophores from porifera and eumetazoa. The C-terminal half of the DG core protein is highly-conserved, yet the N-terminal region, that includes the laminin-binding region, has undergone major lineage-specific divergences. Phylogenetic analysis based on the C-terminal IG2_MAT_NU region identified three distinct clades corresponding to deuterostomes, arthropods, and mollusks/early-diverging metazoans. Whereas the glycosyltransferases that modify α-DG are also present in choanoflagellates, the DG-binding proteins dystrophin and laminin originated at the base of the metazoa, and DG-associated sarcoglycan is restricted to cnidarians and bilaterians. These findings implicate extensive functional diversification of DG within invertebrate lineages and identify the laminin-DG-dystrophin axis as a conserved adhesion system that evolved subsequent to integrin-ECM adhesion, likely to enhance the functional complexity of cell-BM interactions in early metazoans.

  13. The evolution of the dystroglycan complex, a major mediator of muscle integrity.

    Science.gov (United States)

    Adams, Josephine C; Brancaccio, Andrea

    2015-08-28

    Basement membrane (BM) extracellular matrices are crucial for the coordination of different tissue layers. A matrix adhesion receptor that is important for BM function and stability in many mammalian tissues is the dystroglycan (DG) complex. This comprises the non-covalently-associated extracellular α-DG, that interacts with laminin in the BM, and the transmembrane β-DG, that interacts principally with dystrophin to connect to the actin cytoskeleton. Mutations in dystrophin, DG, or several enzymes that glycosylate α-DG underlie severe forms of human muscular dystrophy. Nonwithstanding the pathophysiological importance of the DG complex and its fundamental interest as a non-integrin system of cell-ECM adhesion, the evolution of DG and its interacting proteins is not understood. We analysed the phylogenetic distribution of DG, its proximal binding partners and key processing enzymes in extant metazoan and relevant outgroups. We identify that DG originated after the divergence of ctenophores from porifera and eumetazoa. The C-terminal half of the DG core protein is highly-conserved, yet the N-terminal region, that includes the laminin-binding region, has undergone major lineage-specific divergences. Phylogenetic analysis based on the C-terminal IG2_MAT_NU region identified three distinct clades corresponding to deuterostomes, arthropods, and mollusks/early-diverging metazoans. Whereas the glycosyltransferases that modify α-DG are also present in choanoflagellates, the DG-binding proteins dystrophin and laminin originated at the base of the metazoa, and DG-associated sarcoglycan is restricted to cnidarians and bilaterians. These findings implicate extensive functional diversification of DG within invertebrate lineages and identify the laminin-DG-dystrophin axis as a conserved adhesion system that evolved subsequent to integrin-ECM adhesion, likely to enhance the functional complexity of cell-BM interactions in early metazoans.

  14. Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly.

    Science.gov (United States)

    AhYoung, Andrew P; Jiang, Jiansen; Zhang, Jiang; Khoi Dang, Xuan; Loo, Joseph A; Zhou, Z Hong; Egea, Pascal F

    2015-06-23

    Membrane contact sites (MCS) between organelles are proposed as nexuses for the exchange of lipids, small molecules, and other signals crucial to cellular function and homeostasis. Various protein complexes, such as the endoplasmic reticulum-mitochondrial encounter structure (ERMES), function as dynamic molecular tethers between organelles. Here, we report the reconstitution and characterization of subcomplexes formed by the cytoplasm-exposed synaptotagmin-like mitochondrial lipid-binding protein (SMP) domains present in three of the five ERMES subunits--the soluble protein Mdm12, the endoplasmic reticulum (ER)-resident membrane protein Mmm1, and the mitochondrial membrane protein Mdm34. SMP domains are conserved lipid-binding domains found exclusively in proteins at MCS. We show that the SMP domains of Mdm12 and Mmm1 associate into a tight heterotetramer with equimolecular stoichiometry. Our 17-Å-resolution EM structure of the complex reveals an elongated crescent-shaped particle in which two Mdm12 subunits occupy symmetric but distal positions at the opposite ends of a central ER-anchored Mmm1 homodimer. Rigid body fitting of homology models of these SMP domains in the density maps reveals a distinctive extended tubular structure likely traversed by a hydrophobic tunnel. Furthermore, these two SMP domains bind phospholipids and display a strong preference for phosphatidylcholines, a class of phospholipids whose exchange between the ER and mitochondria is essential. Last, we show that the three SMP-containing ERMES subunits form a ternary complex in which Mdm12 bridges Mmm1 to Mdm34. Our findings highlight roles for SMP domains in ERMES assembly and phospholipid binding and suggest a structure-based mechanism for the facilitated transport of phospholipids between organelles.

  15. Central complex and mushroom bodies mediate novelty choice behavior in Drosophila.

    Science.gov (United States)

    Solanki, Narendra; Wolf, Reinhard; Heisenberg, Martin

    2015-03-01

    Novelty choice, a visual paired-comparison task, for the fly Drosophila melanogaster is studied with severely restrained single animals in a flight simulator. The virtual environment simulates free flight for rotation in the horizontal plane. The behavior has three functional components: visual azimuth orientation, working memory, and pattern discrimination (perception). Here we study novelty choice in relation to its neural substrate in the brain and show that it requires the central complex and, in particular, the ring neurons of the ellipsoid body. Surprisingly, it also involves the mushroom bodies which are needed specifically in the comparison of patterns of different sizes.

  16. Copper-Mediated Fluorination of Arylboronate Esters. Identification of a Copper(III) Fluoride Complex

    Science.gov (United States)

    Fier, Patrick S.; Luo, Jingwei; Hartwig, John F.

    2013-01-01

    A method for the direct conversion of arylboronate esters to aryl fluorides under mild conditions with readily available reagents is reported. Tandem reactions have also been developed for the fluorination of arenes and aryl bromides through aryl-boronate ester intermediates. Mechanistic studies suggest that this fluorination reaction occurs through facile oxidation of Cu(I) to Cu(III) followed by rate-limiting transmetallation of a bound arylboronate to Cu(III). Fast C-F reductive elimination is proposed to occur from an aryl-copper(III)-fluoride complex. Cu(III) intermediates have been generated independently and identified by NMR spectroscopy and ESI-MS. PMID:23384209

  17. Well-defined silica-supported zirconium–imido complexes mediated heterogeneous imine metathesis

    KAUST Repository

    Hamzaoui, Bilel

    2016-02-15

    Upon prolonged thermal exposure under vacuum, a well-defined single-site surface species [(≡Si-O-)Zr(NEt2)3] (1) evolves into an ethylimido complex [(≡Si-O-)Zr(=NEt)NEt2] (2). Reactions of 2 with an imine substrate result in imido/imine (=NRi, R: Et, Ph) exchange (metathesis) with the formation of [(≡Si-O-)Zr(=NPh)NEt2] (3). Compounds 2 and 3 effectively catalyze imine/imine cross-metathesis and are thus considered as the first heterogeneous catalysts active for imine metathesis. © The Royal Society of Chemistry 2016.

  18. Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Nardou Katya

    2008-06-01

    Full Text Available Abstract Background Histone deacetylase inhibitors (HDACi are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. Results We show here that the HDACi Sodium Butyrate (NaB, suberoylanilide hydroxamic acid (SAHA and Trichostatin A (TSA strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. Conclusion HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

  19. Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores.

    Science.gov (United States)

    Lass, Achim; Zimmermann, Robert; Oberer, Monika; Zechner, Rudolf

    2011-01-01

    Lipolysis is the biochemical pathway responsible for the catabolism of triacylglycerol (TAG) stored in cellular lipid droplets. The hydrolytic cleavage of TAG generates non-esterified fatty acids, which are subsequently used as energy substrates, essential precursors for lipid and membrane synthesis, or mediators in cell signaling processes. Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, it is most abundant, however, in white and brown adipose tissue. Over the last 5years, important enzymes and regulatory protein factors involved in lipolysis have been identified. These include an essential TAG hydrolase named adipose triglyceride lipase (ATGL) [annotated as patatin-like phospholipase domain-containing protein A2], the ATGL activator comparative gene identification-58 [annotated as α/β hydrolase containing protein 5], and the ATGL inhibitor G0/G1 switch gene 2. Together with the established hormone-sensitive lipase [annotated as lipase E] and monoglyceride lipase, these proteins constitute the basic "lipolytic machinery". Additionally, a large number of hormonal signaling pathways and lipid droplet-associated protein factors regulate substrate access and the activity of the "lipolysome". This review summarizes the current knowledge concerning the enzymes and regulatory processes governing lipolysis of fat stores in adipose and non-adipose tissues. Special emphasis will be given to ATGL, its regulation, and physiological function. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Lipolysis – A highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores

    Science.gov (United States)

    Lass, Achim; Zimmermann, Robert; Oberer, Monika; Zechner, Rudolf

    2011-01-01

    Summary Lipolysis is the biochemical pathway responsible for the catabolism of triacylglycerol (TAG) stored in cellular lipid droplets. The hydrolytic cleavage of TAG generates non-esterified fatty acids, which are subsequently used as energy substrates, essential precursors for lipid and membrane synthesis, or mediators in cell signaling processes. Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, it is most abundant, however, in white and brown adipose tissue. Over the last 5 years, important enzymes and regulatory protein factors involved in lipolysis have been identified. These include an essential TAG hydrolase named adipose triglyceride lipase (ATGL) [annotated as patatin-like phospholipase domain-containing protein A2], the ATGL activator comparative gene identification-58 [annotated as α/β hydrolase containing protein 5], and the ATGL inhibitor G0/G1 switch gene 2. Together with the established hormone-sensitive lipase [annotated as lipase E] and monoglyceride lipase, these proteins constitute the basic “lipolytic machinery”. Additionally, a large number of hormonal signaling pathways and lipid droplet-associated protein factors regulate substrate access and the activity of the “lipolysome”. This review summarizes the current knowledge concerning the enzymes and regulatory processes governing lipolysis of fat stores in adipose and non-adipose tissues. Special emphasis will be given to ATGL, its regulation, and physiological function. PMID:21087632

  1. Main Group Lewis Acid-Mediated Transformations of Transition-Metal Hydride Complexes.

    Science.gov (United States)

    Maity, Ayan; Teets, Thomas S

    2016-08-10

    This Review highlights stoichiometric reactions and elementary steps of catalytic reactions involving cooperative participation of transition-metal hydrides and main group Lewis acids. Included are reactions where the transition-metal hydride acts as a reactant as well as transformations that form the metal hydride as a product. This Review is divided by reaction type, illustrating the diverse roles that Lewis acids can play in mediating transformations involving transition-metal hydrides as either reactants or products. We begin with a discussion of reactions where metal hydrides form direct adducts with Lewis acids, elaborating the structure and dynamics of the products of these reactions. The bulk of this Review focuses on reactions where the transition metal and Lewis acid act in cooperation, and includes sections on carbonyl reduction, H2 activation, and hydride elimination reactions, all of which can be promoted by Lewis acids. Also included is a section on Lewis acid-base secondary coordination sphere interactions, which can influence the reactivity of hydrides. Work from the past 50 years is included, but the majority of this Review focuses on research from the past decade, with the intent of showcasing the rapid emergence of this field and the potential for further development into the future.

  2. REST-mediated recruitment of polycomb repressor complexes in mammalian cells

    DEFF Research Database (Denmark)

    Dietrich, Nikolaj; Lerdrup, Mads; Landt, Eskild

    2012-01-01

    Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1......, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest¿/¿ and Eed¿/¿ mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently...... of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest¿/¿ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context...

  3. Drying-Mediated Self-Assembly of Highly Ordered Complex Structures: From Polymers to Nanoparticles

    Science.gov (United States)

    Lin, Zhiqun

    2009-03-01

    Drying of a sessile drop containing nonvolatile solutes readily self-assembles into a number of concentric ``coffee rings'' by repetitive ``stick-slip'' motion of the three-phase contact line. However, due mainly to lack of control over the evaporation process of the drop, the challenge remains to use evaporative self-assembly to rationally ``synthesize'' ``coffee rings'' of different shapes and sizes of high regularity and fidelity. Here, we report a facile, robust, and one-step evaporation method for producing in a precisely controllable manner versatile microstructures possessing high regularity, dispensing with the need for lithographic techniques and externally applied fields. Polymer or nanocrystal solutions are confined in a simple geometry comprised of a curved surface placed upon a flat substrate. By changing the shape of the upper surface of the imposed geometry, the controlled, evaporative self-assembly of polymer or nanocrystal solutions yields a variety of complex, intriguing, and well-ordered structures over large areas. As such, this method represents a significant advance in creating regularly organized, complex structures with potential applications in microelectronics, optoelectronics, and biotechnology, among other areas.

  4. Complex-Mediated Synthesis of Tantalum Oxyfluoride Hierarchical Nanostructures for Highly Efficient Photocatalytic Hydrogen Evolution.

    Science.gov (United States)

    Xu, Leilei; Gong, Haotian; Deng, Li; Long, Fei; Gu, Yu; Guan, Jianguo

    2016-04-13

    In this work, we have, for the first time, developed a facile wet-chemical route to obtain a novel photocatalytic material of tantalum oxyfluoride hierarchical nanostructures composed of amorphous cores and single crystalline TaO2F nanorod shells (ACHNs) by regulating the one-step hydrothermal process of TaF5 in a mixed solution of isopropanol (i-PrOH) and H2O. In this approach, elaborately controlling the reaction temperature and volume ratio of i-PrOH and H2O enabled TaF5 to transform into intermediate coordination complex ions of [TaOF3·2F](2-) and [TaF7](2-), which subsequently produced tantalum oxyfluoride ACHNs via a secondary nucleation and growth due to a stepwise change in hydrolysis rates of the two complex ions. Because of the unique chemical composition, crystal structure and micromorphology, the as-prepared tantalum oxyfluoride ACHNs show a more negative flat band potential, an accelerated charge transfer, and a remarkable surface area of 152.4 m(2) g(-1) contributing to increased surface reaction sites. As a result, they exhibit a photocatalytic activity for hydrogen production up to 1.95 mmol h(-1) g(-1) under the illumination of a simulated solar light without any assistance of co-catalysts, indicating that the as-prepared tantalum oxyfluoride ACHNs are a novel promising photocatalytic material for hydrogen production.

  5. Complex interactions mediate the effects of fish farming on benthic chemistry within a region of Scotland.

    Science.gov (United States)

    Mayor, Daniel J; Solan, Martin

    2011-07-01

    Fish farms typically generate a localised gradient of both organic and inorganic pollutants in the underlying sediments. The factors governing the extent of such impacts remain poorly understood, particularly when multiple sites are considered. We used regression-type techniques to examine the drivers of sediment chemistry patterns around five Scottish fish farms that ranged in size (120-2106 tonnes) and fish species, but were located within farm illustrate that between-site variability can be high, even at this regional-scale. These effects must be accounted for when comparing the effects of fish farming at different locations. All measured chemical parameters declined rapidly as a function of distance from the cage edge, with the rate of decline depending on local current speeds. Only phosphorus concentrations increased directly with farm size. Increasing current speeds at farms carbon in the underlying sediments, whereas the opposite occurred at larger farms. The counterintuitive effect of current speed at farms above the threshold size suggests that the physical properties of the seabed at these locations favour the accumulation of organic wastes and/or that the underlying communities have a lower assimilative capacity. These imply that the environmental efficiency of fish farming activities may be further optimised by taking into account the interaction between current speed, substrate complexity and the functional characteristics of the benthos. Collectively, our analyses demonstrate that the fate of fish farm-derived wastes is complex and highlight the need for site-specific management techniques.

  6. Crystal Structure of the ERp44-Peroxiredoxin 4 Complex Reveals the Molecular Mechanisms of Thiol-Mediated Protein Retention.

    Science.gov (United States)

    Yang, Kai; Li, De-Feng; Wang, Xi'e; Liang, Jinzhao; Sitia, Roberto; Wang, Chih-Chen; Wang, Xi

    2016-10-04

    ERp44 controls the localization and transport of diverse proteins in the early secretory pathway. The mechanisms that allow client recognition and the source of the oxidative power for forming intermolecular disulfides are as yet unknown. Here we present the structure of ERp44 bound to a client, peroxiredoxin 4. Our data reveal that ERp44 binds the oxidized form of peroxiredoxin 4 via thiol-disulfide interchange reactions. The structure explains the redox-dependent recognition and characterizes the essential non-covalent interactions at the interface. The ERp44-Prx4 covalent complexes can be reduced by glutathione and protein disulfide isomerase family members in the ER, allowing the two components to recycle. This work provides insights into the mechanisms of thiol-mediated protein retention and indicates the key roles of ERp44 in this biochemical cycle to optimize oxidative folding and redox homeostasis.

  7. TGFbeta-mediated formation of pRb-E2F complexes in human myeloid leukemia cells.

    Science.gov (United States)

    Hu, Xiao Tang

    2008-05-01

    TGFbeta is well known for its inhibitory effect on cell cycle G1 checkpoint kinases. However, its role in the control of pRb-E2F complexes is not well established. TGFbeta inhibits phosphorylation of pRb at several serine and threonine residues and regulates the association of E2F transcription factors with pRb family proteins. Recent studies found that predominantly E2F-4, p130, and histone deacetylase (HDAC) are found to bind to corresponding E2F-responsive promoters in G0/G1 phase. As cells progress through mid-G1, p130-E2F4 complex are replaced by p107-E2F4 followed by activators E2F1, 2, and 3. pRb was not detectable in the promoters containing the E2F-responsive site in cycling cells but was associated with E2F4-p130 complexes or E2F4-p107 complexes during G0/G1 phase. In human myeloid leukemia cell line, MV4-11, TGFbeta upregulated pRb-E2F-4 and p130-E2F-4, and downregulated p107-E2F-4 complexes. However, pRB-E2F1 and pRb-E2F3 complexes were found in proliferating cells but not in TGFbeta arrested G1 cells. In addition, electrophoretic gel mobility shift assay (EMSA) could not detect pRb-E2F DNA-binding activities either in S or G1 phase but exhibited the existence of p107-E2F4 in proliferating cells and p130-E2F4 complexes in TGFbeta-arrested G1 cells, respectively. Our data suggest that p107 and p130, but not pRb, and the repressor E2F, but not activator E2Fs, play a critical role in regulating E2F-responsive gene expression in TGFbeta-mediated cell cycle control in human myeloid leukemia cells.

  8. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    Science.gov (United States)

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-05-11

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E₂, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis.

  9. A component of the transcriptional mediator complex inhibits RAS-dependent vulval fate specification in C. elegans.

    Science.gov (United States)

    Moghal, Nadeem; Sternberg, Paul W

    2003-01-01

    Negative regulation of receptor tyrosine kinase (RTK)/RAS signaling pathways is important for normal development and the prevention of disease in humans. We have used a genetic screen in C. elegans to identify genes that antagonize the activity of activated LET-23, a member of the EGFR family of RTKs. We identified two loss-of-function mutations in dpy-22, previously cloned as sop-1, that promote the ability of activated LET-23 to induce ectopic vulval fates. DPY-22 is a glutamine-rich protein that is most similar to human TRAP230, a component of a transcriptional mediator complex. DPY-22 has previously been shown to regulate WNT responses through inhibition of the beta-catenin-like protein BAR-1. We provide evidence that DPY-22 also inhibits RAS-dependent vulval fate specification independently of BAR-1, and probably regulates the activities of multiple transcription factors during development. Furthermore, we demonstrate that although inhibition of BAR-1-dependent gene expression has been shown to require the C-terminal glutamine-rich region, this region is dispensable for inhibition of RAS-dependent cell differentiation. Thus, the glutamine-rich region contributes to specificity of this class of mediator protein.

  10. Well-defined silica-supported zirconium-imido complexes mediated heterogeneous imine metathesis.

    Science.gov (United States)

    Hamzaoui, Bilel; Pelletier, Jérémie D A; Abou-Hamad, Edy; Basset, Jean-Marie

    2016-03-28

    Upon prolonged thermal exposure under vacuum, a well-defined single-site surface species [([triple bond, length as m-dash]Si-O-)Zr(NEt2)3] () evolves into an ethylimido complex [([triple bond, length as m-dash]Si-O-)Zr([double bond, length as m-dash]NEt)NEt2] (). Reactions of with an imine substrate result in imido/imine ([double bond, length as m-dash]NRi, R: Et, Ph) exchange (metathesis) with the formation of [([triple bond, length as m-dash]Si-O-)Zr([double bond, length as m-dash]NPh)NEt2] (). Compounds and effectively catalyze imine/imine cross-metathesis and are thus considered as the first heterogeneous catalysts active for imine metathesis.

  11. Inter-plant communication through mycorrhizal networks mediates complex adaptive behaviour in plant communities.

    Science.gov (United States)

    Gorzelak, Monika A; Asay, Amanda K; Pickles, Brian J; Simard, Suzanne W

    2015-01-01

    Adaptive behaviour of plants, including rapid changes in physiology, gene regulation and defence response, can be altered when linked to neighbouring plants by a mycorrhizal network (MN). Mechanisms underlying the behavioural changes include mycorrhizal fungal colonization by the MN or interplant communication via transfer of nutrients, defence signals or allelochemicals. We focus this review on our new findings in ectomycorrhizal ecosystems, and also review recent advances in arbuscular mycorrhizal systems. We have found that the behavioural changes in ectomycorrhizal plants depend on environmental cues, the identity of the plant neighbour and the characteristics of the MN. The hierarchical integration of this phenomenon with other biological networks at broader scales in forest ecosystems, and the consequences we have observed when it is interrupted, indicate that underground 'tree talk' is a foundational process in the complex adaptive nature of forest ecosystems.

  12. Sustained immune complex-mediated reduction in CD16 expression after vaccination regulates NK cell function

    Directory of Open Access Journals (Sweden)

    Martin R Goodier

    2016-09-01

    Full Text Available Cross-linking of FcγRIII (CD16 by immune complexes induces antibody dependent cellular cytotoxicity (ADCC by natural killer (NK cells, contributing to control of intracellular pathogens; this pathway can also be targeted for immunotherapy of cancerous or otherwise diseased cells. However, down-regulation of CD16 expression on activated NK cells may limit or regulate this response. Here, we report sustained downregulation of CD16 expression on NK cells in vivo after intramuscular (but not intranasal influenza vaccination. CD16 downregulation persisted for at least 12 weeks after vaccination and was associated with robust enhancement of influenza-specific plasma antibodies after intramuscular (but not intranasal vaccination. This effect could be emulated in vitro by co-culture of NK cells with influenza antigen and immune serum and, consistent with the sustained effects after vaccination, only very limited recovery of CD16 expression was observed during long term in vitro culture of immune complex-treated cells. CD16 downregulation was most marked among normally CD16high CD57+ NK cells, irrespective of NKG2C expression, and was strongly positively associated with degranulation (surface CD107a expression. CD16 downregulation was partially reversed by inhibition of ADAM17 matrix metalloprotease, leading to a sustained increase in both CD107a and CD25(IL-2R expression. Both the degranulation and CD25 responses of CD57+ NK cells were uniquely dependent on TIV-specific IgG. These data support a role for CD16 in early activation of NK cells after vaccination and for CD16 down regulation as a means to modulate NK cell responses and maintain immune homeostasis of both antibody and T cell-dependent pathways.

  13. Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells

    Science.gov (United States)

    Eichholz, Karsten; Bru, Thierry; Tran, Thi Thu Phuong; Fernandes, Paulo; Mennechet, Franck J. D.; Manel, Nicolas; Alves, Paula; Perreau, Matthieu

    2016-01-01

    Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles containing a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health standards. Following repeat exposure to multiple HAdV types, we develop robust and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and persistent HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein containing a caspase activation/recruitment domain) aggregation, inflammasome formation, caspase 1 activation, and IL-1β and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs. PMID:27636895

  14. REST-mediated recruitment of polycomb repressor complexes in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Nikolaj Dietrich

    Full Text Available Polycomb Repressive Complex (PRC 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1, and PRC2 and show that RNF2 and REST co-regulate a number of neuronal genes in human teratocarcinoma cells (NT2-D1. Using NT2-D1 cells as a model of neuronal differentiation, we furthermore showed that retinoic-acid stimulation led to displacement of PRC1 at REST binding sites, reduced H3K27Me3, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest⁻/⁻ and Eed⁻/⁻ mouse embryonic stem (mES cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest⁻/⁻ mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context-dependent functions for PRC1- and PRC2- recruitment, which allows this transcription factor to act both as a recruiter of Polycomb as well as a limiting factor for PRC2 recruitment at CpG islands.

  15. Peptide-based antibodies against glutathione-binding domains suppress superoxide production mediated by mitochondrial complex I.

    Science.gov (United States)

    Chen, Jingfeng; Chen, Chwen-Lih; Rawale, Sharad; Chen, Chun-An; Zweier, Jay L; Kaumaya, Pravin T P; Chen, Yeong-Renn

    2010-01-29

    Complex I (NQR) is a critical site of superoxide (O2-*) production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain ((200)GAGAYICGEETALIESIEGK(219) of 51-kDa protein and (361)VDSDTLCTEEVFPTAGAGTDLR(382) of 75-kDa protein) as chimeric epitopes incorporating a "promiscuous" T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated O2-* generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain ((21)SGDTTAPKKTSFGSLKDFDR(40) of 51-kDa peptide and (100)WNILTNSEKTKKAREGVMEFL(120) of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated generation to a significant level. However, binding of Ab75 inhibited NQR-mediated O2-*generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction.

  16. Forgetting is regulated via Musashi-mediated translational control of the Arp2/3 complex.

    Science.gov (United States)

    Hadziselimovic, Nils; Vukojevic, Vanja; Peter, Fabian; Milnik, Annette; Fastenrath, Matthias; Fenyves, Bank Gabor; Hieber, Petra; Demougin, Philippe; Vogler, Christian; de Quervain, Dominique J-F; Papassotiropoulos, Andreas; Stetak, Attila

    2014-03-13

    A plastic nervous system requires the ability not only to acquire and store but also to forget. Here, we report that musashi (msi-1) is necessary for time-dependent memory loss in C. elegans. Tissue-specific rescue demonstrates that MSI-1 function is necessary in the AVA interneuron. Using RNA-binding protein immunoprecipitation (IP), we found that MSI-1 binds to mRNAs of three subunits of the Arp2/3 actin branching regulator complex in vivo and downregulates ARX-1, ARX-2, and ARX-3 translation upon associative learning. The role of msi-1 in forgetting is also reflected by the persistence of learning-induced GLR-1 synaptic size increase in msi-1 mutants. We demonstrate that memory length is regulated cooperatively through the activation of adducin (add-1) and by the inhibitory effect of msi-1. Thus, a GLR-1/MSI-1/Arp2/3 pathway induces forgetting and represents a novel mechanism of memory decay by linking translational control to the structure of the actin cytoskeleton in neurons.

  17. AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust.

    Science.gov (United States)

    Lin, Ya-Huei; Currinn, Heather; Pocha, Shirin Meher; Rothnie, Alice; Wassmer, Thomas; Knust, Elisabeth

    2015-12-15

    Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutation of AP-2 causes expansion of the Crumbs-positive plasma membrane domain and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking down both AP-2 and Stardust leads to the retention of Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.

  18. The simple neuroendocrine-immune regulatory network in oyster Crassostrea gigas mediates complex functions

    Science.gov (United States)

    Liu, Zhaoqun; Wang, Lingling; Zhou, Zhi; Sun, Ying; Wang, Mengqiang; Wang, Hao; Hou, Zhanhui; Gao, Dahai; Gao, Qiang; Song, Linsheng

    2016-05-01

    The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of the host. In the present study, the bioinformatical analysis of the transcriptomic data from oyster Crassostrea gigas and further biological validation revealed that oyster TNF (CgTNF-1 CGI_10018786) could activate the transcription factors NF-κB and HSF (heat shock transcription factor) through MAPK signaling pathway, and then regulate apoptosis, redox reaction, neuro-regulation and protein folding in oyster haemocytes. The activated immune cells then released neurotransmitters including acetylcholine, norepinephrine and [Met5]-enkephalin to regulate the immune response by arising the expression of three TNF (CGI_10005109, CGI_10005110 and CGI_10006440) and translocating two NF-κB (Cgp65, CGI_10018142 and CgRel, CGI_10021567) between the cytoplasm and nuclei of haemocytes. Neurotransmitters exhibited the immunomodulation effects by influencing apoptosis and phagocytosis of oyster haemocytes. Acetylcholine and norepinephrine could down-regulate the immune response, while [Met5]-enkephalin up-regulate the immune response. These results suggested that the simple neuroendocrine-immune regulatory network in oyster might be activated by oyster TNF and then regulate the immune response by virtue of neurotransmitters, cytokines and transcription factors.

  19. The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination.

    Science.gov (United States)

    O'Donnell, Lara; Panier, Stephanie; Wildenhain, Jan; Tkach, Johnny M; Al-Hakim, Abdallah; Landry, Marie-Claude; Escribano-Diaz, Cristina; Szilard, Rachel K; Young, Jordan T F; Munro, Meagan; Canny, Marella D; Kolas, Nadine K; Zhang, Wei; Harding, Shane M; Ylanko, Jarkko; Mendez, Megan; Mullin, Michael; Sun, Thomas; Habermann, Bianca; Datti, Alessandro; Bristow, Robert G; Gingras, Anne-Claude; Tyers, Michael D; Brown, Grant W; Durocher, Daniel

    2010-11-24

    Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. MicroProtein-mediated recruitment of CONSTANS into a TOPLESS trimeric complex represses flowering in Arabidopsis

    DEFF Research Database (Denmark)

    Graeff, Moritz; Straub, Daniel; Eguen, Tenai E.

    2016-01-01

    Arabidopsis thaliana microProteins, miP1a and miP1b, physically interact with CONSTANS (CO) a potent regulator of flowering time. The miP1a/b-type microProteins evolved in dicotyledonous plants and have an additional carboxy-terminal PF(V/L)FL motif. This motif enables miP1a/b microProteins to interact......MicroProteins are short, single domain proteins that act by sequestering larger, multi-domain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two...... with TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins. Interaction of CO with miP1a/b/TPL causes late flowering due to a failure in the induction of FLOWERING LOCUS T (FT) expression under inductive long day conditions. Both miP1a and miP1b are expressed in vascular tissue, where CO and FT are active. Genetically...

  1. Hepatocyte gene transfer mediated by stable polyplexes based on MPP-containing DNA complexes

    Institute of Scientific and Technical Information of China (English)

    Bao-Feng Yu; Wan-I Li; Xiao-Nian Hu; Yue-Hong Zhang; Bo Niu; Jun Xie

    2009-01-01

    BACKGROUND: In the field of gene therapy, viral vectors as delivery tools have a number of disadvantages for medical application. This study aimed to explore a novel nonviral vector as a vehicle for gene therapy. METHODS: Transvector-rpE-MPP and EGFP (enhanced green fluorescent protein) were used as the gene transfer carrier and the reporter gene, respectively. Polyplexes which integrate transvector-rpE-MPP, the object gene, and EGFP were formed. The optimal charge ratio, stability, and transduction capacity of the polyplexes in mouse hepatocytes in vitro and in mouse liver in vivo were investigated. The polyplexes of transvector-rpE-MPP and pcDNA3-EGFP, with charge ratios of 0, 0.25, 0.5, 0.75, 1 and 1.5 were compared to determine the optimal charge ratio. RESULTS:  Polyplexes with charge ratios of 1∶1 were most stable; pcDNA3-EGFP in these complexes resisted digestion by DNase Ⅰ and blood plasma. On the other hand, pcDNA3-EGFP alone was digested. Fluorescence analysis indicated that transvector-rpE-MPP successfully delivered the reporter gene EGFP into hepatocytes and that EGFP expression was detected in hepatocyte cultures and in liver tissue. CONCLUSION: These results have laid a foundation for further study of a novel nonviral gene delivery system.

  2. Sme4 coiled-coil protein mediates synaptonemal complex assembly, recombinosome relocalization, and spindle pole body morphogenesis.

    Science.gov (United States)

    Espagne, Eric; Vasnier, Christelle; Storlazzi, Aurora; Kleckner, Nancy E; Silar, Philippe; Zickler, Denise; Malagnac, Fabienne

    2011-06-28

    We identify a large coiled-coil protein, Sme4/PaMe4, that is highly conserved among the large group of Sordariales and plays central roles in two temporally and functionally distinct aspects of the fungal sexual cycle: first as a component of the meiotic synaptonemal complex (SC) and then, after disappearing and reappearing, as a component of the spindle pole body (SPB). In both cases, the protein mediates spatial juxtaposition of two major structures: linkage of homolog axes through the SC and a change in the SPB from a planar to a bent conformation. Corresponding mutants exhibit defects, respectively, in SC and SPB morphogenesis, with downstream consequences for recombination and astral-microtubule nucleation plus postmeiotic nuclear migration. Sme4 is also required for reorganization of recombination complexes in which Rad51, Mer3, and Msh4 foci relocalize from an on-axis position to a between-axis (on-SC) position concomitant with SC installation. Because involved recombinosome foci represent total recombinational interactions, these dynamics are irrespective of their designation for maturation into cross-overs or noncross-overs. The defined dual roles for Sme4 in two different structures that function at distinct phases of the sexual cycle also provide more functional links and evolutionary dynamics among the nuclear envelope, SPB, and SC.

  3. Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL–p97 complex

    Science.gov (United States)

    Braunstein, Ilana; Zach, Lolita; Allan, Susanne; Kalies, Kai-Uwe; Stanhill, Ariel

    2015-01-01

    The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97–AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97’s role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin. PMID:26337389

  4. Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

    Science.gov (United States)

    Wan, Fengyi; Anderson, D Eric; Barnitz, Robert A; Snow, Andrew; Bidere, Nicolas; Zheng, Lixin; Hegde, Vijay; Lam, Lloyd T; Staudt, Louis M; Levens, David; Deutsch, Walter A; Lenardo, Michael J

    2007-11-30

    NF-kappaB is a DNA-binding protein complex that transduces a variety of activating signals from the cytoplasm to specific sets of target genes. To understand the preferential recruitment of NF-kappaB to specific gene regulatory sites, we used NF-kappaB p65 in a tandem affinity purification and mass spectrometry proteomic screen. We identified ribosomal protein S3 (RPS3), a KH domain protein, as a non-Rel subunit of p65 homodimer and p65-p50 heterodimer DNA-binding complexes that synergistically enhances DNA binding. RPS3 knockdown impaired NF-kappaB-mediated transcription of selected p65 target genes but not nuclear shuttling or global protein translation. Rather, lymphocyte-activating stimuli caused nuclear translocation of RPS3, parallel to p65, to form part of NF-kappaB bound to specific regulatory sites in chromatin. Thus, RPS3 is an essential but previously unknown subunit of NF-kappaB involved in the regulation of key genes in rapid cellular activation responses. Our observations provide insight into how NF-kappaB selectively controls gene expression.

  5. Cytotoxicity of Manganese (III) Complex in Human Breast Adenocarcinoma Cell Line Is Mediated by the Generation of Reactive Oxygen Species Followed by Mitochondrial Damage.

    Science.gov (United States)

    Al-Anbaky, Qudes; Al-Karakooly, Zeiyad; Kilaparty, Surya P; Agrawal, Megha; Albkuri, Yahya M; RanguMagar, Ambar B; Ghosh, Anindya; Ali, Nawab

    2016-11-01

    Manganese (Mn) complexes are widely studied because of their important catalytic properties in synthetic and biochemical reactions. A Mn (III) complex of an amidoamine ligand was synthesized using a tetradentate amidoamine ligand. In this study, the Mn (III) complex was evaluated for its biological activity by measuring its cytotoxicity in human breast adenocarcinoma cell line (MCF-7). Cytotoxic effects of the Mn (III) complex were determined using established biomarkers in an attempt to delineate the mechanism of action and the utility of the complex as a potential anticancer drug. The Mn (III) complex induces cell death in a dose- and time-dependent manner as shown by microculture tetrazolium assay, a measure of cytotoxic cell death. Our results demonstrated that cytotoxic effects were significantly increased at higher concentrations of Mn (III) complex and with longer time of treatment. The IC50 (Inhibitor concentration that results in 50% cell death) value of Mn (III) complex in MCF-7 cells was determined to be 2.5 mmol/L for 24 hours of treatment. In additional experiments, we determined the Mn (III) complex-mediated cell death was due to both apoptotic and nonspecific necrotic cell death mechanisms. This was assessed by ethidium bromide/acridine orange staining and flow cytometry techniques. The Mn (III) complex produced reactive oxygen species (ROS) triggering the expression of manganese superoxide dismutase 1 and ultimately damaging the mitochondrial function as is evident by a decline in mitochondrial membrane potential. Treatment of the cells with free radical scavenger, N, N-dimethylthiourea decreased Mn (III) complex-mediated generation of ROS and attenuated apoptosis. Together, these results suggest that the Mn (III) complex-mediated MCF-7 cell death utilizes combined mechanism involving apoptosis and necrosis perhaps due to the generation of ROS.

  6. BCNU-induced gR2 defect mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium.

    Science.gov (United States)

    Kang, Patrick T; Chen, Chwen-Lih; Ren, Pei; Guarini, Giacinta; Chen, Yeong-Renn

    2014-06-15

    A deficiency of mitochondrial glutathione reductase (or GR2) is capable of adversely affecting the reduction of GSSG and increasing mitochondrial oxidative stress. BCNU [1,3-bis (2-chloroethyl)-1-nitrosourea] is an anticancer agent and known inhibitor of cytosolic GR ex vivo and in vivo. Here we tested the hypothesis that a BCNU-induced GR2 defect contributes to mitochondrial dysfunction and subsequent impairment of heart function. Intraperitoneal administration of BCNU (40 mg/kg) specifically inhibited GR2 activity by 79.8 ± 2.7% in the mitochondria of rat heart. However, BCNU treatment modestly enhanced the activities of mitochondrial Complex I and other ETC components. The cardiac function of BCNU-treated rats was analyzed by echocardiography, revealing a systolic dysfunction associated with decreased ejection fraction, decreased cardiac output, and an increase in left ventricular internal dimension and left ventricular volume in systole. The respiratory control index of isolated mitochondria from the myocardium was moderately decreased after BCNU treatment, whereas NADH-linked uncoupling of oxygen consumption was significantly enhanced. Extracellular flux analysis to measure the fatty acid oxidation of myocytes indicated a 20% enhancement after BCNU treatment. When the mitochondria were immunoblotted with antibodies against GSH and UCP3, both protein S-glutathionylation of Complex I and expression of UCP3 were significantly up-regulated. Overexpression of SOD2 in the myocardium significantly reversed BCNU-induced GR2 inhibition and mitochondrial impairment. In conclusion, BCNU-mediated cardiotoxicity is characterized by the GR2 deficiency that negatively regulates heart function by impairing mitochondrial integrity, increasing oxidative stress with Complex I S-glutathionylation, and enhancing uncoupling of mitochondrial respiration.

  7. A theoretical analysis of the extraction of heterocyclic organic compounds from an organic phase using chemically mediated electrochemically modulated complexation in ion exchange polymer beads

    Energy Technology Data Exchange (ETDEWEB)

    Ozekin, K.; Noble, R.D.; Koval, C.A.

    1991-01-01

    A cyclical electrochemical process for the removal of heterocyclic organic compounds (pollutants) from an organic solvent using an ion-exchange polymer is analyzed. In this analysis, there are three main steps: In the first step, the polymer beads containing the active form of the complexing agent are contacted with the contaminated (feed) hydrocarbon phase. The pollutant diffuses into the beads and binds with the complexing agent which is in the reduced state. It is a fast reversible reaction. For the second step, the beads which contain a pollutant are contacted with a waste (receiving) phase and a chemical mediator is then used to oxidize the complexing agent and to reduce its affinity towards the pollutant so that it can be released. The oxidation of the complexing agent is an irreversible reaction. This is a moving boundary problem with countercurrent diffusion. For each mole of mediator that goes into the bead, one mole of pollutant exits since each complexing agent binds one pollutant. In the third step, the waste hydrocarbon phase is removed and a second chemical mediator is then used to reduce the complexing agent. The reduction of the complexing agent is also an irreversible reaction. Partial differential equations are used to analyze this process. 26 refs., 9 figs.

  8. Complexity

    CERN Document Server

    Gershenson, Carlos

    2011-01-01

    The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.

  9. Cyclin-dependent kinase-mediated phosphorylation of RBP1 and pRb promotes their dissociation to mediate release of the SAP30·mSin3·HDAC transcriptional repressor complex.

    Science.gov (United States)

    Suryadinata, Randy; Sadowski, Martin; Steel, Rohan; Sarcevic, Boris

    2011-02-18

    Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G(1)-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G(1)-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G(1) into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

  10. Circadian and diurnal variation of circulating immune complexes, complement-mediated solubilization, and the complement split product C3d in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Petersen, Ivan; Baatrup, Gunnar; Brandslund, I;

    1986-01-01

    Nine patients with active classical rheumatoid arthritis (ARA criteria) were studied with reference to circadian variation of immunological and clinical parameters. Complement-mediated solubilization (CMS) of immune complexes (IC) and the level of circulating IC were found to be inversely related...

  11. Predator attack rate evolution in space: the role of ecology mediated by complex emergent spatial structure and self-shading.

    Science.gov (United States)

    Messinger, Susanna M; Ostling, Annette

    2013-11-01

    Predation interactions are an important element of ecological communities. Population spatial structure has been shown to influence predator evolution, resulting in the evolution of a reduced predator attack rate; however, the evolutionary role of traits governing predator and prey ecology is unknown. The evolutionary effect of spatial structure on a predator's attack rate has primarily been explored assuming a fixed metapopulation spatial structure, and understood in terms of group selection. But endogenously generated, emergent spatial structure is common in nature. Furthermore, the evolutionary influence of ecological traits may be mediated through the spatial self-structuring process. Drawing from theory on pathogens, the evolutionary effect of emergent spatial structure can be understood in terms of self-shading, where a voracious predator limits its long-term invasion potential by reducing local prey availability. Here we formalize the effects of self-shading for predators using spatial moment equations. Then, through simulations, we show that in a spatial context self-shading leads to relationships between predator-prey ecology and the predator's attack rate that are not expected in a non-spatial context. Some relationships are analogous to relationships already shown for host-pathogen interactions, but others represent new trait dimensions. Finally, since understanding the effects of ecology using existing self-shading theory requires simplifications of the emergent spatial structure that do not apply well here, we also develop metrics describing the complex spatial structure of the predator and prey populations to help us explain the evolutionary effect of predator and prey ecology in the context of self-shading. The identification of these metrics may provide a step towards expansion of the predictive domain of self-shading theory to more complex spatial dynamics.

  12. pRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption.

    Science.gov (United States)

    Seifried, L A; Talluri, S; Cecchini, M; Julian, L M; Mymryk, J S; Dick, F A

    2008-05-01

    Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how E1A affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that E1A is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability.

  13. The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803

    Institute of Scientific and Technical Information of China (English)

    MA WeiMin; WEI LanZhen; WANG QuanXi

    2008-01-01

    The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem Ⅰ (PSI) and photosystem Ⅱ (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactivation mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the extent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and mediumcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.

  14. The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactiva-tion mutants △ndhB (M55) and △ndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETRPSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55 ℃ for 10 min significantly inhibited PSII-driven ETR (ETRPSII) in the WT, M55 and D1/D2 cells, and the ex-tent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETRPSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETRPSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETRPSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and medi-umcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETRPSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.

  15. Dissection of the complex phenotype in cuticular mutants of Arabidopsis reveals a role of SERRATE as a mediator.

    Directory of Open Access Journals (Sweden)

    Derry Voisin

    2009-10-01

    Full Text Available Mutations in LACERATA (LCR, FIDDLEHEAD (FDH, and BODYGUARD (BDG cause a complex developmental syndrome that is consistent with an important role for these Arabidopsis genes in cuticle biogenesis. The genesis of their pleiotropic phenotypes is, however, poorly understood. We provide evidence that neither distorted depositions of cutin, nor deficiencies in the chemical composition of cuticular lipids, account for these features, instead suggesting that the mutants alleviate the functional disorder of the cuticle by reinforcing their defenses. To better understand how plants adapt to these mutations, we performed a genome-wide gene expression analysis. We found that apparent compensatory transcriptional responses in these mutants involve the induction of wax, cutin, cell wall, and defense genes. To gain greater insight into the mechanism by which cuticular mutations trigger this response in the plants, we performed an overlap meta-analysis, which is termed MASTA (MicroArray overlap Search Tool and Analysis, of differentially expressed genes. This suggested that different cell integrity pathways are recruited in cesA cellulose synthase and cuticular mutants. Using MASTA for an in silico suppressor/enhancer screen, we identified SERRATE (SE, which encodes a protein of RNA-processing multi-protein complexes, as a likely enhancer. In confirmation of this notion, the se lcr and se bdg double mutants eradicate severe leaf deformations as well as the organ fusions that are typical of lcr and bdg and other cuticular mutants. Also, lcr does not confer resistance to Botrytis cinerea in a se mutant background. We propose that there is a role for SERRATE-mediated RNA signaling in the cuticle integrity pathway.

  16. Complex

    African Journals Online (AJOL)

    CLEMENT O BEWAJI

    Schiff bases and their complex compounds have been studied for their .... establishing coordination of the N–(2 – hydroxybenzyl) - L - α - valine Schiff base ..... (1967); “Spectrophotometric Identification of Organic Compounds”, Willey, New.

  17. Immune Complex Mediated Glomerulonephritis with Acute Thrombotic Microangiopathy following Newly Detected Hepatitis B Virus Infection in a Kidney Transplant Recipient

    Directory of Open Access Journals (Sweden)

    Tracey Salter

    2016-01-01

    Full Text Available Hepatitis B virus (HBV presents a risk to patients and staff in renal units. To minimise viral transmission, there are international and UK guidelines recommending HBV immunisation for patients commencing renal replacement therapy (RRT and HBV surveillance in kidney transplant recipients. We report the case of a 56-year-old male who was immunised against HBV before starting haemodialysis. He received a deceased donor kidney transplant three years later, at which time there was no evidence of HBV infection. After a further six years he developed an acute kidney injury; allograft biopsy revealed an acute thrombotic microangiopathy (TMA with glomerulitis, peritubular capillaritis, and C4d staining. Due to a “full house” immunoprofile, tests including virological screening were undertaken, which revealed acute HBV infection. Entecavir treatment resulted in an improvement in viral load and kidney function. HBV genotyping demonstrated a vaccine escape mutant, suggesting “past resolved” infection that reactivated with immunosuppression, though posttransplant acquisition cannot be excluded. This is the first reported case of acute HBV infection associated with immune complex mediated glomerulonephritis and TMA. Furthermore, it highlights the importance of HBV surveillance in kidney transplant recipients, which although addressed by UK guidelines is not currently practiced in all UK units.

  18. The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription–replication conflicts

    Science.gov (United States)

    Herrera-Moyano, Emilia; Mergui, Xénia; García-Rubio, María L.; Barroso, Sonia; Aguilera, Andrés

    2014-01-01

    FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP–chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription–replication conflicts, likely associated with a specific chromatin organization. PMID:24636987

  19. The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription-replication conflicts.

    Science.gov (United States)

    Herrera-Moyano, Emilia; Mergui, Xénia; García-Rubio, María L; Barroso, Sonia; Aguilera, Andrés

    2014-04-01

    FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP-chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription-replication conflicts, likely associated with a specific chromatin organization.

  20. Delivery of short interfering ribonucleic acid-complexed magnetic nanoparticles in an oscillating field occurs via caveolae-mediated endocytosis.

    Directory of Open Access Journals (Sweden)

    Jenson Lim

    Full Text Available Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA against green fluorescent protein (GFP or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway.

  1. Immune Complex Mediated Glomerulonephritis with Acute Thrombotic Microangiopathy following Newly Detected Hepatitis B Virus Infection in a Kidney Transplant Recipient

    Science.gov (United States)

    Burton, Hannah; Douthwaite, Sam; Newsholme, William; Horsfield, Catherine

    2016-01-01

    Hepatitis B virus (HBV) presents a risk to patients and staff in renal units. To minimise viral transmission, there are international and UK guidelines recommending HBV immunisation for patients commencing renal replacement therapy (RRT) and HBV surveillance in kidney transplant recipients. We report the case of a 56-year-old male who was immunised against HBV before starting haemodialysis. He received a deceased donor kidney transplant three years later, at which time there was no evidence of HBV infection. After a further six years he developed an acute kidney injury; allograft biopsy revealed an acute thrombotic microangiopathy (TMA) with glomerulitis, peritubular capillaritis, and C4d staining. Due to a “full house” immunoprofile, tests including virological screening were undertaken, which revealed acute HBV infection. Entecavir treatment resulted in an improvement in viral load and kidney function. HBV genotyping demonstrated a vaccine escape mutant, suggesting “past resolved” infection that reactivated with immunosuppression, though posttransplant acquisition cannot be excluded. This is the first reported case of acute HBV infection associated with immune complex mediated glomerulonephritis and TMA. Furthermore, it highlights the importance of HBV surveillance in kidney transplant recipients, which although addressed by UK guidelines is not currently practiced in all UK units.

  2. Neuronal Fc gamma receptor I as a novel mediator for IgG immune complex-induced peripheral sensitization

    Institute of Scientific and Technical Information of China (English)

    Lintao Qu

    2012-01-01

    Chronic pain often accompanies immune-related diseases with an elevated level of IgG immune complex (IgG-IC) in the serum and/or the affected tissues though the underlying mechanisms are largely unknown. Fc gamma receptors (FcγRs), known as the receptors for the Fc domain of immunoglobulin G (IgG), are typically expressed on immune cells. A general consensus is that the activation of FcγRs by IgG-IC in such immune cells induces the release of proinflammatory cytokines from the immune cells, which may contribute to the IgG-IC-mediated peripheral sensitization. In addition to the immune cells, recent studies have revealed that FcγRI, but not FcγRII and FcγRIII, is also expressed in a subpopulation of primary sensory neurons. Moreover, IgG-IC directly excites the primary sensory neurons through neuronal FcγRI. These findings indicate that neuronal FcγRI provides a novel direct linkage between immunoglobulin and primary sensory neurons, which may be a novel target for the treatment of pain in the immune-related disorders. In this review, we summarize the expression pattern, functions, and the associated cellular signaling of FcγRs in the primary sensory neurons.

  3. Molecular mechanisms involved in TFF3 peptide-mediated modulation of the E-cadherin/catenin cell adhesion complex.

    Science.gov (United States)

    Meyer zum Büschenfelde, Dirk; Hoschützky, Heinz; Tauber, Rudolf; Huber, Otmar

    2004-05-01

    TFF3 is a member of the TFF-domain peptide family which is constitutively expressed in mucous epithelial tissues where it acts as a motogenic factor and plays an important role during epithelial restitution after wounding and during inflammation. In contrast to these beneficial functions, TFFs were also reported to be involved in cell scattering and tumor invasion. These changes in epithelial cell morphology and motility are associated with a modulation of cell contacts. In this respect, we here investigated the E-cadherin/catenin cell adhesion complex in FLAG-hTFF3-transfected HT29/B6 and MDCK cells. In hTFF3-transfected cells the amount of E-cadherin is reduced with a concomitant reduction of alpha- and beta-catenin levels. On one hand, E-cadherin expression is lowered at the transcriptional level as shown by multiplex RT-PCR analysis. This decrease does not depend on differences in the promoter methylation status as shown by methylation-specific PCR. On the other hand, pulse-chase experiments showed a reduction in the E-cadherin half-life in hTFF3-transfected cells reflecting increased E-cadherin degradation. In summary, hTFF3 induces transcriptional and posttranslational processes resulting in a modulation of E-cadherin-mediated cell-cell contacts that may play an important role in the paradoxical benefical and pathogenic function of TFF peptides.

  4. Thermodynamic consequences of disrupting a water-mediated hydrogen bond network in a protein:pheromone complex.

    Science.gov (United States)

    Sharrow, Scott D; Edmonds, Katherine A; Goodman, Michael A; Novotny, Milos V; Stone, Martin J

    2005-01-01

    The mouse pheromones (+/-)-2-sec-butyl-4,5-dihydrothiazole (SBT) and 6-hydroxy-6-methyl-3-heptanone (HMH) bind into an occluded hydrophobic cavity in the mouse major urinary protein (MUP-1). Although the ligands are structurally unrelated, in both cases binding is accompanied by formation of a similar buried, water-mediated hydrogen bond network between the ligand and several backbone and side chain groups on the protein. To investigate the energetic contribution of this hydrogen bond network to ligand binding, we have applied isothermal titration calorimetry to measure the binding thermodynamics using several MUP mutants and ligand analogs. Mutation of Tyr-120 to Phe, which disrupts a hydrogen bond from the phenolic hydroxyl group of Tyr-120 to one of the bound water molecules, results in a substantial loss of favorable binding enthalpy, which is partially compensated by a favorable change in binding entropy. A similar thermodynamic effect was observed when the hydrogen bonded nitrogen atom of the heterocyclic ligand was replaced by a methyne group. Several other modifications of the protein or ligand had smaller effects on the binding thermodynamics. The data provide supporting evidence for the role of the hydrogen bond network in stabilizing the complex.

  5. Methyl 6-Amino-6-deoxy-d-pyranoside-Conjugated Platinum(II) Complexes for Glucose Transporter (GLUT)-Mediated Tumor Targeting: Synthesis, Cytotoxicity, and Cellular Uptake Mechanism.

    Science.gov (United States)

    Li, Taoli; Gao, Xiangqian; Yang, Liu; Shi, Yunli; Gao, Qingzhi

    2016-05-19

    Methyl 6-aminodeoxy-d-pyranoside-derived platinum(II) glycoconjugates were designed and synthesized based on the clinical drug oxaliplatin for glucose transporter (GLUT)-mediated tumor targeting. In addition to a substantial improvement in water solubility, the conjugates exhibited cytotoxicity similar to or higher than that of oxaliplatin in six different human cancer cell lines. GLUT-mediated transport of the complexes was investigated with a cell-based fluorescence competition assay and GLUT-inhibitor-mediated cytotoxicity analysis in a GLUT-overexpressing human colorectal adenocarcinoma (HT29) cell line. The antitumor effect of the aminodeoxypyranoside-conjugated platinum(II) complexes was found to depend significantly on the GLUT inhibitor, and the cellular uptake of the molecules was regulated by GLUT-mediated transport. The results from this study demonstrate the potential advantages of aminodeoxypyranosides as sugar motifs for glycoconjugation for Warburg-effect-targeted drug design. These fundamental results also support the potential of aminodeoxypyranoside-conjugated platinum(II) complexes as lead compounds for further preclinical evaluation.

  6. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Directory of Open Access Journals (Sweden)

    Chenggang Lu

    2015-12-01

    Full Text Available Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s. In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC, a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs, spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  7. The Mediator complex of Caenorhabditis elegans: insights into the developmental and physiological roles of a conserved transcriptional coregulator

    National Research Council Canada - National Science Library

    Grants, Jennifer M; Goh, Grace Y S; Taubert, Stefan

    2015-01-01

    .... Here, we review the current knowledge of Mediator subunit function in the nematode Caenorhabditis elegans, a metazoan in which established and emerging genetic technologies facilitate the study...

  8. Exploration of CH···π mediated stacking interactions in saccharide: aromatic residue complexes through conformational sampling.

    Science.gov (United States)

    Kumari, Manju; Sunoj, Raghavan B; Balaji, Petety V

    2012-11-01

    Saccharides interact with aromatic residues mostly through CH···π mediated stacking interactions. The energetics of such interactions depends upon the mutual position-orientations (POs) of the two moieties. The POs found in the crystal structures are only a subset of the various possible ways of interaction. Hence, potential energy surfaces of saccharide-aromatic residue complexes have been explored by mixed Monte Carlo multiple minimum/low mode sampling. The saccharides considered in this study are α/β-D-glucose, β-D-galactose, α-D-mannose, and α/β-L-fucose. p-Hydroxytoluene, toluene, and 3-methylindole were used as analogs of tyrosine, phenylalanine, and tryptophan, respectively. The saccharides interact from either above or below the π-cloud of an aromatic ring but not along the edges. The POs preferred by different saccharides, both in the preferred chair and skew-boat forms, for interacting with different aromatic amino acid residue analogs have been identified. Aromatic residues can interact with the same -CH group in many POs but not so with the -OH groups. Changes in the configurations of pyranose ring carbon atoms cause remarkable changes in stacking preferences. β-D-Galactose and β-L-fructose interact only through their b- and a-faces, respectively. Saccharides use a wide variety of apolar patches for stacking against aromatic residues and these have been analyzed in detail. As many as four -CH groups can simultaneously participate in CH···π interactions, especially with 3-methylindole owing to its larger surface area.

  9. SUPERKILLER Complex Components Are Required for the RNA Exosome-Mediated Control of Cuticular Wax Biosynthesis in Arabidopsis Inflorescence Stems.

    Science.gov (United States)

    Zhao, Lifang; Kunst, Ljerka

    2016-06-01

    ECERIFERUM7 (CER7)/AtRRP45B core subunit of the exosome, the main cellular 3'-to-5' exoribonuclease, is a positive regulator of cuticular wax biosynthesis in Arabidopsis (Arabidopsis thaliana) inflorescence stems. CER7-dependent exosome activity determines stem wax load by controlling transcript levels of the wax-related gene CER3 Characterization of the second-site suppressors of the cer7 mutant revealed that small interfering RNAs (siRNAs) are direct effectors of CER3 expression. To explore the relationship between the exosome and posttranscriptional gene silencing (PTGS) in regulating CER3 transcript levels, we investigated two additional suppressor mutants, wax restorer1 (war1) and war7. We show that WAR1 and WAR7 encode Arabidopsis SUPERKILLER3 (AtSKI3) and AtSKI2, respectively, components of the SKI complex that associates with the exosome during cytoplasmic 3'-to-5' RNA degradation, and that CER7-dependent regulation of wax biosynthesis also requires participation of AtSKI8. Our study further reveals that it is the impairment of the exosome-mediated 3'-5' decay of CER3 transcript in the cer7 mutant that triggers extensive production of siRNAs and efficient PTGS of CER3. This identifies PTGS as a general mechanism for eliminating highly abundant endogenous transcripts that is activated when 3'-to-5' mRNA turnover by the exosome is disrupted. Diminished efficiency of PTGS in ski mutants compared with cer7, as evidenced by lower accumulation of CER3-related siRNAs, suggests that reduced amounts of CER3 transcript are available for siRNA synthesis, possibly because CER3 mRNA that does not interact with SKI is degraded by 5'-to-3' XRN4 exoribonuclease.

  10. GR SUMOylation and formation of an SUMO-SMRT/NCoR1-HDAC3 repressing complex is mandatory for GC-induced IR nGRE-mediated transrepression

    Science.gov (United States)

    Hua, Guoqiang; Paulen, Laetitia; Chambon, Pierre

    2016-01-01

    Unique among the nuclear receptor superfamily, the glucocorticoid (GC) receptor (GR) can exert three distinct transcriptional regulatory functions on binding of a single natural (cortisol in human and corticosterone in mice) and synthetic [e.g., dexamethasone (Dex)] hormone. The molecular mechanisms underlying GC-induced positive GC response element [(+)GRE]-mediated activation of transcription are partially understood. In contrast, these mechanisms remain elusive for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression and for tethered indirect transrepression that is mediated by DNA-bound NF-κB/activator protein 1 (AP1)/STAT3 activators and instrumental in GC-induced anti-inflammatory activity. We demonstrate here that SUMOylation of lysine K293 (mouse K310) located within an evolutionary conserved sequence in the human GR N-terminal domain allows the formation of a GR-small ubiquitin-related modifiers (SUMOs)-NCoR1/SMRT-HDAC3 repressing complex mandatory for GC-induced IR nGRE-mediated direct repression in vitro, but does not affect transactivation. Importantly, these results were validated in vivo: in K310R mutant mice and in mice ablated selectively for nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors in skin keratinocytes, Dex-induced direct repression and the formation of repressing complexes on IR nGREs were impaired, whereas transactivation was unaffected. In mice selectively ablated for histone deacetylase 3 (HDAC3) in skin keratinocytes, GC-induced direct repression, but not bindings of GR and of corepressors NCoR1/SMRT, was abolished, indicating that HDAC3 is instrumental in IR nGRE-mediated repression. Moreover, we demonstrate that the binding of HDAC3 to IR nGREs in vivo is mediated through interaction with SMRT/NCoR1. We also show that the GR ligand binding domain (LBD) is not required for SMRT-mediated

  11. GR SUMOylation and formation of an SUMO-SMRT/NCoR1-HDAC3 repressing complex is mandatory for GC-induced IR nGRE-mediated transrepression.

    Science.gov (United States)

    Hua, Guoqiang; Paulen, Laetitia; Chambon, Pierre

    2016-02-02

    Unique among the nuclear receptor superfamily, the glucocorticoid (GC) receptor (GR) can exert three distinct transcriptional regulatory functions on binding of a single natural (cortisol in human and corticosterone in mice) and synthetic [e.g., dexamethasone (Dex)] hormone. The molecular mechanisms underlying GC-induced positive GC response element [(+)GRE]-mediated activation of transcription are partially understood. In contrast, these mechanisms remain elusive for GC-induced evolutionary conserved inverted repeated negative GC response element (IR nGRE)-mediated direct transrepression and for tethered indirect transrepression that is mediated by DNA-bound NF-κB/activator protein 1 (AP1)/STAT3 activators and instrumental in GC-induced anti-inflammatory activity. We demonstrate here that SUMOylation of lysine K293 (mouse K310) located within an evolutionary conserved sequence in the human GR N-terminal domain allows the formation of a GR-small ubiquitin-related modifiers (SUMOs)-NCoR1/SMRT-HDAC3 repressing complex mandatory for GC-induced IR nGRE-mediated direct repression in vitro, but does not affect transactivation. Importantly, these results were validated in vivo: in K310R mutant mice and in mice ablated selectively for nuclear receptor corepressor 1 (NCoR1)/silencing mediator for retinoid or thyroid-hormone receptors (SMRT) corepressors in skin keratinocytes, Dex-induced direct repression and the formation of repressing complexes on IR nGREs were impaired, whereas transactivation was unaffected. In mice selectively ablated for histone deacetylase 3 (HDAC3) in skin keratinocytes, GC-induced direct repression, but not bindings of GR and of corepressors NCoR1/SMRT, was abolished, indicating that HDAC3 is instrumental in IR nGRE-mediated repression. Moreover, we demonstrate that the binding of HDAC3 to IR nGREs in vivo is mediated through interaction with SMRT/NCoR1. We also show that the GR ligand binding domain (LBD) is not required for SMRT-mediated

  12. A clinico-radiological phenotype of voltage-gated potassium channel complex antibody-mediated disorder presenting with seizures and basal ganglia changes.

    Science.gov (United States)

    Hacohen, Yael; Wright, Sukhvir; Siddiqui, Ata; Pandya, Nikki; Lin, Jean-Pierre; Vincent, Angela; Lim, Ming

    2012-12-01

    In childhood, central nervous system (CNS) presentations associated with antibodies to voltage-gated potassium channel (VGKC) complex include limbic encephalitis, status epilepticus, epileptic encephalopathy, and autistic regression. We report the cases of two individuals (a 6-year-old male and an 11-year-old female) who presented with an acute-onset explosive seizure disorder with positive VGKC complex antibodies and bilateral basal ganglia changes on magnetic resonance imaging (MRI). Both patients made a complete clinical recovery, without immunotherapy, with resolution of the MRI changes and normalization of the antibody levels. Extended antibody testing, including testing for leucine-rich glioma-inactivated 1 (LGI1), contactin-associated protein 2, and contactin-2 was negative. This could suggest that the clinico-radiological phenotype in our patients may in fact be associated with a novel autoreactive target(s) within the VGKC complex, as may be the case in other children with VGKC complex-mediated CNS disorders.

  13. Childhood trauma and complex posttraumatic stress disorder symptoms in older adults: A study of direct effects and social-interpersonal factors as potential mediators.

    Science.gov (United States)

    Krammer, Sandy; Kleim, Birgit; Simmen-Janevska, Keti; Maercker, Andreas

    2016-01-01

    Childhood traumatic events may lead to long-lasting psychological effects and contribute to the development of complex posttraumatic sequelae. These might be captured by the diagnostic concept of complex posttraumatic stress disorder (CPTSD) as an alternative to classic posttraumatic stress disorder (PTSD). CPTSD comprises a further set of symptoms in addition to those of PTSD, namely, changes in affect, self, and interpersonal relationships. Previous empirical research on CPTSD has focused on middle-aged adults but not on older adults. Moreover, predictor models of CPTSD are still rare. The current study investigated the association between traumatic events in childhood and complex posttraumatic stress symptoms in older adults. The mediation of this association by 2 social-interpersonal factors (social acknowledgment as a survivor and dysfunctional disclosure) was investigated. These 2 factors focus on the perception of acknowledgment by others and either the inability to disclose traumatic experiences or the ability to do so only with negative emotional reactions. A total of 116 older individuals (age range = 59-98 years) who had experienced childhood traumatic events completed standardized self-report questionnaires indexing childhood trauma, complex trauma sequelae, social acknowledgment, and dysfunctional disclosure of trauma. The results showed that traumatic events during childhood were associated with later posttraumatic stress symptoms but with classic rather than complex symptoms. Social acknowledgment and dysfunctional disclosure partially mediated this relationship. These findings suggest that childhood traumatic stress impacts individuals across the life span and may be associated with particular adverse psychopathological consequences.

  14. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  15. leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3.

    Science.gov (United States)

    Saxena, Neeraj K; Vertino, Paula M; Anania, Frank A; Sharma, Dipali

    2007-05-01

    Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes.

  16. Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo.

    Science.gov (United States)

    Ward, Christopher M; Pechar, Michal; Oupicky, David; Ulbrich, Karel; Seymour, Leonard W

    2002-01-01

    Gene delivery vectors based on poly(L-lysine) and DNA (pLL/DNA complexes) have limited use for targeted systemic application in vivo since they bind cells and proteins non-specifically. In this study we have attempted to form folate-targeted vectors with extended systemic circulation by surface modification of pLL/DNA complexes with hydrophilic polymers. pLL/DNA complexes were stabilised by surface modification with a multivalent reactive polymer based on alternating segments of poly(ethylene glycol) and tripeptides bearing reactive ester groups. Folate moieties were incorporated into the vectors either by direct attachment of folate to the polymer or via intermediate poly(ethylene glycol) spacers of 800 and 3400 Da. Polymer-coated complexes show similar morphology to uncoated complexes, their zeta potential is decreased towards zero, serum protein binding is inhibited and aqueous solubility is substantially increased. Intravenous (i.v.) administration to mice of coated complexes produced extended systemic circulation, with up to 2000-fold more DNA measured in the bloodstream after 30 min compared with simple pLL/DNA complexes. In further contrast to simple pLL/DNA complexes, coated complexes do not bind blood cells in vivo. Folate receptor targeting is shown to mediate targeted association with HeLa cells in vitro, leading to increased transgene expression. We demonstrate for the first time that DNA uptake via the folate receptor is dependent on pEG spacer length, with the transgene expression relatively independent of the level of internalised DNA. We show increased systemic circulation, decreased blood cell and protein binding, and folate-targeted transgene expression using pLL/DNA complexes surface-modified with a novel multireactive hydrophilic polymer. This work provides the basis for the development of plasma-circulating targeted vectors for in vivo applications. Copyright 2002 John Wiley & Sons, Ltd.

  17. An oligogalacturonide-derived molecular probe demonstrates the dynamics of calcium-mediated pectin complexation in cell walls of tip-growing structures

    DEFF Research Database (Denmark)

    Mravec, Jozef; Kracun, Stjepan Kresimir; Rydahl, Maja Gro

    2017-01-01

    Pectic homogalacturonan (HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell......-mediated binding of fluorescently tagged long oligogalacturonides (OGs) with endogenous de-esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested....... Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip-growing structures....

  18. Nuclear export signal-interacting protein forms complexes with lamin A/C-Nups to mediate the CRM1-independent nuclear export of large hepatitis delta antigen.

    Science.gov (United States)

    Huang, Cheng; Jiang, Jia-Yin; Chang, Shin C; Tsay, Yeou-Guang; Chen, Mei-Ru; Chang, Ming-Fu

    2013-02-01

    Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

  19. Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases

    Science.gov (United States)

    Shin, Heungsop; Shin, Hwa-Soo; Guo, Zibiao; Blancaflor, Elison B.; Masson, Patrick H.; Chen, Rujin

    2005-01-01

    Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.

  20. The TWD40-2 protein and the AP2 complex cooperate in the clathrin-mediated endocytosis of cellulose synthase to regulate cellulose biosynthesis.

    Science.gov (United States)

    Bashline, Logan; Li, Shundai; Zhu, Xiaoyu; Gu, Ying

    2015-10-13

    Cellulose biosynthesis is performed exclusively by plasma membrane-localized cellulose synthases (CESAs). Therefore, the trafficking of CESAs to and from the plasma membrane is an important mechanism for regulating cellulose biosynthesis. CESAs were recently identified as cargo proteins of the classic adaptor protein 2 (AP2) complex of the clathrin-mediated endocytosis (CME) pathway. The AP2 complex of the CME pathway is conserved in yeast, animals, and plants, and has been well-characterized in many systems. In contrast, the recently discovered TPLATE complex (TPC), which is proposed to function as a CME adaptor complex, is only conserved in plants and a few other eukaryotes. In this study, we discovered that the TWD40-2 protein, a putative member of the TPC, is also important for the endocytosis of CESAs. Genetic analysis between TWD40-2 and AP2M of the AP2 complex revealed that the roles of TWD40-2 in CME are both distinct from and cooperative with the AP2 complex. Loss of efficient CME in twd40-2-3 resulted in the unregulated overaccumulation of CESAs at the plasma membrane. In seedlings of twd40-2-3 and other CME-deficient mutants, a direct correlation was revealed between endocytic deficiency and cellulose content deficiency, highlighting the importance of controlled CESA endocytosis in regulating cellulose biosynthesis.

  1. Immobilized surfactant-nanotube complexes support selectin-mediated capture of viable circulating tumor cells in the absence of capture antibodies.

    Science.gov (United States)

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-10-01

    The metastatic spread of tumor cells from the primary site to anatomically distant organs leads to a poor patient prognosis. Increasing evidence has linked adhesive interactions between circulating tumor cells (CTCs) and endothelial cells to metastatic dissemination. Microscale biomimetic flow devices hold promise as a diagnostic tool to isolate CTCs and develop metastatic therapies, utilizing E-selectin (ES) to trigger the initial rolling adhesion of tumor cells under flow. To trigger firm adhesion and capture under flow, such devices also typically require antibodies against biomarkers thought to be expressed on CTCs. This approach is challenged by the fact that CTCs are now known to exhibit heterogeneous expression of conventional biomarkers. Here, we describe surfactant-nanotube complexes to enhance ES-mediated capture and isolation of tumor cells without the use of capture antibodies. While the majority of tumor cells exhibited weaker rolling adhesion on halloysite nanotubes (HNT) coated with ES, HNT functionalization with the sodium dodecanoate (NaL) surfactant induced a switch to firm cellular adhesion under flow. Conversely, surfactant-nanotube complexes significantly reduced the number of primary human leukocytes captured via ES-mediated adhesion under flow. The switch in tumor cell adhesion was exploited to capture and isolate tumor cells in the absence of EpCAM antibodies, commonly utilized as the gold standard for CTC isolation. Additionally, HNT-NaL complexes were shown to capture tumor cells with low to negligible EpCAM expression, that are not efficiently captured using conventional approaches.

  2. Cyclodextrin-mediated enantioseparation of phenylalanine amide derivatives and amino alcohols by capillary electrophoresis-role of complexation constants and complex mobilities.

    Science.gov (United States)

    Aranyi, Anita; Péter, Antal; Ilisz, István; Fülöp, Ferenc; Scriba, Gerhard K E

    2014-10-01

    The separation of the enantiomers of phenylalanine amide and N-methyl derivatives as well as some amino alcohols was studied by CE in acidic BGEs using CDs as chiral selectors. The native CDs displayed only low chiral recognition ability at a concentration of 15 mg/mL in 20 mM sodium phosphate buffer, pH 2.5. In contrast, the analyte enantiomers were separated in the presence of randomly sulfated CDs under reversed polarity of the applied voltage. Sulfated β-CD proved to be the most universal selector resulting in the enantioseparation of all analytes. Opposite enantiomer migration order depending on the size of the CD cavity was observed for phenylalanine amide and 2-amino-2-phenylethanol. The R-enantiomers migrated first in the presence of sulfated α-CD and γ-CD while the S-enantiomers were detected first in the presence of sulfated β-CD. The enantioseparations could be rationalized based on analyte complexation by the respective CDs except for 2-amino-2-phenylethanol and sulfated β-CD where essentially equal complexation constants were derived for the enantiomers. In this case, the migration behavior could be attributed to the mobilities of the enantiomer-CD complexes adding another example to the CE-specific phenomenon of enantioseparations based primarily on complex mobilities.

  3. Penetratin-Mediated Transepithelial Insulin Permeation: Importance of Cationic Residues and pH for Complexation and Permeation

    DEFF Research Database (Denmark)

    Kristensen, Mie; Franzyk, Henrik; Klausen, M. T.

    2015-01-01

    Penetratin is a widely used carrier peptide showing promising potential for mucosal delivery of therapeutic proteins. In the present study, the importance of specific penetratin residues and pH was investigated with respect to complexation with insulin and subsequent transepithelial insulin...... permeation. Besides penetratin, three analogues were studied. The carrier peptide-insulin complexes were characterized in terms of size and morphology at pH 5, 6.5, and 7.4 by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. At pH 7.4 mainly very large complexes were...... present, while much smaller complexes dominated at pH 5. Presence of arginine residues in the carrier peptide proved to be a prerequisite for complexation with insulin as well as for enhanced transepithelial insulin permeation in vitro. Rearrangement of tryptophan residues resulted in significantly...

  4. Immune-mediated steroid-responsive epileptic spasms and epileptic encephalopathy associated with VGKC-complex antibodies.

    Science.gov (United States)

    Suleiman, Jehan; Brenner, Tanja; Gill, Deepak; Troedson, Christopher; Sinclair, Adriane J; Brilot, Fabienne; Vincent, Angela; Lang, Bethan; Dale, Russell C

    2011-11-01

    Autoantibodies that bind to voltage-gated potassium-channel complex proteins (VGKC-complex antibodies) occur frequently in adults with limbic encephalitis presenting with cognitive impairment and seizures. Recently, VGKC-complex antibodies have been described in a few children with limbic encephalitis, and children with unexplained encephalitis presenting with status epilepticus. We report a case of infantile-onset epileptic spasms and developmental delay compatible with epileptic encephalopathy. Our patient was a female infant, aged 4 months at presentation. She had evidence of immune activation in the central nervous system with elevated cerebrospinal fluid neopterin and mirrored oligoclonal bands, which prompted testing for autoantibodies. VGKC-complex antibodies were elevated (201 pmol/L, normalVGKC-complex antibodies might represent a marker of immune therapy responsiveness in a subgroup of patients with infantile epileptic encephalopathy.

  5. Oxidovanadium(IV/V) complexes as new redox mediators in dye-sensitized solar cells: a combined experimental and theoretical study.

    Science.gov (United States)

    Apostolopoulou, Andigoni; Vlasiou, Manolis; Tziouris, Petros A; Tsiafoulis, Constantinos; Tsipis, Athanassios C; Rehder, Dieter; Kabanos, Themistoklis A; Keramidas, Anastasios D; Stathatos, Elias

    2015-04-20

    Corrosiveness is one of the main drawbacks of using the iodide/triiodide redox couple in dye-sensitized solar cells (DSSCs). Alternative redox couples including transition metal complexes have been investigated where surprisingly high efficiencies for the conversion of solar to electrical energy have been achieved. In this paper, we examined the development of a DSSC using an electrolyte based on square pyramidal oxidovanadium(IV/V) complexes. The oxidovanadium(IV) complex (Ph4P)2[V(IV)O(hybeb)] was combined with its oxidized analogue (Ph4P)[V(V)O(hybeb)] {where hybeb(4-) is the tetradentate diamidodiphenolate ligand [1-(2-hydroxybenzamido)-2-(2-pyridinecarboxamido)benzenato}and applied as a redox couple in the electrolyte of DSSCs. The complexes exhibit large electron exchange and transfer rates, which are evident from electron paramagnetic resonance spectroscopy and electrochemistry, rendering the oxidovanadium(IV/V) compounds suitable for redox mediators in DSSCs. The very large self-exchange rate constant offered an insight into the mechanism of the exchange reaction most likely mediated through an outer-sphere exchange mechanism. The [V(IV)O(hybeb)](2-)/[V(V)O(hybeb)](-) redox potential and the energy of highest occupied molecular orbital (HOMO) of the sensitizing dye N719 and the HOMO of [V(IV)O(hybeb)](2-) were calculated by means of density functional theory electronic structure calculation methods. The complexes were applied as a new redox mediator in DSSCs, while the cell performance was studied in terms of the concentration of the reduced and oxidized form of the complexes. These studies were performed with the commercial Ru-based sensitizer N719 absorbed on a TiO2 semiconducting film in the DSSC. Maximum energy conversion efficiencies of 2% at simulated solar light (AM 1.5; 1000 W m(-2)) with an open circuit voltage of 660 mV, a short-circuit current of 5.2 mA cm(-2), and a fill factor of 0.58 were recorded without the presence of any additives in the

  6. Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells.

    Science.gov (United States)

    Weiler, Andrea M; Das, Arpita; Akinyosoye, Oluwasayo; Cui, Sherry; O'Connor, Shelby L; Scheef, Elizabeth A; Reed, Jason S; Panganiban, Antonito T; Sacha, Jonah B; Rakasz, Eva G; Friedrich, Thomas C; Maness, Nicholas J

    2015-12-04

    Nef-specific CD8(+) T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef165-173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T170I, unexpectedly and uniquely degraded Nef's major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication.

  7. Solvent-mediated crystal-to-crystal interconversion between discrete lanthanide complexes and one-dimensional coordination polymers and selective sensing for small molecules.

    Science.gov (United States)

    Wu, Jin-Ji; Ye, Yu-Xin; Qiu, Ying-Yu; Qiao, Zheng-Ping; Cao, Man-Li; Ye, Bao-Hui

    2013-06-03

    Two isostructural 1D coordination polymers {[Ln(OAc)2(H2O)(OBPT)]·3H2O}n (HOBPT = 4,6-bis(2-pyridyl)-1,3,5-triazin-2-ol, Ln = Eu(3+), 1; Tb(3+), 3) and two discrete complexes [Ln(OAc)2(DMF)2(OBPT)] (Ln = Eu(3+), 2; Tb(3+), 4) have been synthesized in H2O-MeOH or DMF solvents, respectively. Their structures were identified by powder X-ray diffraction. Single-crystal X-ray studies for complexes 1 and 2 revealed that the coordination geometries of the Eu(3+) ions are similar and can be described as a distorted tricapped trigonal prism with six oxygen atoms and three nitrogen atoms. The difference between them is that one aqua ligand and one oxygen atom from the OBPT ligand complete the coordination sphere in complex 1, whereas two DMF molecules complete the coordination sphere in complex 2. Interestingly, the solvent-mediated, reversible crystal-to-crystal transformation between them was achieved by immersing the crystalline samples in the corresponding solvent (H2O or DMF) or by exposing them to solvent vapor. Complex 1 shows a highly selective luminescence enhancement in response to DMF in comparison to that observed in response to other examined solvents such as acetone, ethyl acetate, ethanol, acetonitrile, methanol, and THF.

  8. Distinct complexes of yeast Snx4 family SNX-BARs mediate retrograde trafficking of Snc1 and Atg27.

    Science.gov (United States)

    Ma, Mengxiao; Burd, Christopher G; Chi, Richard J

    2017-02-01

    The yeast SNX4 sub-family of sorting nexin containing a Bin-Amphiphysin-Rvs domain (SNX-BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4-Atg20 and Snx4-Snx41. Each heterodimer coats an endosome-derived tubule that mediates retrograde sorting of distinct cargo; the v-SNARE, Snc1, is a cargo of the Snx4-Atg20 pathway, and Snx4-Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5-Vps17 retromer SNX-BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer-coated tubules, promotes fission of Snx4-Atg20 coated tubules. The results indicate that the yeast SNX-BAR proteins coat 3 distinct types of endosome-derived carriers that mediate endosome-to-Golgi retrograde trafficking.

  9. eEF1A Mediates the Nuclear Export of SNAG-Containing Proteins via the Exportin5-Aminoacyl-tRNA Complex

    Directory of Open Access Journals (Sweden)

    José Manuel Mingot

    2013-11-01

    Full Text Available Exportin5 mediates the nuclear export of double-stranded RNAs, including pre-microRNAs, adenoviral RNAs, and tRNAs. When tRNAs are aminoacylated, the Exportin5-aminoacyl (aa-tRNA complex recruits and coexports the translation elongation factor eEF1A. Here, we show that eEF1A binds to Snail transcription factors when bound to their main target, the E-cadherin promoter, facilitating their export to the cytoplasm in association with the aa-tRNA-Exportin5 complex. Snail binds to eEF1A through the SNAG domain, a protein nuclear export signal present in several transcription factor families, and this binding is regulated by phosphorylation. Thus, we describe a nuclear role for eEF1A and provide a mechanism for protein nuclear export that attenuates the activity of SNAG-containing transcription factors.

  10. eEF1A mediates the nuclear export of SNAG-containing proteins via the Exportin5-aminoacyl-tRNA complex.

    Science.gov (United States)

    Mingot, José Manuel; Vega, Sonia; Cano, Amparo; Portillo, Francisco; Nieto, M Angela

    2013-11-14

    Exportin5 mediates the nuclear export of double-stranded RNAs, including pre-microRNAs, adenoviral RNAs, and tRNAs. When tRNAs are aminoacylated, the Exportin5-aminoacyl (aa)-tRNA complex recruits and coexports the translation elongation factor eEF1A. Here, we show that eEF1A binds to Snail transcription factors when bound to their main target, the E-cadherin promoter, facilitating their export to the cytoplasm in association with the aa-tRNA-Exportin5 complex. Snail binds to eEF1A through the SNAG domain, a protein nuclear export signal present in several transcription factor families, and this binding is regulated by phosphorylation. Thus, we describe a nuclear role for eEF1A and provide a mechanism for protein nuclear export that attenuates the activity of SNAG-containing transcription factors.

  11. The Meckel syndrome- associated protein MKS1 functionally interacts with components of the BBSome and IFT complexes to mediate ciliary trafficking and hedgehog signaling

    Science.gov (United States)

    Barrington, Chloe L.; Katsanis, Nicholas

    2017-01-01

    The importance of primary cilia in human health is underscored by the link between ciliary dysfunction and a group of primarily recessive genetic disorders with overlapping clinical features, now known as ciliopathies. Many of the proteins encoded by ciliopathy-associated genes are components of a handful of multi-protein complexes important for the transport of cargo to the basal body and/or into the cilium. A key question is whether different complexes cooperate in cilia formation, and whether they participate in cilium assembly in conjunction with intraflagellar transport (IFT) proteins. To examine how ciliopathy protein complexes might function together, we have analyzed double mutants of an allele of the Meckel syndrome (MKS) complex protein MKS1 and the BBSome protein BBS4. We find that Mks1; Bbs4 double mutant mouse embryos exhibit exacerbated defects in Hedgehog (Hh) dependent patterning compared to either single mutant, and die by E14.5. Cells from double mutant embryos exhibit a defect in the trafficking of ARL13B, a ciliary membrane protein, resulting in disrupted ciliary structure and signaling. We also examined the relationship between the MKS complex and IFT proteins by analyzing double mutant between Mks1 and a hypomorphic allele of the IFTB component Ift172. Despite each single mutant surviving until around birth, Mks1; Ift172avc1 double mutants die at mid-gestation, and exhibit a dramatic failure of cilia formation. We also find that Mks1 interacts genetically with an allele of Dync2h1, the IFT retrograde motor. Thus, we have demonstrated that the MKS transition zone complex cooperates with the BBSome to mediate trafficking of specific trans-membrane receptors to the cilium. Moreover, the genetic interaction of Mks1 with components of IFT machinery suggests that the transition zone complex facilitates IFT to promote cilium assembly and structure. PMID:28291807

  12. DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yong; Fang, Shi-ji [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China); Zhu, Li-juan [Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and Institute of Neuroscience, Soochow University, Suzhou, Jiangsu 215021 (China); Zhu, Lun-qing, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children’s Bone Diseases, The Children’s Hospital Affiliated to Soochow University, Suzhou, Jiangsu 215000 (China); Zhou, Xiao-zhong, E-mail: zhouxz@suda.edu.cn [The Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou 215000 (China)

    2014-10-24

    Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

  13. Pdx1 and BETA2/NeuroD1 participate in a transcriptional complex that mediates short-range DNA looping at the insulin gene.

    Science.gov (United States)

    Babu, Daniella A; Chakrabarti, Swarup K; Garmey, James C; Mirmira, Raghavendra G

    2008-03-28

    The activity of the insulin gene, Ins, in islet beta cells is thought to arise in part from the synergistic action of the transcription factors Pdx1 and BETA2/NeuroD1. We asked how the binding of these factors to A and E elements many tens or hundreds of base pairs upstream of the start site could influence activity of transcriptional machinery. We therefore tested the hypothesis that the complex of Pdx1 and BETA2/NeuroD1 maintains a DNA conformation such that distal regions of the gene are brought into proximity of the promoter and coding region. We show by coimmunoprecipitation that Pdx1 and BETA2/NeuroD1 exist within a complex and that the two physically interact with one another in this complex as assessed by fluorescence resonance energy transfer. Consistent with this interaction, we found that the two factors simultaneously occupy the same fragment of the Ins gene in beta cell lines using the chromatin immunoprecipitation/re-chromatin immunoprecipitation assay. Using a modification of the chromosome conformation capture assay in vitro and in beta cells, we observed that Pdx1 and BETA2/NeuroD1 mediate looping of a segment of Ins that brings EcoRI sites located at -623 and +761 bp (relative to the transcriptional start site) in proximity to one another. This looping appears to be dependent in vitro upon an intact A3 binding element, but not upon the E2 element. Based on our findings, we propose a model whereby Pdx1 and BETA2/NeuroD1 physically interact to form a nucleoprotein complex on the Ins gene that mediates formation of a short DNA loop. Our results suggest that such short loop conformations may be a general mechanism to permit interactions between transcription factors and basal transcriptional machinery.

  14. Iridium mediated phenolic O-H activation and cyclometalation of 2-(naphthyl-1'-azo)-4-methylphenol - Formation of organoiridium complexes

    Indian Academy of Sciences (India)

    Rama Acharyya; Shie-Ming Peng; Gene-Hsiang Lee; Samaresh Bhattacharya

    2009-07-01

    Reaction of 2-(naphthyl-1'-azo)-4-methylphenol with [Ir(PPh3)3Cl] in refluxing ethanol in the presence of a base (NEt3) affords an organoiridium complex of type [Ir(PPh3)2(L)(H)], where L represents the coordinated 2-(naphthyl-1'-azo)-4-methylphenolate ligand. A similar reaction carried out in toluene affords the [Ir(PPh3)2(L)(H)] complex along with a similar complex of type [Ir(PPh3)2(L)Cl]. Structures of both the [Ir(PPh3)2(L)(H)] and [Ir(PPh3)2(L)Cl] complexes have been determined by X-ray crystallography. In both the complexes, 2-(naphthyl-1'-azo)-4-methylphenol is coordinated to iridium, via C-H activation at the 2' position of the naphthyl ring, as a dianionic tridentate C, N, O-donor and the two triphenylphosphines are trans. The organoiridium complexes show intense MLCT transitions in the visible region. Cyclic voltammetry on the [Ir(PPh3)2(L)(H)] and [Ir(PPh3)2(L)Cl] complexes shows a reversible Ir(III)-Ir(IV) oxidation respectively at 0.55 and 0.73 V vs SCE. An irreversible oxidation of the coordinated 2-(naphthyl-1'-azo)-4-methylphenolate ligand is observed above 1.0 V vs SCE and an irreversible reduction of the same is observed near -1.0 V vs SCE.

  15. Cooperative Transcriptional Activation of Antimicrobial Genes by STAT and NF-κB Pathways by Concerted Recruitment of the Mediator Complex

    Directory of Open Access Journals (Sweden)

    Sebastian Wienerroither

    2015-07-01

    Full Text Available The transcriptional response to infection with the bacterium Listeria monocytogenes (Lm requires cooperative signals of the type I interferon (IFN-I-stimulated JAK-STAT and proinflammatory NF-κB pathways. Using ChIP-seq analysis, we define genes induced in Lm-infected macrophages through synergistic transcriptional activation by NF-κB and the IFN-I-activated transcription factor ISGF3. Using the Nos2 and IL6 genes as prime examples of this group, we show that NF-κB functions to recruit enzymes that establish histone marks of transcriptionally active genes. In addition, NF-κB regulates transcriptional elongation by employing the mediator kinase module for the recruitment of the pTEFb complex. ISGF3 has a major role in associating the core mediator with the transcription start as a prerequisite for TFIID and RNA polymerase II (Pol II binding. Our data suggest that the functional cooperation between two major antimicrobial pathways is based on promoter priming by NF-κB and the engagement of the core mediator for Pol II binding by ISGF3.

  16. Evidence for a Precursor Complex in C-H Hydrogen Atom Transfer Reactions Mediated by a Manganese(IV) Oxo Complex

    OpenAIRE

    Garcia Bosch, Isaac; Company Casadevall, Anna; Cady, Clyde W.; Styring, Stenbjörn; Browne, Wesley R; Ribas Salamaña, Xavi; Costas Salgueiro, Miquel

    2011-01-01

    HAT trick: [MnIV(OH)2(H,MePytacn)]2+ (A) and [MnIV(O)(OH)(H,MePytacn)]+ (B) differ in their reactions with CH bonds: compound A engages in typical single-step hydrogen atom transfer (HAT) reactions, whereas B first forms a substrate–B encounter complex (C; see scheme). This equilibrium alters the relative CH reactivity from that expected from CH bond dissociation energies Aquest mateix article està publicat a l'edició alemanya d''Angewandte Chemie' (ISSN 0044-8249, EISSN 1521-3757), 2011, ...

  17. Charge mediation by ruthenium poly(pyridine) complexes in 'second-generation' glucose biosensors based on carboxymethylated beta-cyclodextrin polymer membranes.

    Science.gov (United States)

    Kosela, Edyta; Elzanowska, Hanna; Kutner, Wlodzimierz

    2002-04-01

    Four different poly(pyridine) complexes of ruthenium, viz. Ru(II)(trpy)(phen)(OH(2))](2+) (1), trans-[Ru(III)(2,2'bpy)(2)(OH(2))(OH)](2+) (2), [(2,2'bpy)(2)(OH)Ru(III)ORu(III)(OH)(2,2'bpy)(2)](4+) (3), and [Ru(II)(4,4'bpy)(NH(3))(5)](2+) (4) (2,2'bpy=2,2'-bipyridine, 4,4'bpy=4,4'-bipyridine, trpy=2,2',2"-terpyridine, phen=1,10-phenanthroline), were tested as non-physiological charge mediators of 'second-generation' glucose biosensors. The membranes for these biosensors were prepared by casting anionic carboxymethylated beta-cyclodextrin polymer films (beta-CDPA) directly onto the Pt or glassy carbon (GC) disk electrodes. Simultaneously, glucose oxidase (GOD) was immobilized in the films by covalent bonding and the Ru complexes were incorporated both by inclusion in the beta-CD molecular cavities and by ion exchange at the fixed carboxymethyl cation-exchange sites. The leakage of the mediator from the polymer has been minimized by adopting a suitable pre-treatment procedure. The biosensors catalytic activities increased in the order 1inclusion complex with beta-CD, the biosensor sensitivity was the highest and equal to 7.2 micro A mM(-1) cm(-2), detectability was as low as 1 mM, but the linear concentration range was limited only to 4 mM. In contrast, for complexes 2 and 3 the sensitivity was 0.4 and 3.2 micro A mM(-1) cm(-2), while the linear concentration range extended up to at least 24 and 14 mM glucose, respectively. Even though some common interfering substances, such as ascorbate, paracetamol or urea, are oxidized at potentials close to those of the Ru complex redox couples, their electro-oxidation currents at physiological concentrations are insignificant compared to those due to the biocatalytic oxidation of glucose. The biosensor response to glucose is reversible as demonstrated by the inhibition of GOD activity by Cu(II). That is, the Cu(II) concentration required to inhibit by half the response to glucose of the biosensor containing complex 2 was 1.0 m

  18. Zn(II-Chlorido complexes of phytohormone kinetin and its derivatives modulate expression of inflammatory mediators in THP-1 cells.

    Directory of Open Access Journals (Sweden)

    Jan Hošek

    Full Text Available Kinetin (N6-furfuryladenine belongs to a group of plant growth hormones involved in cell division, differentiation and other physiological processes. One of the possible ways to obtain biologically active compounds is to complex biologically relevant natural compounds to suitable metal atoms. In this work, two structural groups of Zn(II complexes [Zn(L(n2Cl2]·Solv (1-5 and [Zn(HL(nCl3] · xL(n (6-7; n=1-5, Solv=CH3OH for 1 and 2H2O for 2; x =1 for 6 and 2 for 7; involving a phytohormone kinetin and its derivatives (L(n were evaluated for their ability to modulate secretion of tumour necrosis factor (TNF-α, interleukin (IL-1β and matrix metalloproteinase (MMP-2 in a lipopolysaccharide (LPS-activated macrophage-like THP-1 cell model. The penetration of the complexes to cells was also detected. The mechanism of interactions of the zinc(II complexes with a fluorescent sensor N-(6-methoxy-8-quinolyl-p-toluene sulphonamide (TSQ and sulfur-containing biomolecules (l-cysteine and reduced glutathione was studied by electrospray-ionization mass spectrometry and flow-injection analysis with fluorescence detection. The present study showed that the tested complexes exhibited a low cytotoxic effect on the THP-1 cell line (IC50>40 µM, apart from complex 4, with an IC50=10.9 ± 1.1 µM. Regarding the inflammation-related processes, the Zn(II complexes significantly decreased IL-1β production by a factor of 1.47-2.22 compared with the control (DMSO, but did not affect TNF-α and MMP-2 secretions. However, application of the Zn(II complexes noticeably changed the pro-MMP-2/MMP-2 ratio towards a higher amount of maturated MMP-2, when they induced a 4-times higher production of maturated MMP-2 in comparison with the vehicle-treated cells under LPS stimulation. These results indicated that the complexes are able to modulate an inflammatory response by influencing secretion and activity of several inflammation-related cytokines and enzymes.

  19. Spectrophotometric determination of Mercury (II by simultaneous micelle mediated extraction through ternary complex formation in water samples

    Directory of Open Access Journals (Sweden)

    Farzin Nekouei

    2014-06-01

    Full Text Available In this study, a micelle mediated extraction procedure for preconcentration of trace quantities of Hg(II as a prior step to its simultaneous spectrophotometric determination has been developed. The method is based on a ternary ion-association of Hg(II, Xylidyl Blue (XB and cationic surfactant (CTAB. Major factors affecting the efficiency of the method has been studied. The limit of detection (LOD under optimum conditions based on 3Sb was 4.65 ng mL-1. The proposed method has been applied for determination of trace amount of mercury in water samples with satisfactory results.

  20. Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex

    Science.gov (United States)

    Nyikó, Tünde; Kerényi, Farkas; Szabadkai, Levente; Benkovics, Anna H.; Major, Péter; Sonkoly, Boglárka; Mérai, Zsuzsanna; Barta, Endre; Niemiec, Emilia; Kufel, Joanna; Silhavy, Dániel

    2013-01-01

    Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3′untranslated region (UTR). In yeasts, unusually long 3′UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3′UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3′UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD. PMID:23666629

  1. The Dream About the Magic Silver Bullet – the Complexity of Designing for Tablet-Mediated Learning

    DEFF Research Database (Denmark)

    Jahnke, Isa; Svendsen, Niels Vandel; Johansen, Simon Kristoffer

    2014-01-01

    of reducing it. The teachers must learn how to navigate during the breakdowns before media tablets reduce complexity and reach a state in which the tablets take part in the classroom ecology as functional organs. The teachers have to deal with complex situations during class in situ. In order to be able......In this paper, we report three cases of the integration of technology, such as web-enabled media tablets in Scandinavian schools. Both qualitative and quantitative data have been applied. A daily challenge for teachers is to coordinate their group of students in a way that enables collaborative...... learning. We report the gaps and interrelations between the dreams and the practice of the teachers. They dream about an interconnected praxis – the magic silver bullet – and establish their visions of inter- connectivity because of their breakdown experiences of media tablets aiding complexity instead...

  2. Upstream promoter sequences and αCTD mediate stable DNA wrapping within the RNA polymerase–promoter open complex

    Science.gov (United States)

    Cellai, Sara; Mangiarotti, Laura; Vannini, Nicola; Naryshkin, Nikolai; Kortkhonjia, Ekaterine; Ebright, Richard H; Rivetti, Claudio

    2007-01-01

    We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP–promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP α-subunit carboxy-terminal domain and on the presence and length of the RNAP α-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP–promoter open complex at the λ PR promoter is not a general feature of RNAP–promoter open complexes. PMID:17290289

  3. Upstream promoter sequences and alphaCTD mediate stable DNA wrapping within the RNA polymerase-promoter open complex.

    Science.gov (United States)

    Cellai, Sara; Mangiarotti, Laura; Vannini, Nicola; Naryshkin, Nikolai; Kortkhonjia, Ekaterine; Ebright, Richard H; Rivetti, Claudio

    2007-03-01

    We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP-promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP alpha-subunit carboxy-terminal domain and on the presence and length of the RNAP alpha-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP-promoter open complex at the lambda P(R) promoter is not a general feature of RNAP-promoter open complexes.

  4. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity

    OpenAIRE

    Nuñez, James K.; Kranzusch, Philip J.; Noeske, Jonas; Wright, Addison V.; Davies, Christopher W; Doudna, Jennifer A.

    2014-01-01

    The initial stage of CRISPR–Cas immunity involves the acquisition of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved amongst all CRISPR–Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å resolution crystal structure of the Cas1–Cas2 complex. M...

  5. Pivotal Role for a Tail Subunit of the RNA Polymerase II Mediator Complex CgMed2 in Azole Tolerance and Adherence in Candida glabrata

    Science.gov (United States)

    Borah, Sapan; Shivarathri, Raju; Srivastava, Vivek Kumar; Ferrari, Sélène; Sanglard, Dominique

    2014-01-01

    Antifungal therapy failure can be associated with increased resistance to the employed antifungal agents. Candida glabrata, the second most common cause of invasive candidiasis, is intrinsically less susceptible to the azole class of antifungals and accounts for 15% of all Candida bloodstream infections. Here, we show that C. glabrata MED2 (CgMED2), which codes for a tail subunit of the RNA polymerase II Mediator complex, is required for resistance to azole antifungal drugs in C. glabrata. An inability to transcriptionally activate genes encoding a zinc finger transcriptional factor, CgPdr1, and multidrug efflux pump, CgCdr1, primarily contributes to the elevated susceptibility of the Cgmed2Δ mutant toward azole antifungals. We also report for the first time that the Cgmed2Δ mutant exhibits sensitivity to caspofungin, a constitutively activated protein kinase C-mediated cell wall integrity pathway, and elevated adherence to epithelial cells. The increased adherence of the Cgmed2Δ mutant was attributed to the elevated expression of the EPA1 and EPA7 genes. Further, our data demonstrate that CgMED2 is required for intracellular proliferation in human macrophages and modulates survival in a murine model of disseminated candidiasis. Lastly, we show an essential requirement for CgMed2, along with the Mediator middle subunit CgNut1 and the Mediator cyclin-dependent kinase/cyclin subunit CgSrb8, for the high-level fluconazole resistance conferred by the hyperactive allele of CgPdr1. Together, our findings underscore a pivotal role for CgMed2 in basal tolerance and acquired resistance to azole antifungals. PMID:25070095

  6. The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Kuksin Dmitry

    2005-04-01

    Full Text Available Abstract Background Simian virus 40 (SV40 enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi. Results We confirmed our earlier finding that SV40 co localizes with the Golgi marker β-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide. Conclusion The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with β-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.

  7. The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum.

    Science.gov (United States)

    Norkin, Leonard C; Kuksin, Dmitry

    2005-04-19

    Simian virus 40 (SV40) enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi. We confirmed our earlier finding that SV40 co localizes with the Golgi marker beta-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide. The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with beta-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.

  8. A βPIX-PAK2 complex confers protection against Scrib-dependent and cadherin-mediated apoptosis

    DEFF Research Database (Denmark)

    Frank, Scott R; Bell, Jennifer H; Frödin, Morten;

    2012-01-01

    During epithelial morphogenesis, a complex comprising the βPIX (PAK-interacting exchange factor β) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian ortholog of the Drosophila polarity determinant and tumor suppressor Scribble, binds βPIX directly. ...

  9. Synthesis, Crystal Structure of Ruthenium 1,2-Naphthoquinone-1-oxime Complex and Its Mediated C-C Coupling Reactions of Terminal Alkynes

    Institute of Scientific and Technical Information of China (English)

    SUN, Ke; WONG, Wing-Tak; LIU, Xiao-Xia; ZHANG, Bao-Yan

    2003-01-01

    Substituted decarbonylation reaction of ruthenium 1,2-naphthoquinone-1-oxime (1-nqo) complex, cis-, cis-[Ru{ η2-N(O)C10-H6O}2(CO)2] (1), with acetonitrile gave cis-, cis-[Ru { η2-N(O)C10H6O}2(CO)(NCMe)] (2). Complex 2 was fully characterized by 1H NMR, FAB MS, IR spectra and single crystal X-ray analysis. Complex 2 maintains the coordination structure of 1 with the two naphthoquinonic oxygen atoms, as well as the two oximato nitrogen atoms located cis to each other, showing that there is no ligand rearrangement of the 1-nqo ligands during the substitution reaction. The carbonyl group originally trans to the naphthoquinonic oxygen in one 1-nqo ligand is left in its original position [O(5)-Ru-C(1), 174.0(6)°], while the other one originally trans to the oximato group of the other 1-nqo llgand is substituted by NCMe [N(1)-Ru-N(3), 170.6(6)°].This shows that the carbonyl trans to oximato group is more labile than the one trans to naphthoquinonic O atom towards substitution. This is probably due to the comparatively stronger π back bonding from ruthenium metal to the carbonyl group trans to naphthoquinonic O atom, than the one trans to oximato group, resulting in the comparatively weaker Ru-CO bond for the latter and consequently easier replacement of this carbonyl. Selected coupling of phenylacetylene mediated by 2 gave a single trans-dimerization product 3, while 2 mediated coupling reaction of methyl propiolate produced three products:one trans-dimerization product 4 and two cyclotrimeric products 5 and 6.

  10. c-Myb protein interacts with Rcd-1, a component of the CCR4 transcription mediator complex.

    Science.gov (United States)

    Haas, Martin; Siegert, Michaela; Schürmann, André; Sodeik, Beate; Wolfes, Heiner

    2004-06-29

    Transcriptional initiation of eukaryotic genes depends on the cooperative interaction of various transcription factors. Using the yeast two-hybrid assay, we have identified the murine Rcd-1 protein as a cofactor of the c-myb proto-oncogene product. Rcd-1 is evolutionarily conserved among many species, and moreover the yeast homologue CAF40 is part of the carbon catabolite repressor protein transcriptional mediator thought to be involved in the negative regulation of genes transcribed by RNA polymerase II. Rcd-1 is located mainly in the nucleus, and it interacts with c-Myb both in vitro and in vivo. The activation of the myeloid c-myb-specific mim-1 promoter is repressed by Rcd-1. Interestingly, rcd-1 is an erythropoietin regulated gene, which also represses the action of the AP-1 transcription factor on its target genes.

  11. Cubilin, the endocytic receptor for intrinsic factor-vitamin B(12) complex, mediates high-density lipoprotein holoparticle endocytosis.

    Science.gov (United States)

    Hammad, S M; Stefansson, S; Twal, W O; Drake, C J; Fleming, P; Remaley, A; Brewer, H B; Argraves, W S

    1999-08-31

    Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B(12). Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL(2), HDL(3), apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.

  12. KRAS protein stability is regulated through SMURF2: UBCH5 complex-mediated β-TrCP1 degradation.

    Science.gov (United States)

    Shukla, Shirish; Allam, Uday Sankar; Ahsan, Aarif; Chen, Guoan; Krishnamurthy, Pranathi Meda; Marsh, Katherine; Rumschlag, Matthew; Shankar, Sunita; Whitehead, Christopher; Schipper, Matthew; Basrur, Venkatesha; Southworth, Daniel R; Chinnaiyan, Arul M; Rehemtulla, Alnawaz; Beer, David G; Lawrence, Theodore S; Nyati, Mukesh K; Ray, Dipankar

    2014-02-01

    Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.

  13. KRAS Protein Stability Is Regulated through SMURF2: UBCH5 Complex-Mediated β-TrCP1 Degradation

    Directory of Open Access Journals (Sweden)

    Shirish Shukla

    2014-02-01

    Full Text Available Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2 and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1. Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.

  14. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    Science.gov (United States)

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  15. An Elmo-Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion.

    Science.gov (United States)

    Toret, Christopher P; Collins, Caitlin; Nelson, W James

    2014-12-08

    Cell-cell contact formation is a dynamic process requiring the coordination of cadherin-based cell-cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell-cell adhesion identified an Elmo-Dock complex. This was unexpected as Elmo-Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell-cell contacts in Madin-Darby canine kidney cells. At cell-cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell-cell adhesion. Upon completion of cell-cell adhesion, Elmo2 and Dock1 no longer localize to cell-cell contacts and are not required subsequently for the maintenance of cell-cell adhesion. These studies show that Elmo-Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell-cell adhesion. © 2014 Toret et al.

  16. MiRNA-Mediated Regulation of the SWI/SNF Chromatin Remodeling Complex Controls Pluripotency and Endodermal Differentiation in Human ESCs.

    Science.gov (United States)

    Wade, Staton L; Langer, Lee F; Ward, James M; Archer, Trevor K

    2015-10-01

    MicroRNAs and chromatin remodeling complexes represent powerful epigenetic mechanisms that regulate the pluripotent state. miR-302 is a strong inducer of pluripotency, which is characterized by a distinct chromatin architecture. This suggests that miR-302 regulates global chromatin structure; however, a direct relationship between miR-302 and chromatin remodelers has not been established. Here, we provide data to show that miR-302 regulates Brg1 chromatin remodeling complex composition in human embryonic stem cells (hESCs) through direct repression of the BAF53a and BAF170 subunits. With the subsequent overexpression of BAF170 in hESCs, we show that miR-302's inhibition of BAF170 protein levels can affect the expression of genes involved in cell proliferation. Furthermore, miR-302-mediated repression of BAF170 regulates pluripotency by positively influencing mesendodermal differentiation. Overexpression of BAF170 in hESCs led to biased differentiation toward the ectoderm lineage during EB formation and severely hindered directed definitive endoderm differentiation. Taken together, these data uncover a direct regulatory relationship between miR-302 and the Brg1 chromatin remodeling complex that controls gene expression and cell fate decisions in hESCs and suggests that similar mechanisms are at play during early human development.

  17. Multiple domains of Stardust differentially mediate localisation of the Crumbs-Stardust complex during photoreceptor development in Drosophila.

    Science.gov (United States)

    Bulgakova, Natalia A; Kempkens, Ozlem; Knust, Elisabeth

    2008-06-15

    Drosophila Stardust (Sdt), a member of the MAGUK family of scaffolding proteins, is a constituent of the evolutionarily conserved Crumbs-Stardust (Crb-Sdt) complex that controls epithelial cell polarity in the embryo and morphogenesis of photoreceptor cells. Although apical localisation is a hallmark of the complex in all cell types and in all organisms analysed, only little is known about how individual components are targeted to the apical membrane. We have performed a structure-function analysis of Sdt by constructing transgenic flies that express altered forms of Sdt to determine the roles of individual domains for localisation and function in photoreceptor cells. The results corroborate the observation that the organisation of the Crb-Sdt complex is differentially regulated in pupal and adult photoreceptors. In pupal photoreceptors, only the PDZ domain of Sdt - the binding site of Crb - is required for apical targeting. In adult photoreceptors, by contrast, targeting of Sdt to the stalk membrane, a distinct compartment of the apical membrane between the rhabdomere and the zonula adherens, depends on several domains, and seems to be a two-step process. The N-terminus, including the two ECR domains and a divergent N-terminal L27 domain that binds the multi-PDZ domain protein PATJ in vitro, is necessary for targeting the protein to the apical pole of the cell. The PDZ-, the SH3- and the GUK-domains are required to restrict the protein to the stalk membrane. Drosophila PATJ or Drosophila Lin-7 are stabilised whenever a Sdt variant that contains the respective binding site is present, independently of where the variant is localised. By contrast, only full-length Sdt, confined to the stalk membrane, stabilises and localises Crb, although only in reduced amounts. The amount of Crumbs recruited to the stalk membrane correlates with its length. Our results highlight the importance of the different Sdt domains and point to a more intricate regulation of the Crb

  18. The P2X7 receptor–pannexin-1 complex decreases muscarinic acetylcholine receptor–mediated seizure susceptibility in mice

    OpenAIRE

    Kim, Ji-Eun; Kang, Tae-Cheon

    2011-01-01

    Pannexin-1 (Panx1) plays a role in the release of ATP and glutamate in neurons and astrocytes. Panx1 can be opened at the resting membrane potential by extracellular ATP via the P2X7 receptor (P2X7R). Panx1 opening has been shown to induce neuronal death and aberrant firing, but its role in neuronal activity has not been established. Here, we report the role of the P2X7R-Panx1 complex in regulating muscarinic acetylcholine 1 (M1) receptor function. P2X7R knockout (P2X7–/–) mice showed greater...

  19. Arabidopsis COMPASS-like complexes mediate histone H3 lysine-4 trimethylation to control floral transition and plant development.

    Directory of Open Access Journals (Sweden)

    Danhua Jiang

    2011-03-01

    Full Text Available Histone H3 lysine-4 (H3K4 methylation is associated with transcribed genes in eukaryotes. In Drosophila and mammals, both di- and tri-methylation of H3K4 are associated with gene activation. In contrast to animals, in Arabidopsis H3K4 trimethylation, but not mono- or di-methylation of H3K4, has been implicated in transcriptional activation. H3K4 methylation is catalyzed by the H3K4 methyltransferase complexes known as COMPASS or COMPASS-like in yeast and mammals. Here, we report that Arabidopsis homologs of the COMPASS and COMPASS-like complex core components known as Ash2, RbBP5, and WDR5 in humans form a nuclear subcomplex during vegetative and reproductive development, which can associate with multiple putative H3K4 methyltransferases. Loss of function of ARABIDOPSIS Ash2 RELATIVE (ASH2R causes a great decrease in genome-wide H3K4 trimethylation, but not in di- or mono-methylation. Knockdown of ASH2R or the RbBP5 homolog suppresses the expression of a crucial Arabidopsis floral repressor, FLOWERING LOCUS C (FLC, and FLC homologs resulting in accelerated floral transition. ASH2R binds to the chromatin of FLC and FLC homologs in vivo and is required for H3K4 trimethylation, but not for H3K4 dimethylation in these loci; overexpression of ASH2R causes elevated H3K4 trimethylation, but not H3K4 dimethylation, in its target genes FLC and FLC homologs, resulting in activation of these gene expression and consequent late flowering. These results strongly suggest that H3K4 trimethylation in FLC and its homologs can activate their expression, providing concrete evidence that H3K4 trimethylation accumulation can activate eukaryotic gene expression. Furthermore, our findings suggest that there are multiple COMPASS-like complexes in Arabidopsis and that these complexes deposit trimethyl but not di- or mono-methyl H3K4 in target genes to promote their expression, providing a molecular explanation for the observed coupling of H3K4 trimethylation (but not H3

  20. T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompatibility complex class II-bound peptides

    DEFF Research Database (Denmark)

    Larsen, S L; Pedersen, L O; Buus, S;

    1996-01-01

    the exopeptidase Aminopeptidase N (APN, CD13) as one of the enzymes involved in the observed cell-surface antigen processing. The NH2-terminal end of the longer peptide could, even while bound to major histocompatibility complex (MHC) class II molecules, be digested by APN with dramatic consequences for T cell...... antigen recognition. This could be demonstrated both in cell-free systems using purified reagents and in cellular systems. Thus, MHC class II and APN may act in concert to generate the final T cell epitopes....

  1. A loop-mediated isothermal amplification (LAMP) method for the identification of species within the Echinococcus granulosus complex.

    Science.gov (United States)

    Wassermann, Marion; Mackenstedt, Ute; Romig, Thomas

    2014-02-24

    To facilitate the specific identification of Echinococcus spp. isolates in endemic countries, a LAMP (loop-mediated isothermal amplification) assay was developed to detect the various agents known to cause cystic echinococcosis (E. granulosus s.s., E. equinus, E. ortleppi, E. canadensis and E. felidis). The infectivity of the different species and the severity of the disease in humans and livestock vary significantly among those species, and correct molecular identification of large numbers of field isolates is crucial to understand their epidemiology. However, funding constraints in many CE endemic countries often prevent PCR-based screening of field isolates. The LAMP method allows the amplification of DNA fragments under isothermal conditions which can be achieved using an ordinary waterbath, and the detection of amplification products only requires a UV light source. In the present study a LAMP assay was developed which allows the detection and differentiation of the 5 CE causing Echinococcus species. The diagnostic power was adjusted to species level, i.e. intraspecific strains (G1-3 within E. granulosus s.s., G6-10 within E. canadensis) are not discriminated. Wherever this would be necessary for epidemiological purposes, the method can be adjusted according to local requirements. The sensitivity of the assay was tested down to one fiftieth of a single protoscolex or egg, respectively. The present study describes a fast and simple method for the differentiation of CE causing Echinococcus species which can facilitate epidemiological studies in endemic countries.

  2. The anaphase-promoting complex protein 5 (AnapC5 associates with A20 and inhibits IL-17-mediated signal transduction.

    Directory of Open Access Journals (Sweden)

    Allen W Ho

    Full Text Available IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7, which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3, a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways.

  3. P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.

    Directory of Open Access Journals (Sweden)

    Olle Dahlberg

    2015-02-01

    Full Text Available Positive Transcription Elongation Factor b (P-TEFb is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II into active elongation. It can assemble into a larger Super Elongation Complex (SEC consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3' end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.

  4. Optic atrophy 1 mediates coenzyme Q-responsive regulation of respiratory complex IV activity in brain mitochondria.

    Science.gov (United States)

    Takahashi, Kazuhide; Ohsawa, Ikuroh; Shirasawa, Takuji; Takahashi, Mayumi

    2017-11-01

    The oxygen consumption rate (OCR) in brain mitochondria is significantly lower in aged mice than in young mice, and the reduced OCR is rescued by administration of water-solubilized CoQ10 to aged mice via drinking water. However, the mechanism behind this remains unclear. Here, we show that the activity of respiratory complex IV (CIV) in brain mitochondria declined in aged mice than in young mice, with no significant change in individual respiratory complex levels and their supercomplex assembly. Reduced CIV activity in the aged mice coincided with reduced binding of optic atrophy 1 (OPA1) to CIV. Both reduced activity and OPA1 binding of CIV were rescued by water-solubilized CoQ10 administration to aged mice via drinking water. OCR and the activity and OPA1 binding of CIV in isolated brain mitochondria from aged mice were restored by incubation with CoQ10, but not in the presence of 15-deoxy-prostaglandin J2, an inhibitor of a GTPase effector domain-containing GTPase such as OPA1 and DRP1. By contrast, the CoQ10-responsive restoration of OCR in the isolated mitochondria was not inhibited by Mdivi-1, a selective inhibitor of DRP1. Thus, we propose a novel function of OPA1 in regulating the CIV activity in brain mitochondria in response to CoQ10. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Electron-transfer-mediated binding of optically active cobalt(III) complexes to horse heart cytochrome c.

    Science.gov (United States)

    Scholten, Ulrich; Merchán, Alejandro Castillejo; Bernauer, Klaus

    2005-03-22

    Optically active cobalt(II) complexes are used as reducing agents in the electron-transfer reaction involving horse heart cytochrome c. Analysis of the circular dichroism (CD) spectra of reaction products indicates that the corresponding cobalt(III) species of both enantiomers of [CoII(alamp)] (H2alamp=N,N'-[(pyridine-2,6-diyl)bis(methylene)]-bis[alanine]) are partly attached to the protein during electron transfer by coordination to an imidazole unit of one of the histidine residues. His-26 and His-33 are both solvent exposed, and the results suggest that one of these histidine residues acts as a bridge in the electron transfer to and from the haem iron of cytochrome c. The reaction is enantioselective: the ratio of the relative reactivity at 15 degrees C is 2.9 in favour of the R,R-enantiomer. A small induced CD activity in the haem chromophore reveals that some structural changes in the protein occur consecutively with the binding of the cobalt(III) complex.

  6. Trauma typology as a risk factor for aggression and self-harm in a complex PTSD population: the mediating role of alterations in self-perception.

    Science.gov (United States)

    Dyer, Kevin F W; Dorahy, Martin J; Shannon, Maria; Corry, Mary

    2013-01-01

    This study examined the role of prolonged, repeated traumatic experiences such as childhood and sectarian trauma in the development of posttraumatic aggression and self-harm. Forty-four adult participants attending therapy for complex trauma in Northern Ireland were obtained via convenience sampling. When social desirability was controlled, childhood emotional and physical neglect were significant correlates of posttraumatic hostility and history of self-harm. These relationships were mediated by alterations in self-perception (e.g., shame, guilt). Severity of sectarian-related experiences was not related to self-destructive behaviors. Moreover, none of the trauma factors were related to overt aggressive behavior. The findings have implications for understanding risk factors for posttraumatic aggression and self-harm, as well as their treatment.

  7. Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex.

    Science.gov (United States)

    Dutta, Arijit; Eckelmann, Bradley; Adhikari, Sanjay; Ahmed, Kazi Mokim; Sengupta, Shiladitya; Pandey, Arvind; Hegde, Pavana M; Tsai, Miaw-Sheue; Tainer, John A; Weinfeld, Michael; Hegde, Muralidhar L; Mitra, Sankar

    2017-03-17

    Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

    Science.gov (United States)

    Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

    2007-08-15

    Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

  9. Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53

    Science.gov (United States)

    Martinez-Zapien, Denise; Ruiz, Francesc Xavier; Poirson, Juline; Mitschler, André; Ramirez-Ramos, Juan; Forster, Anne; Cousido-Siah, Alexandra; Masson, Murielle; Pol, Scott Vande; Podjarny, Alberto; Travé, Gilles; Zanier, Katia

    2015-01-01

    Summary The p53 pro-apoptotic tumor suppressor is mutated or functionally altered in most cancers. In epithelial tumors induced by “high-risk” mucosal Human Papillomaviruses (hrm-HPVs), including human cervical carcinoma and a growing number of head-and-neck cancers 1, p53 is degraded by the viral oncoprotein E6 2. In this process, E6 binds to a short LxxLL consensus sequence within the cellular ubiquitin ligase E6AP 3. Subsequently, the E6/E6AP heterodimer recruits and degrades p53 4. Neither E6 nor E6AP are separately able to recruit p53 3,5, and the precise mode of assembly of E6, E6AP and p53 is unknown. Here, we solved the crystal structure of a ternary complex comprising full-length HPV16 E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6-p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumor suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against HPV-mediated oncogenesis. PMID:26789255

  10. The DinB•RecA complex of Escherichia coli mediates an efficient and high-fidelity response to ubiquitous alkylation lesions.

    Science.gov (United States)

    Cafarelli, Tiziana M; Rands, Thomas J; Godoy, Veronica G

    2014-03-01

    Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases.

  11. Investigation of the receptor-mediated endocytosis of transcobalamin/intrinsic factor-vitamin B12 complexes

    DEFF Research Database (Denmark)

    Beedholm, Rasmus; Grissom, Charles B.; Fedosov, Sergey N.

    receptor structure. This receptor is suggested to be regulated by the vitamin B12 level in the cells, which is interesting in relation to cancer growth. The cellular endocytosis of TC- B12 complex by this unknown receptor is being investigated, using confocal microscopy. Fluorescently labeled B12 molecules...... (Oregon green linked to B12) have been synthesized to determine the B12 uptake level in normal and various tumour-derived cells (e.g. Hela cells from cervix epithelioid carcinoma and BN- cells from rat yolk sac sarcoma). Costaining of the B12 binders has been performed using fluorescently labelled...... secondary antibodies recognising primary antibodies against IF and TC. The data show a cell growth-regulated uptake of free fluorescent B12 but a strong inducement of uptake by TC and IF. After uptake the B12 fluorochrome colocalizes with the B12 binders. ...

  12. CR2-mediated activation of the complement alternative pathway results in formation of membrane attack complexes on human B lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, C H; Marquart, H V; Prodinger, W M;

    2001-01-01

    Normal human B lymphocytes activate the alternative pathway of complement via complement receptor type 2 (CR2, CD21), that binds hydrolysed C3 (iC3) and thereby promotes the formation of a membrane-bound C3 convertase. We have investigated whether this might lead to the generation of a C5...... convertase and consequent formation of membrane attack complexes (MAC). Deposition of C3 fragments and MAC was assessed on human peripheral B lymphocytes in the presence of 30% autologous serum containing 4.4 mM MgCl2/20 mM EGTA, which abrogates the classical pathway of complement without affecting...... the alternative pathway. Blockade of the CR2 ligand-binding site with the monoclonal antibody FE8 resulted in 56 +/- 13% and 71 +/- 9% inhibition of the C3-fragment and MAC deposition, respectively, whereas the monoclonal antibody HB135, directed against an irrelevant CR2 epitope, had no effect. Blockade...

  13. The origin of room temperature ferromagnetism mediated by Co–VZn complexes in the ZnO grain boundary

    KAUST Repository

    Devi, Assa Aravindh Sasikala

    2016-05-20

    Ferromagnetism in polycrystalline ZnO doped with Co has been observed to be sustainable in recent experiments. We use first-principle calculations to show that Co impurities favorably substitute at the grain boundary (GB) rather than in the bulk. We reveal that room-temperature ferromagnetism (RTFM) at the Co-doped ZnO GB in the presence of Zn vacancies is due to ferromagnetic exchange coupling of a pair of closely associated Co atoms in the GB, with a ferromagnetic exchange coupling energy of ∼300 meV, which is in contrast to a previous study that suggested the O vacancy-Co complex induced ferromagnetism. Electronic structure analysis was used to predict the exchange coupling mechanism, showing that the hybridization of O p states with Co and Zn d states enhances the magnetic polarization originating from the GB. Our results indicate that RTFM originates from Co clusters at interfaces or in GBs. © 2016 The Royal Society of Chemistry.

  14. Rare ecomorphological convergence on a complex adaptive landscape: Body size and diet mediate evolution of jaw shape in squirrels (Sciuridae).

    Science.gov (United States)

    Zelditch, Miriam Leah; Ye, Ji; Mitchell, Jonathan S; Swiderski, Donald L

    2017-03-01

    Convergence is widely regarded as compelling evidence for adaptation, often being portrayed as evidence that phenotypic outcomes are predictable from ecology, overriding contingencies of history. However, repeated outcomes may be very rare unless adaptive landscapes are simple, structured by strong ecological and functional constraints. One such constraint may be a limitation on body size because performance often scales with size, allowing species to adapt to challenging functions by modifying only size. When size is constrained, species might adapt by changing shape; convergent shapes may therefore be common when size is limiting and functions are challenging. We examine the roles of size and diet as determinants of jaw shape in Sciuridae. As expected, size and diet have significant interdependent effects on jaw shape and ecomorphological convergence is rare, typically involving demanding diets and limiting sizes. More surprising is morphological without ecological convergence, which is equally common between and within dietary classes. Those cases, like rare ecomorphological convergence, may be consequences of evolving on an adaptive landscape shaped by many-to-many relationships between ecology and function, many-to-one relationships between form and performance, and one-to-many relationships between functionally versatile morphologies and ecology. On complex adaptive landscapes, ecological selection can yield different outcomes. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

  15. Solvent-mediated pseudo-quadruple hydrogen-bond motifs in three lamotrigine-carboxylic acid complexes.

    Science.gov (United States)

    Sridhar, Balasubramanian; Nanubolu, Jagadeesh Babu; Ravikumar, Krishnan

    2013-10-01

    Lamotrigine, an antiepileptic drug, has been complexed with three aromatic carboxylic acids. All three compounds crystallize with the inclusion of N,N-dimethylformamide (DMF) solvent, viz. lamotriginium [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazin-2-ium] 4-iodobenzoate N,N-dimethylformamide monosolvate, C9H8Cl2N5(+)·C7H4IO2(-)·C3H7NO, (I), lamotriginium 4-methylbenzoate N,N-dimethylformamide monosolvate, C9H7Cl2N5(+)·C8H8O2(-)·C3H7NO, (II), and lamotriginium 3,5-dinitro-2-hydroxybenzoate N,N-dimethylformamide monosolvate, C9H8Cl2N5(+)·C7H3N2O7(-)·C3H7NO, (III). In all three structures, proton transfer takes place from the acid to the lamotrigine molecule. However, in (I) and (II), the acidic H atom is disordered over two sites and there is only partial transfer of the H atom from O to N. In (III), the corresponding H atom is ordered and complete proton transfer has occurred. Lamotrigine-lamotrigine, lamotrigine-acid and lamotrigine-solvent interactions are observed in all three structures and they thereby exhibit isostructurality. The DMF solvent extends the lamotrigine-lamotrigine dimers into a pseudo-quadruple hydrogen-bonding motif.

  16. Histidine availability is decisive in ROS-mediated cytotoxicity of copper complexes of Aβ1-16 peptide.

    Science.gov (United States)

    Ginotra, Yamini P; Ramteke, Shefali N; Walke, Gulshan R; Rapole, Srikanth; Kulkarni, Prasad P

    2016-01-01

    The binding of metal ions to Aβ peptide plays an important role in the etiology of AD. Copper coordinates chiefly to His residues and produces reactive oxygen species (ROS) upon redox cycling. ROS builds enormous burden on the normal functioning of neuronal cells and results into deleterious effects. Recently, two structurally distinct copper binding sites with contrasting redox properties were characterized. Here, we demonstrate for the first time the effect of binding of two equivalents of Cu(2+) on redox properties and cytotoxicity of Aβ peptide. Our electrochemical data and ascorbate consumption assay suggest that in the presence of two equivalents of copper; Aβ peptide has higher propensity of H2O2 generation. The oxidation of Aβ1-16 peptide due to both gamma radiolysis and metal catalyzed oxidation in the presence of two equivalents of copper is inhibited confirming the binding of both equivalents of copper to peptide. The electrochemical and cytotoxicity study shows that negative shift in the reduction potential is reflected as slightly higher cytotoxicity in SH-SY5Y cell lines for Aβ1-16-Cu(2+) (1:2) complex.

  17. Dysphagia-gastroesophageal reflux complex: complications due to dysfunction of solitary tract nucleus-mediated vago-vagal reflex.

    Science.gov (United States)

    Saito, Y; Kawashima, Y; Kondo, A; Chikumaru, Y; Matsui, A; Nagata, I; Ohno, K

    2006-06-01

    We report on the complication of gastroesophageal reflux (GER) in four patients with lower brainstem dysfunction. These patients suffered from perinatal asphyxia, cerebellar hemorrhage, or congenital dysphagia of unknown origin and showed facial nerve palsy, inspiratory stridor due to vocal cord paralysis, central sleep apnea, and dysphagia, in various combinations. Naso-intestinal tube feeding was introduced in all of the patients due to recurrent vomiting and aspiration pneumonia resulting from GER. T2-weighted magnetic resonance (MR) imaging revealed symmetrical high intensity lesions in the tegmentum of the lower pons and the medulla oblongata in two of the patients, and pontomedullary atrophy in another patient. In normal subjects, lower esophageal sphincter contraction is provoked by distension of the gastric wall, through a vago-vagal reflex. Since this reflex arc involves the solitary tract nucleus, where the swallowing center is located, the association of dysphagia and GER in the present patients is thought to result from the lesions in the tegmentum of medulla oblongata. We propose the term "dysphagia-GER complex" to describe the disturbed motility of the upper digestive tract due to lower brainstem involvement. In children with brainstem lesions, neurological assessment of GER is warranted, in addition to the examination of other signs of brainstem dysfunction, including dysphagia and respiratory disturbance.

  18. Rmi1 functions in S phase-mediated cohesion establishment via a pathway involving the Ctf18-RFC complex and Mrc1.

    Science.gov (United States)

    Lai, Mong Sing; Seki, Masayuki; Tada, Shusuke; Enomoto, Takemi

    2012-10-26

    Saccharomyces cerevisiae RecQ helicase (Sgs1) combines with DNA topoisomerase III (Top3) and RecQ-mediated genome instability 1 (Rmi1) to form an evolutionarily conserved complex that is required for processing homologous recombination intermediates and restarting collapsed replication forks. It was previously reported that Rmi1 contributes to sister chromatid cohesion; however, the underlying molecular mechanism has been unclear. In the present study, Rmi1 was found to be enriched at the region close to an early-firing replication origin when replication forks were arrested near their origins in the presence of hydroxyurea. Genetic analyses revealed that Rmi1 promoted sister chromatid cohesion in a process that was distinct from both the cohesion establishment pathway involving Ctf4, Csm3, and Chl1 and the pathway involving the acetylation of Smc3. On the other hand, Rmi1 seemed to function in a pathway involving the Ctf18-RFC complex and Mrc1, which were previously predicted to regulate leading-strand DNA replication.

  19. Magnetism mediated by a majority of [Fe³⁺ + VO²⁻] complexes in Fe-doped CeO₂ nanoparticles.

    Science.gov (United States)

    Paidi, V K; Ferreira, N S; Goltz, D; van Lierop, J

    2015-08-26

    We examine the role of Fe(3+) and vacancies (V(O)) on the magnetism of Fe-doped CeO2 nanoparticles. Magnetic nanoparticles of Ce(100-x)Fe(x)O2 (x  =  0, 0.26, 1.82, 2.64, 5.26, 6.91, and 7.22) were prepared by a co-precipitation method, and their structural, compositional and magnetic properties were investigated. The CeO2 nanoparticles had a mixed valance of Ce(4+) and Ce(3+) ions, and doping introduced Fe(3+) ions. The decrease in Ce(3+) and increase in Fe(3+) concentrations indicated the presence of more [Fe(3+) + V(O)(2-)] complexes with Fe loading in the particles. Charge neutralization, Fe(3+) + V(O)(2-) + 2Ce(4+) ↔ 2Ce(3+) + Fe(3+), identified the impact of V(O) on the magnetism, where our results suggest that the Fe-doped CeO2 nanoparticle magnetism is mediated by a majority of [Fe(3+) + V(O)(2-)]-Ce(3+) -[Fe(3+) + V(O)(2-)] complexes.

  20. Antibody repertoire complexity and effector cell biology determined by assays for IgE-mediated basophil and T-cell activation.

    Science.gov (United States)

    Lund, Gitte; Willumsen, Nicholas; Holm, Jens; Christensen, Lars Harder; Würtzen, Peter Adler; Lund, Kaare

    2012-09-28

    Effector cell activation and T-cell activation, the latter mediated by facilitated antigen presentation, are immunological mechanisms that play crucial roles in the manifestation and maintenance of allergic disease. In addition to their relevance for the pathogenesis of allergy in-vivo, in-vitro assays based on these immunological mechanisms have been established and used for diagnostics, for monitoring the progression of disease and for the effect of specific immunotherapy as well as for basic research purposes. Here we review different parameters that affect effector cell activation and facilitated antigen uptake and presentation, including assay designs, readout parameters and critical experimental conditions. Central to the two immunological mechanisms is complex formation between allergen-specific IgE, allergen, and cell surface-anchored immunoglobulin receptor; the high affinity IgE-receptor FcεRI on basophils and mast cells, and the low affinity IgE-receptor FcεRII (CD23) on B-cells. Accordingly, the effect of IgE repertoire complexity and allergen diversity on effector cell and facilitated antigen presentation is discussed in detail.

  1. Dual-channel detection of metallothioneins and mercury based on a mercury-mediated aptamer beacon using thymidine-mercury-thymidine complex as a quencher.

    Science.gov (United States)

    Chen, Si-Han; Wang, Yong-Sheng; Chen, Yun-Sheng; Tang, Xian; Cao, Jin-Xiu; Li, Ming-Hui; Wang, Xiao-Feng; Zhu, Yu-Feng; Huang, Yan-Qin

    2015-01-01

    A novel dual-channel strategy for the detection of metallothioneins (MTs) and Hg(2+) has been developed based on a mercury-mediated aptamer beacon (MAB) using thymidine-mercury-thymidine complex as a quencher for the first time. In the presence of Hg(2+), the T-rich oligonucleotide with a 6-carboxyfluorescein (TRO-FAM) can form an aptamer beacon via the formation of T-Hg(2+)-T base pairs, which results in a fluorescence quenching of the sensing system owing to the fluorescence resonance energy transfer (FRET) from the fluorophore of FAM to the terminated T-Hg(2+)-T base pair. The addition of MTs into this solution leads to the disruption of the T-Hg(2+)-T complex, resulting in an increase of the fluorescent signal of the system. In the optimizing condition, ΔF was directly proportional to the concentrations ranging from 5.63 nM to 0.275 μM for MTs, and 14.2 nM to 0.30 μM for Hg(2+) with the detection limits of 1.69 nM and 4.28 nM, respectively. The proposed dual-channel method avoids the label steps of a quencher in common molecular beacon strategies, without tedious procedure or the requirement of sophisticated equipment, and is rapid, inexpensive and sensitive.

  2. Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

    Science.gov (United States)

    Lacruz, Rodrigo S; Brookes, Steven J; Wen, Xin; Jimenez, Jaime M; Vikman, Susanna; Hu, Ping; White, Shane N; Lyngstadaas, S Petter; Okamoto, Curtis T; Smith, Charles E; Paine, Michael L

    2013-03-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data

  3. Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Wei, E-mail: hongwei@tijmu.edu.cn [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Li, Jinru; Wang, Bo [College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Chen, Linfeng [Department of Medical Oncology, Harvard Medical School, Dana Farber Cancer Institute, Boston, 02115 MA (United States); Niu, Wenyan; Yao, Zhi [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); Baniahmad, Aria, E-mail: aban@mti.uni-jena.de [Institute for Human Genetics, Jena University Hospital, 07740 Jena (Germany)

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by which Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

  4. Electrophilic Pt(II) complexes: precision instruments for the initiation of transformations mediated by the cation-olefin reaction.

    Science.gov (United States)

    Felix, Ryan J; Munro-Leighton, Colleen; Gagné, Michel R

    2014-08-19

    A discontinuity exists between the importance of the cation-olefin reaction as the principal C-C bond forming reaction in terpene biosynthesis and the synthetic tools for mimicking this reaction under catalyst control; that is, having the product identity, stereochemistry, and functionality under the control of a catalyst. The main reason for this deficiency is that the cation-olefin reaction starts with a reactive intermediate (a carbocation) that reacts exothermically with an alkene to reform the reactive intermediate; not to mention that reactive intermediates can also react in nonproductive fashions. In this Account, we detail our efforts to realize catalyst control over this most fundamental of reactions and thereby access steroid like compounds. Our story is organized around our progress in each component of the cascade reaction: the metal controlled electrophilic initiation, the propagation and termination of the cyclization (the cyclase phase), and the turnover deplatinating events. Electrophilic Pt(II) complexes efficiently initiate the cation-olefin reaction by first coordinating to the alkene with selection rules that favor less substituted alkenes over more substituted alkenes. In complex substrates with multiple alkenes, this preference ensures that the least substituted alkene is always the better ligand for the Pt(II) initiator, and consequently the site at which all electrophilic chemistry is initiated. This control element is invariant. With a suitably electron deficient ligand set, the catalyst then activates the coordinated alkene to intramolecular addition by a second alkene, which initiates the cation-olefin reaction cascade and generates an organometallic Pt(II)-alkyl. Deplatination by a range of mechanisms (β-H elimination, single electron oxidation, two-electron oxidation, etc.) provides an additional level of control that ultimately enables A-ring functionalizations that are orthogonal to the cyclase cascade. We particularly focus on

  5. The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Jordi Solana

    Full Text Available Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

  6. The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

    Science.gov (United States)

    Solana, Jordi; Gamberi, Chiara; Mihaylova, Yuliana; Grosswendt, Stefanie; Chen, Chen; Lasko, Paul; Rajewsky, Nikolaus; Aboobaker, A Aziz

    2013-01-01

    Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

  7. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

    Science.gov (United States)

    Guillonneau, Maëva; Paris, François; Dutoit, Soizic; Estephan, Hala; Bénéteau, Elise; Huot, Jacques; Corre, Isabelle

    2016-08-01

    Oxidative stress is a leading cause of endothelial dysfunction. The p38 MAPK pathway plays a determinant role in allowing cells to cope with oxidative stress and is tightly regulated by a balanced interaction between p38 protein and its interacting partners. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in HUVECs. Coimmunoprecipitation and microscopic analyses confirmed the existence of a cytosolic nucleophosmin (NPM)/p38 interaction in basal condition. Oxidative stress, which was generated by exposure to 500 µM H2O2, induces a rapid dephosphorylation of NPM at T199 that depends on phosphatase PP2A, another partner of the NPM/p38 complex. Blocking PP2A activity leads to accumulation of NPM-pT199 and to an increased association of NPM with p38. Concomitantly to its dephosphorylation, oxidative stress promotes translocation of NPM to the nucleus to affect the DNA damage response. Dephosphorylated NPM impairs the signaling of oxidative stress-induced DNA damage via inhibition of the phosphorylation of ataxia-telangiectasia mutated and DNA-dependent protein kinase catalytic subunit. Overall, these results suggest that the p38/NPM/PP2A complex acts as a dynamic sensor, allowing endothelial cells to react rapidly to acute oxidative stress.-Guillonneau, M., Paris, F., Dutoit, S., Estephan, H., Bénéteau, E., Huot, J., Corre, I. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.

  8. Mn(2+)-mediated homogeneous Fenton-like reaction of Fe(III)-NTA complex for efficient degradation of organic contaminants under neutral conditions.

    Science.gov (United States)

    Li, Yifan; Sun, Jianhui; Sun, Sheng-Peng

    2016-08-05

    In this work, we report a novel Mn(2+)-mediated Fenton-like process based on Fe(III)-NTA complex that is super-efficient at circumneutral pH range. Kinetics experiments showed that the presence of Mn(2+) significantly enhanced the effectiveness of Fe(III)-NTA complex catalyzed Fenton-like reaction. The degradation rate constant of crotamiton (CRMT), a model compound, by the Fe(III)- NTA_Mn(2+) Fenton-like process was at least 1.6 orders of magnitude larger than that in the absence of Mn(2+). Other metal ions such as Ca(2+), Mg(2+), Co(2+) and Cu(2+) had no impacts or little inhibitory effect on the Fe(III)-NTA complex catalyzed Fenton-like reaction. The generation of hydroxyl radical (HO) and superoxide radical anion (O2(-)) in the Fe(III)-NTA_Mn(2+) Fenton-like process were suggested by radicals scavenging experiments. The degradation efficiency of CRMT was inhibited significantly (approximately 92%) by the addition of HO scavenger 2-propanol, while the addition of O2(-) scavenger chloroform resulted in 68% inhibition. Moreover, the results showed that other chelating agents such as EDTA- and s,s-EDDS-Fe(III) catalyzed Fenton-like reactions were also enhanced significantly by the presence of Mn(2+). The mechanism involves an enhanced generation of O2(-) from the reactions of Mn(2+)-chelates with H2O2, indirectly promoting the generation of HO by accelerating the reduction rate of Fe(III)-chelates to Fe(II)- chelates. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Nanosilver-penetrated polyion graphene complex membrane for mediator-free amperometric immunoassay of alpha-fetoprotein using nanosilver-coated silica nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Tang Juan [Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108 (China); Tang Dianping, E-mail: dianping.tang@fzu.edu.c [Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108 (China); Su Biling; Li Qunfang; Qiu Bin [Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108 (China); Chen Guonan, E-mail: gnchen@fzu.edu.c [Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108 (China)

    2011-04-15

    Research highlights: {yields} We fabricate a polyion graphene complex membrane-based immunosensing platform for sensitive electrochemical immunoassay of alpha-fetoprotein. {yields} Nanosilver-coated silica nanocomposites as bionanolabels. {yields} Graphene nanosheets, single-stranded DNA and silver nanoparticles as matrices. {yields} Direct electron transfer without electron mediator. {yields} Analysis of real samples and method comparison. - Abstract: A facile and sensitive mediator-free electrochemical immunosensor for detection of alpha-fetoprotein (AFP) was designed by using nanosilver-coated silica nanoparticles (Ag-SiO{sub 2}) as bionanolabels. To construct such an electrochemical immunosensor, silver ions/single-stranded DNA/graphene nanosheets were initially immobilized on a gold electrode in turn, then silver ions were in situ reduced to silver nanoparticles with the aid of NaBH{sub 4}, and anti-AFP antibodies conjugated to silver nanoparticles were used. In the presence of AFP analyte, the sandwiched immunocomplex was formed on the electrode surface by using horseradish peroxidase-anti-AFP conjugate-labeled Ag-SiO{sub 2} (HRP-anti-AFP-Ag-SiO{sub 2}) as secondary antibodies. Compared with pure silver nanoparticles, Ag-SiO{sub 2} nanocomposites could provide a large room for the immobilization of HRP-anti-AFP, and improve the electrochemical responses of the immunosensor. Meanwhile, the presence of highly conductive graphene nanosheets and silver nanoparticles provided a good pathway for electron transfer. Under optimal conditions, the immunosensor exhibited good electrochemical responses toward AFP ranging from 0.3 to 200 ng/mL with a detection limit (LOD) of 0.05 ng/mL (at 3{sigma}) in pH 6.0 PBS-H{sub 2}O{sub 2} system. Intra- and inter-assay displayed good precisions with coefficient of variation below 9.5%. In addition, the method was evaluated with 23 clinical serum samples, receiving good correlation with results from commercially available

  10. TPR domain of NrfG mediates complex formation between heme lyase and formate-dependent nitrite reductase in Escherichia coli O157:H7.

    Science.gov (United States)

    Han, Dohyun; Kim, Kyunggon; Oh, Jongkil; Park, Jungeun; Kim, Youngsoo

    2008-02-15

    Escherichia coli synthesize C-type cytochromes only during anaerobic growth in media supplemented with nitrate and nitrite. The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two separate periplasmic enzymes, nitrate reductase and nitrite reductase. The nitrite reductase involved, NrfA, contains cytochrome C and is synthesized coordinately with a membrane-associated cytochrome C, NrfB, during growth in the presence of nitrite or in limiting nitrate concentrations. The genes NrfE, NrfF, and NrfG are required for the formate-dependent nitrite reduction pathway, which involves at least two C-type cytochrome proteins, NrfA and NrfB. The NrfE, NrfF, and NrfG genes (heme lyase complex) are involved in the maturation of a special C-type cytochrome, apocytochrome C (apoNrfA), to cytochrome C (NrfA) by transferring a heme to the unusual heme binding motif of the Cys-Trp-Ser-Cys-Lys sequence in apoNrfA protein. Thus, in order to further investigate the roles of NrfG in the formation of heme lyase complex (NrfEFG) and in the interaction between heme lyase complex and formate-dependent nitrite reductase (NrfA), we determined the crystal structure of NrfG at 2.05 A. The structure of NrfG showed that the contact between heme lyase complex (NrfEFG) and NrfA is accomplished via a TPR domain in NrfG which serves as a binding site for the C-terminal motif of NrfA. The portion of NrfA that binds to TPR domain of NrfG has a unique secondary motif, a helix followed by about a six-residue C-terminal loop (the so called "hook conformation"). This study allows us to better understand the mechanism of special C-type cytochrome assembly during the maturation of formate-dependent nitrite reductase, and also adds a new TPR binding conformation to the list of TPR-mediated protein-protein interactions.

  11. Inhibitory effect of 1,2,4,5-tetramethoxybenzene on mast cell-mediated allergic inflammation through suppression of IκB kinase complex

    Energy Technology Data Exchange (ETDEWEB)

    Je, In-Gyu [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Choi, Hyun Gyu [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Kim, Hui-Hun; Lee, Soyoung; Choi, Jin Kyeong [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, Sung-Wan; Kim, Duk-Sil [Department of Thoracic and Cardiovascular Surgery, CHA Gumi Medical Center, CHA University, Gumi 730-040 (Korea, Republic of); Kwon, Taeg Kyu [Department of Immunology, School of Medicine, Keimyung University, Daegu 704-701 (Korea, Republic of); Shin, Tae-Yong [College of Pharmacy, Woosuk University, Jeonju 565-701 (Korea, Republic of); Park, Pil-Hoon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Khang, Dongwoo, E-mail: dkhang@gachon.ac.kr [Department of Molecular Medicine, School of Medicine, Gachon University, Incheon 406-840 (Korea, Republic of); Kim, Sang-Hyun, E-mail: shkim72@knu.ac.kr [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    2015-09-01

    As the importance of allergic disorders such as atopic dermatitis and allergic asthma, research on potential drug candidates becomes more necessary. Mast cells play an important role as initiators of allergic responses through the release of histamine; therefore, they should be the target of pharmaceutical development for the management of allergic inflammation. In our previous study, anti-allergic effect of extracts of Amomum xanthioides was demonstrated. To further investigate improved candidates, 1,2,4,5-tetramethoxybenzene (TMB) was isolated from methanol extracts of A. xanthioides. TMB dose-dependently attenuated the degranulation of mast cells without cytotoxicity by inhibiting calcium influx. TMB decreased the expression of pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin (IL)-4 at both the transcriptional and translational levels. Increased expression of these cytokines was caused by translocation of nuclear factor-κB into the nucleus, and it was hindered by suppressing activation of IκB kinase complex. To confirm the effect of TMB in vivo, the ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) and IgE-mediated passive cutaneous anaphylaxis (PCA) models were used. In the ASA model, hypothermia was decreased by oral administration of TMB, which attenuated serum histamine, OVA-specific IgE, and IL-4 levels. Increased pigmentation of Evans blue was reduced by TMB in a dose-dependent manner in the PCA model. Our results suggest that TMB is a possible therapeutic candidate for allergic inflammatory diseases that acts through the inhibition of mast cell degranulation and expression of pro-inflammatory cytokines. - Highlights: • TMB reduced the degranulation of mast cells. • TMB inhibited the production of pro-inflammatory cytokines. • TMB suppressed both active and passive anaphylaxis. • Anti-allergic inflammatory effects of TMB might be due to the blocking IKK complex. • TMB might be a candidate for the treatment of

  12. Localization of nucleoporin Tpr to the nuclear pore complex is essential for Tpr mediated regulation of the export of unspliced RNA.

    Science.gov (United States)

    Rajanala, Kalpana; Nandicoori, Vinay Kumar

    2012-01-01

    Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts.

  13. BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in arabidopsis.

    Directory of Open Access Journals (Sweden)

    Rafal Archacki

    Full Text Available SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.

  14. Phosphorylation of the PDH complex precedes HIF-1-mediated effects and PDK1 upregulation during the first hours of hypoxic treatment in HCC cells

    Directory of Open Access Journals (Sweden)

    Zimmer AD

    2016-08-01

    Full Text Available Andreas David Zimmer, Geoffroy Walbrecq, Ines Kozar, Iris Behrmann, Claude Haan Life Sciences Research Unit, University of Luxembourg, Belvaux, Luxembourg Abstract: The pyruvate dehydrogenase complex (PDC is an important gatekeeper enzyme connecting glycolysis to the tricarboxylic acid (TCA cycle and oxidative phosphorylation (OXPHOS. Thereby, it has a strong impact on the glycolytic flux as well as the metabolic phenotype of a cell. PDC activity is regulated via reversible phosphorylation of three serine residues on the pyruvate dehydrogenase (PDH E1α subunit. Phosphorylation of any of these residues by the PDH kinases (PDKs leads to a strong decrease in PDC activity. Under hypoxia, the inactivation of the PDC has been described to be dependent on the hypoxia-inducible factor 1 (HIF-1-induced PDK1 protein upregulation. In this study, we show in two hepatocellular carcinoma cell lines (HepG2 and JHH-4 that, during the adaptation to hypoxia, PDH is already phosphorylated at time points preceding HIF-1-mediated transcriptional events and PDK1 protein upregulation. Using siRNAs and small molecule inhibitor approaches, we show that this inactivation of PDC is independent of HIF-1α expression but that the PDKs need to be expressed and active. Furthermore, we show that reactive oxygen species might be important for the induction of this PDH phosphorylation since it correlates with the appearance of an altered redox state in the mitochondria and is also inducible by H2O2 treatment under normoxic conditions. Overall, these results show that neither HIF-1 expression nor PDK1 upregulation is necessary for the phosphorylation of PDH during the first hours of the adaptation to hypoxia. Keywords: pyruvate dehydrogenase complex, pyruvate dehydrogenase kinase, hypoxia metabolism, glycolytic switch, radical oxygen species

  15. Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells.

    Science.gov (United States)

    Leclerc, Denis; Beauseigle, Diane; Denis, Jérome; Morin, Hélène; Paré, Christine; Lamarre, Alain; Lapointe, Réjean

    2007-02-01

    The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-T-lymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201+ antigen-presenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8+ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigen-specific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I cross-presentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.

  16. The bHLH factors Dpn and members of the E(spl complex mediate the function of Notch signalling regulating cell proliferation during wing disc development

    Directory of Open Access Journals (Sweden)

    Beatriz P. San Juan

    2012-05-01

    The Notch signalling pathway plays an essential role in the intricate control of cell proliferation and pattern formation in many organs during animal development. In addition, mutations in most members of this pathway are well characterized and frequently lead to tumour formation. The Drosophila imaginal wing discs have provided a suitable model system for the genetic and molecular analysis of the different pathway functions. During disc development, Notch signalling at the presumptive wing margin is necessary for the restricted activation of genes required for pattern formation control and disc proliferation. Interestingly, in different cellular contexts within the wing disc, Notch can either promote cell proliferation or can block the G1-S transition by negatively regulating the expression of dmyc and bantam micro RNA. The target genes of Notch signalling that are required for these functions have not been identified. Here, we show that the Hes vertebrate homolog, deadpan (dpn, and the Enhancer-of-split complex (E(splC genes act redundantly and cooperatively to mediate the Notch signalling function regulating cell proliferation during wing disc development.

  17. Hume-Rothery stabilisation mechanism and d-states-mediated Fermi surface-Brillouin zone interactions in structurally complex metallic alloys

    Science.gov (United States)

    Mizutani, U.; Inukai, M.; Sato, H.

    2011-07-01

    The stability of Co2Zn11 and Al8V5 gamma-brasses, both of which are composed of a transition metal element and polyvalent elements Zn or Al, can be discussed in terms of d-states-mediated Fermi surface-Brillouin zone (FsBz) interactions in the context of first-principles full-potential linearised augmented plane wave (FLAPW) band calculations. A FsBz-induced pseudogap is revealed in the FLAPW-Fourier spectrum, though it is hidden behind a much larger d-band in the total density of states. The stability range of three families of complex metallic alloys (CMAs) that include gamma-brasses, RT-, MI- and Tsai-type 1/1-1/1-1/1 approximants and 2/1-2/1-2/1 approximant, each of which is characterised by ? = 18, 50 and 125, respectively, can be well scaled in terms of the number of electrons per unit cell (e/uc) given by the product of the number of atoms per unit cell and the e/a value determined by the Hume-Rothery plot on the basis of the FLAPW-Fourier method. This is taken as the evidence for the justification of the Hume-Rothery stabilisation mechanism for all these CMAs having a pseudogap at the Fermi level.

  18. Synergistic inhibitory effect of nicotine plus oral contraceptive on mitochondrial complex-IV is mediated by estrogen receptor-β in female rats.

    Science.gov (United States)

    Raval, Ami P; Dave, Kunjan R; Saul, Isabel; Gonzalez, Gabriel J; Diaz, Francisca

    2012-04-01

    Chronic nicotine and oral contraceptive (NOC) exposure caused significant loss of hippocampal membrane-bound estrogen receptor-beta (ER-β) in female rats compared with exposure to nicotine alone. Mitochondrial ER-β regulates estrogen-mediated mitochondrial structure and function; therefore, investigating the impact of NOC on mitochondrial ER-β and its function could help delineate the harmful synergism between nicotine and OC. In this study, we tested the hypothesis that NOC-induced loss of mitochondrial ER-β alters the oxidative phosphorylation system protein levels and mitochondrial respiratory function. This hypothesis was tested in hippocampal mitochondria isolated from female rats exposed to saline, nicotine, OC or NOC for 16 days. NOC decreased the mitochondrial ER-β protein levels and reduced oxygen consumption and complex IV (CIV) activity by 34% and 26% compared with saline- or nicotine-administered groups, respectively. We also observed significantly low protein levels of all mitochondrial-encoded CIV subunits after NOC as compared with the nicotine or saline groups. Similarly, the silencing of ER-β reduced the phosphorylation of cyclic-AMP response element binding protein, and also reduced levels of CIV mitochondrial-encoded subunits after estrogen stimulation. Overall, these results suggest that mitochondrial ER-β loss is responsible for mitochondrial malfunction after NOC.

  19. The Schizosaccharomyces pombe Mediator

    DEFF Research Database (Denmark)

    Venturi, Michela

    In the past several years great attention has been dedicated to the characterization of the Mediator complex in a different range of model organisms. Mediator is a conserved co-activator complex involved in transcriptional regulation and it conveys signals from regulatory transcription factors......+ is a nonessential gene, while deletion of med11+ resulted in unviable cells. These results are in line with those obtained in S. cerevisiae. Isolation of S. pombe Mediator by the tandem affinity purification method and Co-IP experiments lead to the conclusion that Med9 and Med11 might not belong to the Mediator...... complex, but our results did not exclude it completely either. Our attempts to demonstrate the presence of these two subunits in the Mediator complex remain inconclusive primarily due to the lack of proper expression of the tagged versions of the proteins. However, we have paved a way to further...

  20. The Schizosaccharomyces pombe Mediator

    DEFF Research Database (Denmark)

    Venturi, Michela

    , Schizosaccharomyces pombe and mammalian Mediator. In our study, we have taken the S. pombe Mediator into consideration and characterized genetically and biochemically two subunits already know in S. cerevisiae, Med9 and Med11, but still not identified in the S. pombe Mediator. Genetic analysis has shown that med9...... complex, but our results did not exclude it completely either. Our attempts to demonstrate the presence of these two subunits in the Mediator complex remain inconclusive primarily due to the lack of proper expression of the tagged versions of the proteins. However, we have paved a way to further...

  1. Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

    Science.gov (United States)

    Quinn, Sierra N; Graves, Sarai H; Dains-McGahee, Clayton; Friedman, Emilee M; Hassan, Humma; Witkowski, Piotr; Sabbatini, Maria E

    2017-04-01

    Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc.

  2. PAH-CALUX, an optimized bioassay for AhR-mediated hazard identification of polycyclic aromatic hydrocarbons (PAHs) as individual compounds and in complex mixtures.

    Science.gov (United States)

    Pieterse, B; Felzel, E; Winter, R; van der Burg, B; Brouwer, A

    2013-10-15

    Polycyclic aromatic hydrocarbons (PAHs) represent a class of ubiquitously occurring environmental compounds that are implicated in a wide range of toxicological effects. Routine measurement of PAH contamination generally involves chemical analytical analysis of a selected group of representatives, for example, EPA-16, which may result in underestimation of the PAH-related toxicity of a sample. Many high molecular weight PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a nuclear receptor that mediates toxic effects related to these compounds. Making use of this property we developed a PAH CALUX assay, a mammalian, H4IIe- cell-based reporter assay for the hazard identification of total PAH mixtures. The PAH CALUX reporter cell line allows for specific, rapid (4 h exposure time) and reliable quantification of AhR-induced luciferase induction relative to benzo[a]pyrene (BaP), which is used as a positive reference PAH congener. Full dose response relationships with inductions over 100-fold were reached within only 2 h of exposure to BaP. The PAH CALUX is highly sensitive, that is, using a 4 h exposure time, a limit of detection (LOD) of 5.2 × 10(-11) M BaP was achieved, and highly accurate, that is, a repeatability of 5.9% and a reproducibility of 6.6% were established. Screening of a selection of PAHs that were prioritized by the European Union and/or the U.S. Environmental Protection Agency showed that the PAH CALUX bioassay has a high predictability, particularly for carcinogenic PAHs. Experiments with synthetic mixtures and reference materials containing complex PAH mixtures show the suitability of the assay for these types of applications. Moreover, the presented results suggest that application of the PAH CALUX will result in a lower risk of underestimation of the toxicity of a sample than chemical analytical approaches that focus on a limited set of prioritized compounds.

  3. Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway.

    Science.gov (United States)

    Hsu, Shih-Che; Huang, Shih-Ming; Chen, Ann; Sun, Chiao-Yin; Lin, Shih-Hua; Chen, Jin-Shuen; Liu, Shu-Ting; Hsu, Yu-Juei

    2014-08-01

    The Klotho gene functions as an aging suppressor gene. Evidence from animal models suggests that induction of Klotho expression may be a potential treatment for age-associated diseases. However, the molecular mechanism involved in regulating renal Klotho gene expression remains unclear. In this study, we determined that resveratrol, a natural polyphenol, induced renal Klotho expression both in vivo and in vitro. In the mouse kidney, resveratrol administration markedly increased both Klotho mRNA and protein expression. In resveratrol-treated NRK-52E cells, increased Klotho expression was accompanied by the upregulation and nuclear translocation of activating transcription factor 3 (ATF3) and c-Jun. ATF3 or c-Jun overexpression enhanced the transcriptional activation of Klotho. Conversely, resveratrol-induced Klotho expression was attenuated in the presence of dominant-negative ATF3 or c-Jun. Coimmunoprecipitation and a chromatin immunoprecipitation assay revealed that ATF3 physically interacted with c-Jun and that the ATF3/c-Jun complex directly bound to the Klotho promoter through ATF3- and AP-1-binding elements. c-Jun cotransfection augmented the effects of ATF3 on Klotho transcription in vitro. Although Sirtuin 1 mRNA expression was induced by resveratrol and involved in regulating Klotho mRNA expression, it was not the primary cause for the aforementioned ATF3/c-Jun pathway. In summary, resveratrol enhances the renal expression of the anti-aging Klotho gene, and the transcriptional factors ATF3 and c-Jun functionally interact and coordinately regulate the resveratrol-mediated transcriptional activation of Klotho.

  4. The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.

    Directory of Open Access Journals (Sweden)

    Simon A Johnston

    Full Text Available The lysis of infected cells by disease-causing microorganisms is an efficient but risky strategy for disseminated infection, as it exposes the pathogen to the full repertoire of the host's immune system. Cryptococcus neoformans is a widespread fungal pathogen that causes a fatal meningitis in HIV and other immunocompromised patients. Following intracellular growth, cryptococci are able to escape their host cells by a non-lytic expulsive mechanism that may contribute to the invasion of the central nervous system. Non-lytic escape is also exhibited by some bacterial pathogens and is likely to facilitate long-term avoidance of the host immune system during latency. Here we show that phagosomes containing intracellular cryptococci undergo repeated cycles of actin polymerisation. These actin 'flashes' occur in both murine and human macrophages and are dependent on classical WASP-Arp2/3 complex mediated actin filament nucleation. Three dimensional confocal imaging time lapse revealed that such flashes are highly dynamic actin cages that form around the phagosome. Using fluorescent dextran as a phagosome membrane integrity probe, we find that the non-lytic expulsion of Cryptococcus occurs through fusion of the phagosome and plasma membranes and that, prior to expulsion, 95% of phagosomes become permeabilised, an event that is immediately followed by an actin flash. By using pharmacological agents to modulate both actin dynamics and upstream signalling events, we show that flash occurrence is inversely related to cryptococcal expulsion, suggesting that flashes may act to temporarily inhibit expulsion from infected phagocytes. In conclusion, our data reveal the existence of a novel actin-dependent process on phagosomes containing cryptococci that acts as a potential block to expulsion of Cryptococcus and may have significant implications for the dissemination of, and CNS invasion by, this organism.

  5. An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription.

    Directory of Open Access Journals (Sweden)

    Reza Djavadian

    2016-06-01

    Full Text Available Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit "true" late kinetics-their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these "βγ" genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.

  6. Kaposi's sarcoma-associated herpesvirus Lana-1 is a major activator of the serum response element and mitogen-activated protein kinase pathways via interactions with the Mediator complex.

    Science.gov (United States)

    Roupelieva, Maria; Griffiths, Samantha J; Kremmer, Elisabeth; Meisterernst, Michael; Viejo-Borbolla, Abel; Schulz, Thomas; Haas, Jürgen

    2010-05-01

    In cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the activation of mitogen-activated protein kinase (MAPK) pathways plays a crucial role early after virus infection as well as during reactivation. In order to systematically identify viral proteins activating MAPK pathways in KSHV-infected cells, a clone collection of KSHV open reading frames (ORFs) was screened for induction of the serum response element (SRE), as SRE is induced by MAPKs. The strongest induction of the SRE was found with ORF73 (latency-associated nuclear antigen 1, or Lana-1), although weaker activation was also found with the kaposin B isoform, ORF54 (dUTPase) and ORF74 (G-protein-coupled receptor). The bipartite SRE is bound by a ternary complex consisting of serum response factor (SRF) and ternary complex factor. Lana-1 bound directly to SRF, but also to the MED25 (ARC92/ACID-1), MED15 (PCQAP) and MED23 (Sur-2) subunits of the Mediator complex, a multi-subunit transcriptional co-activator complex for RNA polymerase II. Lana-1-induced SRE activation was inhibited by the dominant-negative N-terminal domain of the MED25 mediator subunit, suggesting that this subunit mediates Lana-1-induced SRE activation. In summary, these data suggest a model in which Lana-1 acts as an adaptor between the transcription factor SRF and the basal transcriptional machinery.

  7. Meta-metallation of N,N-dimethylaniline: Contrasting direct sodium-mediated zincation with indirect sodiation-dialkylzinc co-complexation

    Directory of Open Access Journals (Sweden)

    David R. Armstrong

    2011-09-01

    Full Text Available Previously we reported that direct zincation of N,N-dimethylaniline by the mixed-metal zincate reagent 1 ((TMEDANa(TMP(t-BuZn(t-Bu surprisingly led to meta-metallation (zincation of the aniline, as manifested in the crystalline complex 2 ((TMEDANa(TMP(m-C6H4-NMe2Zn(t-Bu, and that iodination of these isolated crystals produced the meta-isomer N,N-dimethyl-3-iodoaniline quantitatively. Completing the study here we find that treating the reaction solution with iodine produces a 72% conversion and results in a mixture of regioisomers of N,N-dimethyliodoaniline, with the meta-isomer still the major product (ortho:meta:para ratio, 6:73:21, as determined by NMR. In contrast to this bimetallic method, sodiation of N,N-dimethylaniline with n-BuNa produced the dimeric, ortho-sodiated complex 3 (((TMEDANa(o-C6H4-NMe22, as characterised by X-ray crystallography and NMR. No regioisomers were observed in the reaction solution. Introducing t-Bu2Zn to this reaction solution afforded a cocrystalline product in the solid-state, composed of the bis-anilide 4 ((TMEDANa(o-C6H4-NMe22Zn(t-Bu and the Me2N–C cleavage product 5 ({(TMEDA2Na}+{(t-Bu2Zn2(µ-NMe2}−, which was characterised by X-ray crystallography. NMR studies of the reaction mixture that produces 4 and 5 revealed one additional species, but the mixture as a whole contained only ortho-species and a trace amount of para-species as established by iodine quenching. In an indirect variation of the bimetallic reaction, TMP(H was added at room temperature to the reaction mixture that afforded 4 and 5. This gave the crystalline product 6 ((TMEDANa(TMP(o-C6H4-NMe2Zn(t-Bu, the ortho-isomer of the meta-complex 2, as determined from X-ray crystallographic and NMR data. Monitoring the regioselectivity of the reaction by iodination revealed a 16.6:1.6:1.0 ortho:meta:para ratio. Interestingly, when the TMP(H containing solution was heated under reflux for 18 hours more meta-isomer was produced (corresponding ratio 3

  8. Metal-mediated controllable creation of secondary, tertiary, and quaternary carbon centers: a powerful strategy for the synthesis of iron, cobalt, and copper complexes with in situ generated substituted 1-pyridineimidazo[1,5-a]pyridine ligands.

    Science.gov (United States)

    Chen, Yanmei; Li, Lei; Chen, Zhou; Liu, Yonglu; Hu, Hailiang; Chen, Wenqian; Liu, Wei; Li, Yahong; Lei, Tao; Cao, Yanyuan; Kang, Zhenghui; Lin, Miaoshui; Li, Wu

    2012-09-17

    An efficient strategy for the synthesis of a wide variety of coordination complexes has been developed. The synthetic protocol involves a solvothermal in situ metal-ligand reaction of picolinaldehyde, ammonium acetate, and transition-metal ions, leading to the generation of 12 coordination complexes supported by a novel class of substituted 1-pyridineimidazo[1,5-a]pyridine ligands (L1-L5). The ligands L1-L5 were afforded by metal-mediated controllable conversion of the aldehyde group of picolialdehyde into a ketone and secondary, tertiary, and quaternary carbon centers, respectively. Complexes of various nuclearities were obtained: from mono-, di-, and tetranuclear to 1D chain polymers. The structures of the in situ formed complexes could be controlled rationally via the choice of appropriate starting materials and tuning of the ratio of the starting materials. The plausible mechanisms for the formation of the ligands L1-L5 were proposed.

  9. Technology-Use Mediation

    DEFF Research Database (Denmark)

    Bansler, Jørgen P.; Havn, Erling C.

    2003-01-01

    This study analyzes how a group of ‘mediators’ in a large, multinational company adapted a computer-mediated communication technology (a ‘virtual workspace’) to the organizational context (and vice versa) by modifying features of the technology, providing ongoing support for users, and promoting...... of technology-use mediation is more complex and indeterminate than earlier literature suggests. In particular, we want to draw attention to the fact that advanced computer-mediated communication technologies are equivocal and that technology-use mediation consequently requires ongoing sensemaking (Weick 1995)....... appropriate conventions of use. Our findings corroborate earlier research on technology-use mediation, which suggests that such mediators can exert considerable influence on how a particular technology will be established and used in an organization. However, this study also indicates that the process...

  10. Intercultural Mediation

    OpenAIRE

    Dragos Marian Radulescu; Denisa Mitrut

    2012-01-01

    The Intercultural Mediator facilitates exchanges between people of different socio-cultural backgrounds and acts as a bridge between immigrants and national and local associations, health organizations, services and offices in order to foster integration of every single individual. As the use mediation increases, mediators are more likely to be involved in cross-cultural mediation, but only the best mediators have the opportunity to mediate cross border business disputes or international poli...

  11. Highly water-soluble platinum(II) complexes as GLUT substrates for targeted therapy: improved anticancer efficacy and transporter-mediated cytotoxic properties.

    Science.gov (United States)

    Liu, Pengxing; Lu, Yanhui; Gao, Xiangqian; Liu, Ran; Zhang-Negrerie, Daisy; Shi, Ying; Wang, Yiqiang; Wang, Songqing; Gao, Qingzhi

    2013-03-25

    Glucose-conjugated malonato-platinum(II) complexes are designed and synthesized to target tumor-specific active transporters, namely, glucose transporters (GLUTs); the complexes exhibit much higher aqueous solubility by 150 times, improved potency in cytotoxicities by 10 times, and increased therapeutic index by over 30 fold compared to the newest generation of clinical drugs oxaliplatin.

  12. Zinc(II) complexes containing bis-benzimidazole derivatives as a new class of apoptosis inducers that trigger DNA damage-mediated p53 phosphorylation in cancer cells.

    Science.gov (United States)

    Liu, Shenggui; Cao, Wenqiang; Yu, Lianling; Zheng, Wenjie; Li, Linlin; Fan, Cundong; Chen, Tianfeng

    2013-04-28

    In the present study, two zinc(II) complexes containing bis-benzimidazole derivatives, Zn(bpbp)Cl2 (1) and [Zn(bpbp)2](ClO4)2·CH3CH2OH·H2O (2) (bpbp = 2,6-bis(1-phenyl-1H-benzo[d]imidazol-2-yl)pyridine), have been designed, synthesized and evaluated for their in vitro anticancer activities. The underlying molecular mechanisms through which they caused the cancer cell death were also elucidated. The complexes were identified as potent antiproliferative agents against a panel of five human cancer cell lines by comparing with cisplatin. Complex 2 demonstrated dose-dependent growth inhibition on MCF-7 human breast carcinoma cells with IC50 at 2.9 μM. Despite this potency, the complexes possessed great selectivity between human cancer cells and normal cells. Induction of apoptosis in MCF-7 cells by complex 2 was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Further investigation on intracellular mechanisms revealed that complex 2 was able to induce p53-dependent apoptosis in cancer cells by triggering DNA damage. On the basis of this evidence, we suggest that Zn(II) complexes containing bis-benzimidazole derivatives may be candidates for further evaluation as chemotherapeutic agents for human cancers.

  13. Decreased complement mediated binding of antibody//sup 3/-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, R.P.; Horgan, C.; Buschbacher, R.; Brunner, C.M.; Hess, C.E.; O' Brien, W.M.; Wanebo, H.J.

    1983-06-01

    The complement mediated binding of prepared antibody//sup 3/H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

  14. The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

    Science.gov (United States)

    Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik; Sowd, Gregory A; Qian, Juan; Hao, Caili; Dasgupta, Twishasri; Engelman, Alan N; Skowronski, Jacek

    2016-05-27

    HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin.

  15. Music, Radio, and Mediatization

    DEFF Research Database (Denmark)

    Krogh, Mads; Michelsen, Morten

    2016-01-01

    Mediatization has become a key concept for understanding the relations between media and other cultural and social fields. Contributing to the discussions related to the concept of mediatization, this article discusses how practices of radio and music(al life) influence each other. We follow Deacon......’s and Stanyer’s advice to supplement the concept of mediatization with ‘a series of additional concepts at lower levels of abstraction’ and suggest, in this respect, the notion of heterogeneous milieus of music– radio. Hereby, we turn away from the all-encompassing perspectives related to the concept...... of mediatization where media as such seem to be ascribed agency. Instead, we consider historical accounts of music–radio in order to address the complex non- linearity of concrete processes of mediatization as they take place in the multiple meetings between a decentred notion of radio and musical life....

  16. Electrochemical dehalogenation of chlorinated aromatic hydrocarbons with nickel(II) complexes as mediators in methanol; Elektrochemische Enthalogenierung chlorierter Aromaten mittels Nickel(II)-Komplexen als Mediatoren in Methanol

    Energy Technology Data Exchange (ETDEWEB)

    Nuennecke, D.

    2000-07-01

    The dissertation investigates the electrochemical dechlorination of persistent organic chlorine compounds. An alternative to high-temperature combustion will be developed. The electrochemical dehalogenation reaction was to be made more selective with the aid of so-called mediator substances. A flow cell model was developed for continuous electrolytic cells. [German] Die vorliegende Arbeit beschaeftigt sich mit der elektrochemischen Dechlorierung von persistenten chlororganischen Verbindungen. Es soll eine Alternative zur Hochtemperatureverbrennung erarbeitet werden. Aufgabenstellung der Arbeit war die Erhoehung der Selektivitaet der elektrochemischen Enthalogenierung mithilfe von sog. Mediatoren. Fuer kontinuierlich arbeitenden Elektrolysezellen wurde ein Modell fuer eine Durchflusszelle entwickelt.(uke)

  17. Ruthenium complexes of C,C'-bis(ethynyl)carboranes: An investigation of electronic interactions mediated by spherical pseudo-aromatic spacers

    NARCIS (Netherlands)

    Fox, M.A.; Roberts, R.L.; Baines, T.E.; Le Guennic, B.; Halet, J.-F.; Hartl, F.; Yufit, D.S.; Albesa-Jové, D.; Howard, J.A.K.; Low, P.J.

    2008-01-01

    The complexes [Ru(1-C=C-1,10-C2B8H9)(dppe)Cp*] (3a), [Ru(1-C C-1,12-C2B10H11)(dppe)-Cp*] (3b), [{Ru(dppe)Cp*}(2){mu-1,10-(C C)(2)-1,10-C2B8H8}] (4a) and [{Ru(dppe)Cp*}(2){mu-1,12-(C C)2- 1,12-C2B10-H-10}] (4b), which form a representative series of mono- and bimetallic acetylide complexes featuring

  18. Identification of bolting-related microRNAs and their targets reveals complex miRNA-mediated flowering-time regulatory networks in radish (Raphanus sativus L.).

    Science.gov (United States)

    Nie, Shanshan; Xu, Liang; Wang, Yan; Huang, Danqiong; Muleke, Everlyne M; Sun, Xiaochuan; Wang, Ronghua; Xie, Yang; Gong, Yiqin; Liu, Liwang

    2015-09-15

    MicroRNAs (miRNAs) play vital regulatory roles in plant growth and development. The phase transition from vegetative growth to flowering is crucial in the life cycle of plants. To date, miRNA-mediated flowering regulatory networks remain largely unexplored in radish. In this study, two small RNA libraries from radish leaves at vegetative and reproductive stages were constructed and sequenced by Solexa sequencing. A total of 94 known miRNAs representing 21 conserved and 13 non-conserved miRNA families, and 44 potential novel miRNAs, were identified from the two libraries. In addition, 42 known and 17 novel miRNAs were significantly differentially expressed and identified as bolting-related miRNAs. RT-qPCR analysis revealed that some miRNAs exhibited tissue- or developmental stage-specific expression patterns. Moreover, 154 target transcripts were identified for 50 bolting-related miRNAs, which were predominately involved in plant development, signal transduction and transcriptional regulation. Based on the characterization of bolting-related miRNAs and their target genes, a putative schematic model of miRNA-mediated bolting and flowering regulatory network was proposed. These results could provide insights into bolting and flowering regulatory networks in radish, and facilitate dissecting the molecular mechanisms underlying bolting and flowering time regulation in vegetable crops.

  19. The complexity of trauma types in the lives of women in families referred for family violence: Multiple mediators of mental health.

    Science.gov (United States)

    Banyard, Victoria L; Williams, Linda M; Saunders, Benjamin E; Fitzgerald, Monica M

    2008-10-01

    Responding to calls for further research about the impact of multiple types of trauma across the life span, this study examines the interconnections among types of trauma in childhood and adulthood in a convenience clinical sample of 283 women obtaining social services for family violence. In particular, variables including family of-origin dysfunction and other childhood risk factors, relationship victimization in adulthood, and the presence of adult resources were examined as mediators of links between child maltreatment and adult mental health symptoms. Variables were assessed at different time points, 3 years apart. Path analysis revealed that the conceptual model of multiple pathways between childhood family violence exposure and adult outcomes fit the data well. In particular, the link between child maltreatment and adult trauma symptoms was mediated by more proximal adult sexual and intimate partner violence and its association with childhood risk markers (e.g., negative family environment) and decreased markers of resources. This link was not significant for a more general index of mental health symptoms in adulthood.

  20. The Differential Effects of Two Types of Task Repetition on the Complexity, Accuracy, and Fluency in Computer-Mediated L2 Written Production: A Focus on Computer Anxiety

    Science.gov (United States)

    Amiryousefi, Mohammad

    2016-01-01

    Previous task repetition studies have primarily focused on how task repetition characteristics affect the complexity, accuracy, and fluency in L2 oral production with little attention to L2 written production. The main purpose of the study reported in this paper was to examine the effects of task repetition versus procedural repetition on the…

  1. Strategy to enhance the anticancer efficacy of X-ray radiotherapy in melanoma cells by platinum complexes, the role of ROS-mediated signaling pathways.

    Science.gov (United States)

    Xie, Qiang; Lan, Guoqiang; Zhou, Yangliang; Huang, Jiamin; Liang, Yuanwei; Zheng, Wenjie; Fu, Xiaoyan; Fan, Cundong; Chen, Tianfeng

    2014-11-01

    Radiotherapy plays an important role in treatment of cancers with low toxicity to the surrounding normal tissues. However, it still fails to eradicate hypoxic tumors due to the occurrence of radioresistance. Therefore, the search for new radiation sensitizers is of great significance. Platinum (Pt) complexes have been identified as potential radiation sensitizers to increase the sensitivity of cancer cells to radiotherapy. In the present study, we have synthesized four Pt complexes containing (2 - benzimidazole [4, 5-f] - [1, 10] phenanthroline) ligand and found that they could effectively enhance the X-ray-induced growth inhibition against A375 human melanoma cells through induction of G2/M cell cycle arrest. In contrast, they showed much lower cytotoxicity toward human normal cells. The complexes also dramatically inhibited the TrxR activity and caused intracellular ROS overproduction, due to the Auger electron effect of heavy metal element under X-ray radiation. Excessive ROS triggered DNA damage and activated downstream signaling pathways, including the phosphorylation of p53 and p38MAPK, and down-regulation of phosphorylated AKT and ERK, finally resulted in increase of radiosensitivity and inhibition of tumor reproduction. Taken together, our results suggest that the synthetic Pt complexes could be further developed as sensitizers of X-ray radiotherapy.

  2. The Differential Effects of Two Types of Task Repetition on the Complexity, Accuracy, and Fluency in Computer-Mediated L2 Written Production: A Focus on Computer Anxiety

    Science.gov (United States)

    Amiryousefi, Mohammad

    2016-01-01

    Previous task repetition studies have primarily focused on how task repetition characteristics affect the complexity, accuracy, and fluency in L2 oral production with little attention to L2 written production. The main purpose of the study reported in this paper was to examine the effects of task repetition versus procedural repetition on the…

  3. A complex photoreceptor system mediates the regulation by light of the conidiation genes con-10 and con-6 in Neurospora crassa.

    Science.gov (United States)

    Olmedo, María; Ruger-Herreros, Carmen; Luque, Eva M; Corrochano, Luis M

    2010-04-01

    Genes con-10 and con-6 in Neurospora crassa are activated during conidiation or after illumination of vegetative mycelia. Light activation requires the white-collar complex (WCC), a transcription factor complex composed of the photoreceptor WC-1 and its partner WC-2. We have characterized the photoactivation of con-10 and con-6, and we have identified 300bp required for photoactivation in the con-10 promoter. A complex stimulus-response relationship for con-10 and con-6 photoactivation suggested the activity of a complex photoreceptor system. The WCC is the key element for con-10 activation by light, but we suggest that other photoreceptors, the cryptochrome CRY-1, the rhodopsin NOP-1, and the phytochrome PHY-2, modify the activity of the WCC for con-10 photoactivation, presumably through a repressor. In addition we show that the regulatory protein VE-1 is required for full photocarotenogenesis. We propose that these proteins may modulate the WCC in a gene-specific way.

  4. RELEVANCE OF CLASSIC ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODY (C-ANCA)-MEDIATED INHIBITION OF PROTEINASE 3-ALPHA-1-ANTITRYPSIN COMPLEXATION TO DISEASE-ACTIVITY IN WEGENER-GRANULOMATOSIS

    NARCIS (Netherlands)

    DOLMAN, KM; STEGEMAN, CA; VANDEWIEL, BA; HACK, CE; BORNE, AEGKV; KALLENBERG, CGM; GOLDSCHMEDING, R

    1993-01-01

    In the sera of patients with Wegener's granulomatosis (WG), C-ANCA can be detected that are directed against proteinase 3 (PR3). We have previously observed that C-ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor, alpha1-antitrypsin (alph

  5. The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions

    NARCIS (Netherlands)

    Moser, J; Volker, M; Kool, H; Alekseev, S; Vrieling, H; Yasui, A; van Zeeland, AA; Mullenders, LHF

    2005-01-01

    Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding protei

  6. The calcium-binding protein complex S100A8/A9 has a crucial role in controlling macrophage-mediated renal repair following ischemia/reperfusion

    NARCIS (Netherlands)

    Dessing, M.C.; Tammaro, A.; Pulskens, W.P.C.; Teske, G.J.; Butter, L.M.; Claessen, N.; Eijk, M. van; Poll, T. van der; Vogl, T.; Roth, J.; Florquin, S.; Leemans, J.C.

    2015-01-01

    Upon ischemia/reperfusion (I/R)-induced injury, several damage-associated molecular patterns are expressed including the calcium-binding protein S100A8/A9 complex. S100A8/A9 can be recognized by Toll-like receptor-4 and its activation is known to deleteriously contribute to renal I/R-induced injury.

  7. Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2

    DEFF Research Database (Denmark)

    Cooper, Sarah; Grijzenhout, Anne; Underwood, Elizabeth

    2016-01-01

    The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal...

  8. The calcineurin inhibitor cyclosporin A cyclophilin A complex reduces desensitization of GABA(A)-mediated responses in acutely dissociated rat hippocampal neurons

    NARCIS (Netherlands)

    Martina, M; Mozrzymas, JW; Boddeke, HWGM; Cherubini, E

    1996-01-01

    The effects of the selective inhibitor of calcineurin, cyclosporin A-cyclophilin A (CC) complex on the desensitization kinetics of GABA(A) receptors was studied in acutely dissociated hippocampal neurons, using the patch clamp technique in the whole cell configuration. In control conditions, the dec

  9. Role of Fas-Mediated Apoptosis and Follicle-Stimulating Hormone on the Developmental Capacity of Bovine Cumulus Oocyte Complexes in Vitro

    NARCIS (Netherlands)

    Pomar, F.J.; Roelen, B.A.J.; Slot, K.A.; Tol, van H.T.A.; Colenbrander, B.; Teerds, K.J.

    2004-01-01

    Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling

  10. PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

    Science.gov (United States)

    Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

    2013-06-14

    Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

  11. Microwave-assisted synthesis of arene ruthenium(II) complexes that induce S-phase arrest in cancer cells by DNA damage-mediated p53 phosphorylation.

    Science.gov (United States)

    Wu, Qiong; Fan, Cundong; Chen, Tianfeng; Liu, Chaoran; Mei, Wenjie; Chen, Sidong; Wang, Baoguo; Chen, Yunyun; Zheng, Wenjie

    2013-05-01

    A series of arene ruthenium(II) complexes coordinated by phenanthroimidazole derivates, [(C6H6)Ru(L)Cl]Cl·2H2O (1b L = IP, 2b L = p-NMe2PIP, 3b L = p-MeOPIP, 4b L = p-HOPIP, 5b L = p-COOHPIP, 6b L = p-CF3PIP, 7b L = p-BrPIP) have been synthesized in yields of 89-92% under microwave irradiation in 30 min, and the crystal structure of 1b by XRD gives a typical "piano stool" conformation. The antitumor activity of these complexes against various tumor cells have been evaluated by MTT assay, and the results show that this type of arene Ru(II) complexes exhibit acceptable inhibitory effect against all of these tumor cells, especially osteosarcoma MG-63 cells, but with low toxicity toward HK-2 human normal cells. Studies on the mechanism revealed that cell cycle arrest at S-phase in MG-63 cells induced by the arene Ru(II) complex 2b, which was confirmed by the increase in the percentage of cells at S-phase and down-regulator of cyclin A. The further studies by Comet assay at single cell level indicated that DNA damage in MG-63 cells was triggered by 2b, following with the up-regulation of phosphorylated p53 and histone. The studies by spectroscopy in vitro also indicate that 2b bind to DNA molecule by intercalative mode to disturb the bio-function of tumor cells. In conclusion, the synthetic arene Ru(II) complexes could serve as novel p53 activator with potential application in cancer chemotherapy.

  12. New water-soluble azido- and derived tetrazolato-platinum(II) complexes with PTA. Easy metal-mediated synthesis and isolation of 5-substituted tetrazoles.

    Science.gov (United States)

    Smoleński, Piotr; Mukhopadhyay, Suman; Guedes da Silva, M Fátima C; Charmier, M Adília Januário; Pombeiro, Armando J L

    2008-12-14

    The water-soluble four- and five-coordinate diazido-platinum(II) complexes cis-[Pt(N3)2(PTA)2] (1) (PTA = 1,3,5-triaza-7-phosphaadamantane), cis-[Pt(N3)2(Me-PTA)2]I2 (2) (Me-PTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation) and [Pt(N3)2(PTA)3] (3) were obtained by reactions of cis-[Pt(N3)2(PPh3)2] with PTA or [Me-PTA]I in dichloromethane. [2 + 3] cycloadditions of with organonitriles NCR gave the bis(tetrazolato) complexes trans-[Pt(N4CR)2(PTA)2] (R = Ph (4), 4-ClC6H4 (5) or 3-NC5H4 (6)), the reactions being greatly accelerated by microwave irradiation. 5-R-1H-Tetrazoles N4CR (R = Ph, 4-ClC6H4 and 3-NC5H4) were easily liberated from the tetrazolato complexes and isolated in high yields, in a single-pot process, upon reaction with aqueous diluted HCl, with concomitant formation of the water soluble cis-[Pt(Cl)2(PTA-H)2] complex 7. Alternatively, in a less convenient method, the tetrazoles could be liberated on reaction of 4-6 with propionitrile which also leads to the dicyano trans-[Pt(CN)2(PTA)2] complex 8. The compounds were characterized by IR, 1H, 13C and 31P[1H] NMR spectroscopies, FAB+-MS or ESI-MS, elemental analyses and (for and 4) also by X-ray diffraction.

  13. Stable interaction between the human proliferating cell nuclear antigen loader complex Ctf18-replication factor C (RFC) and DNA polymerase {epsilon} is mediated by the cohesion-specific subunits, Ctf18, Dcc1, and Ctf8.

    Science.gov (United States)

    Murakami, Takeshi; Takano, Ryuji; Takeo, Satoshi; Taniguchi, Rina; Ogawa, Kaori; Ohashi, Eiji; Tsurimoto, Toshiki

    2010-11-05

    One of the proliferating cell nuclear antigen loader complexes, Ctf18-replication factor C (RFC), is involved in sister chromatid cohesion. To examine its relationship with factors involved in DNA replication, we performed a proteomics analysis of Ctf18-interacting proteins. We found that Ctf18 interacts with a replicative DNA polymerase, DNA polymerase ε (pol ε). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ε demonstrated that their binding is direct and mediated by two distinct interactions, one weak and one stable. Three subunits that are specifically required for cohesion in yeast, Ctf18, Dcc1, and Ctf8, formed a trimeric complex (18-1-8) and together enabled stable binding with pol ε. The C-terminal 23-amino acid stretch of Ctf18 was necessary for the trimeric association of 18-1-8 and was required for the stable interaction. The weak interaction was observed with alternative loader complexes including Ctf18-RFC(5), which lacks Dcc1 and Ctf8, suggesting that the common loader structures, including the RFC small subunits (RFC2-5), are responsible for the weak interaction. The two interaction modes, mediated through distinguishable structures of Ctf18-RFC, both occurred through the N-terminal half of pol ε, which includes the catalytic domain. The addition of Ctf18-RFC or Ctf18-RFC(5) to the DNA synthesis reaction caused partial inhibition and stimulation, respectively. Thus, Ctf18-RFC has multiple interactions with pol ε that promote polymorphic modulation of DNA synthesis. We propose that their interaction alters the DNA synthesis mode to enable the replication fork to cooperate with the establishment of cohesion.

  14. Novel High-Viscosity Polyacrylamidated Chitosan for Neural Tissue Engineering: Fabrication of Anisotropic Neurodurable Scaffold via Molecular Disposition of Persulfate-Mediated Polymer Slicing and Complexation

    Directory of Open Access Journals (Sweden)

    Viness Pillay

    2012-10-01

    Full Text Available Macroporous polyacrylamide-grafted-chitosan scaffolds for neural tissue engineering were fabricated with varied synthetic and viscosity profiles. A novel approach and mechanism was utilized for polyacrylamide grafting onto chitosan using potassium persulfate (KPS mediated degradation of both polymers under a thermally controlled environment. Commercially available high molecular mass polyacrylamide was used instead of the acrylamide monomer for graft copolymerization. This grafting strategy yielded an enhanced grafting efficiency (GE = 92%, grafting ratio (GR = 263%, intrinsic viscosity (IV = 5.231 dL/g and viscometric average molecular mass (MW = 1.63 × 106 Da compared with known acrylamide that has a GE = 83%, GR = 178%, IV = 3.901 dL/g and MW = 1.22 × 106 Da. Image processing analysis of SEM images of the newly grafted neurodurable scaffold was undertaken based on the polymer-pore threshold. Attenuated Total Reflectance-FTIR spectral analyses in conjugation with DSC were used for the characterization and comparison of the newly grafted copolymers. Static Lattice Atomistic Simulations were employed to investigate and elucidate the copolymeric assembly and reaction mechanism by exploring the spatial disposition of chitosan and polyacrylamide with respect to the reactional profile of potassium persulfate. Interestingly, potassium persulfate, a peroxide, was found to play a dual role initially degrading the polymers—“polymer slicing”—thereby initiating the formation of free radicals and subsequently leading to synthesis of the high molecular mass polyacrylamide-grafted-chitosan (PAAm-g-CHT—“polymer complexation”. Furthermore, the applicability of the uniquely grafted scaffold for neural tissue engineering was evaluated via PC12 neuronal cell seeding. The novel PAAm-g-CHT exhibited superior neurocompatibility in terms of cell infiltration owing to the anisotropic porous architecture, high molecular mass mediated robustness

  15. Molecular complexes of alprazolam with carboxylic acids, boric acid, boronic acids, and phenols. Evaluation of supramolecular heterosynthons mediated by a triazole ring.

    Science.gov (United States)

    Varughese, Sunil; Azim, Yasser; Desiraju, Gautam R

    2010-09-01

    A series of molecular complexes, both co-crystals and salts, of a triazole drug-alprazolam-with carboxylic acids, boric acid, boronic acids, and phenols have been analyzed with respect to heterosynthons present in the crystal structures. In all cases, the triazole ring behaves as an efficient hydrogen bond acceptor with the acidic coformers. The hydrogen bond patterns exhibited with aromatic carboxylic acids were found to depend on the nature and position of the substituents. Being a strong acid, 2,6-dihydroxybenzoic acid forms a salt with alprazolam. With aliphatic dicarboxylic acids alprazolam forms hydrates and the water molecules play a central role in synthon formation and crystal packing. The triazole ring makes two distinct heterosynthons in the molecular complex with boric acid. Boronic acids and phenols form consistent hydrogen bond patterns, and these are seemingly independent of the substitutional effects. Boronic acids form noncentrosymmetric cyclic synthons, while phenols form O--H...N hydrogen bonds with the triazole ring.

  16. Hemoglobin switching in man and chicken is mediated by a heteromeric complex between the ubiquitous transcription factor CP2 and a developmentally specific protein.

    Science.gov (United States)

    Jane, S M; Nienhuis, A W; Cunningham, J M

    1995-01-03

    The human stage selector protein (SSP) has been implicated in the developmental regulation of the globin genes. Binding of SSP to the stage selector element (SSE) in the proximal gamma-globin promoter is integral to the competitive silencing of a linked beta-promoter in embryonic/fetal stage erythroleukemia (K562) cells. We now report the biochemical purification of SSP from K562 cell nuclear extract and demonstrate that the ubiquitously expressed transcription factor CP2 is pivotal to, but not sufficient for, SSP binding activity. Although addition of anti-CP2 antiserum disrupts the formation of the SSP-SSE complex in the electrophoretic mobility shift assay (EMSA), recombinant CP2 fails to bind to the SSE. Binding of CP2 to the SSE requires a heterodimeric partner present in K562 cells. We have defined the molecular weight of the partner protein as 40-45 kDa in UV and protein cross-linking experiments. An element analogous to the human SSE has previously been demonstrated in the chicken beta A-gene-promoter. The effects of this element are dependent on the binding of the chicken stage selector protein, NF-E4. Comparative studies between human CP2 and chicken NF-E4 demonstrate homology between the protein complexes. SSP binds to the chicken SSE and formation of this complex is ablated by the addition of anti-CP2 antiserum or a monoclonal antibody to NF-E4. Western analysis of partially purified NF-E4 using anti-CP2 antiserum or the NF-E4 monoclonal antibody both demonstrate a dominant band at 66 kDa. Similarly, the NF-E4 antibody recognizes the 66 kDa human CP2 protein in Western analysis of the SSP-SSE complex.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Two Triazole-Based Phosphine Ligands Prepared via Temperature-Mediated Li/H Exchange: Cu(I) and Au(I) Complexes and Structural Studies.

    Science.gov (United States)

    Choubey, Bimba; Radhakrishna, Latchupatula; Mague, Joel T; Balakrishna, Maravanji S

    2016-09-06

    The kinetically favored triazole-based phosphine 1-(2-(diphenylphosphino)phenyl)-4-phenyl-1H-1,2,3-triazole (2, L1) and its thermodynamically preferred isomer, 5-(diphenylphosphino)-1,4-diphenyl-1H-1,2,3-triazole (3, L2), were obtained by the temperature-controlled lithiation of 2-bromotriazole followed by the reaction with chlorodiphenylphosphine. The structures of phosphines 2 and 3 were determined by X-ray diffraction. Upon reaction with late transition-metal derivatives (Cu(I), Ag(I), and Au(I)), phosphines 2 and 3 form complexes with monodentate (Cu(I), Ag(I), and Au(I); κ(1)-P), chelate (Cu(I); κ(2)-P,N), bridged bidentate (Cu(I); μ(2)-P,N), and tridentate (Cu(I); μ(2),κ(2)-P,N,N) modes of coordination. Reactions with copper(I) halides produced mono-, di-, and tetranuclear complexes, whereas the reaction of 2 with [Cu(NCCH3)4]BF4 yielded the binuclear complex [Cu2(CH3CN)2{o-Ph2P(C6H4){1,2,3-N3C(Ph)C(H)}-μ-(κ-P,κ-N),κ-N}2](BF4)2 (10) with the ligand acting as a six-electron donor involving phosphorus and two triazole nitrogen atoms. The copper complexes of 2 and 3 containing rhomboid Cu2X2 units, [(Cu)2(μ-X)2{o-Ph2P(C6H4){1,2,3-N3C(Ph)C(H)}-κ-P}2] (4, X = Cl; 5, X = Br), on treatment with 1,10-phenanthroline and 2,2'-bipyridine gave mixed-ligand complexes of the type [(CuX)(N∩N-κ(2)-N,N){o-Ph2P(C6H4){1,2,3-N3C(Ph)C(H)}-κ-P}] (N∩N = 1,10-phen and 2,2'-bipy; X = Cl, Br, and I).

  18. The novel functions of high-molecular-mass complexes containing insulin receptor substrates in mediation and modulation of insulin-like activities: Emerging concept of diverse function by IRS-associated proteins

    Directory of Open Access Journals (Sweden)

    Fumihiko eHakuno

    2015-05-01

    Full Text Available Insulin-like peptides, such as insulin and insulin-like growth factors (IGFs, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism and decreased catabolism in many cell types and in vivo. In general, insulin or IGFs bind to insulin receptor (IR or IGF-I receptor (IGF-IR, activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAP; IRS-associated protein, which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability and determine intracellular localization of IRSs. In addition, in these complexes we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer we could propose that the IRSome is an important target for treatment of these diseases.

  19. A novel method for the purification of DNA by capturing nucleic acid and magnesium complexes on non-woven fabric filters under alkaline conditions for the gene diagnosis of tuberculosis by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Fukasawa, Tadashi; Oda, Naozumi; Wada, Yasunao; Tamaru, Aki; Fukushima, Yukari; Nakajima, Chie; Suzuki, Yasuhiko

    2010-07-01

    A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg(2+)) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg(2+) complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg(2+) complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg(2+) was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.

  20. Ability of Interleukin-33- and Immune Complex-Triggered Activation of Human Mast Cells to Down-Regulate Monocyte-Mediated Immune Responses.

    Science.gov (United States)

    Rivellese, Felice; Suurmond, Jolien; Habets, Kim; Dorjée, Annemarie L; Ramamoorthi, Nandhini; Townsend, Michael J; de Paulis, Amato; Marone, Gianni; Huizinga, Tom W J; Pitzalis, Costantino; Toes, René E M

    2015-09-01

    Mast cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). In particular, their activation by interleukin-33 (IL-33) has been linked to the development of arthritis in animal models. The aim of this study was to evaluate the functional responses of human mast cells to IL-33 in the context of RA. Human mast cells were stimulated with IL-33 combined with plate-bound IgG or IgG anti-citrullinated protein antibodies (ACPAs), and their effects on monocyte activation were evaluated. Cellular interactions of mast cells in RA synovium were assessed by immunofluorescence analysis, and the expression of messenger RNA (mRNA) for mast cell-specific genes was evaluated in synovial biopsy tissue from patients with early RA who were naive to treatment with disease-modifying antirheumatic drugs. IL-33 induced the up-regulation of Fcγ receptor type IIa and enhanced the activation of mast cells by IgG, including IgG ACPAs, as indicated by the production of CXCL8/IL-8. Intriguingly, mast cell activation triggered with IL-33 and IgG led to the release of mediators such as histamine and IL-10, which inhibited monocyte activation. Synovial mast cells were found in contact with CD14+ monocyte/macrophages. Finally, mRNA levels of mast cell-specific genes were inversely associated with disease severity, and IL-33 mRNA levels showed an inverse correlation with the levels of proinflammatory markers. When human mast cells are activated by IL-33, an immunomodulatory phenotype develops, with human mast cells gaining the ability to suppress monocyte activation via the release of IL-10 and histamine. These findings, together with the presence of synovial mast cell-monocyte interactions and the inverse association between the expression of mast cell genes at the synovial level and disease activity, suggest that these newly described mast cell-mediated inhibitory pathways might have a functional relevance in the pathogenesis of RA. © 2015, American College of Rheumatology.

  1. CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells.

    Science.gov (United States)

    Guo, Na; Zhang, Kui; Lv, Minghua; Miao, Jinlin; Chen, Zhinan; Zhu, Ping

    2015-02-01

    Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4(+) T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data

  2. Isomeric [RuCl2(dmso)2(indazole)2] complexes: ruthenium(II)-mediated coupling reaction of acetonitrile with 1H-indazole.

    Science.gov (United States)

    Reisner, Erwin; Arion, Vladimir B; Rufińska, Anna; Chiorescu, Ion; Schmid, Wolfgang F; Keppler, Bernhard K

    2005-07-21

    Reaction of the antitumor complex trans-[Ru(III)Cl4(Hind)2]- (Hind = indazole) with an excess of dimethyl sulfoxide (dmso) in acetone afforded the complex trans,trans,trans-[Ru(II)Cl2(dmso)2(Hind)2] (1). Two other isomeric compounds trans,cis,cis-[Ru(II)Cl2(dmso)2(Hind)2] (2) and cis,cis,cis-[Ru(II)Cl2(dmso)2(Hind)2] (3) have been obtained on refluxing cis-[Ru(II)Cl(2)(dmso)(4)] with 2 equiv. of indazole in ethanol and methanol, respectively. Isomers 1 and 2 react with acetonitrile yielding the complexes trans-[Ru(II)Cl2(dmso)(Hind){HN=C(Me)ind}].CH3CN (4.CH3CN) and trans,cis-[Ru(II)Cl2(dmso)2{HN=C(Me)ind}].H2O (5.H2O), respectively, containing a cyclic amidine ligand resulting from insertion of the acetonitrile C triple bond N group in the N1-H bond of the N2-coordinated indazole ligand in the nomenclature used for 1H-indazole. These are the first examples of the metal-assisted iminoacylation of indazole. The products isolated have been characterized by elemental analysis, IR spectroscopy, UV-vis spectroscopy, electrospray mass-spectrometry, thermogravimetry, differential scanning calorimetry, 1H NMR spectroscopy, and solid-state 13C CP MAS NMR spectroscopy. The isomeric structures of 1-3 and the presence of a chelating amidine ligand in 4 and 5 have been confirmed by X-ray crystallography. The electrochemical behavior of 1-5 and the formation of 5 have been studied by cyclic voltammetry.

  3. Co-motif discovery identifies an Esrrb-Sox2-DNA ternary complex as a mediator of transcriptional differences between mouse embryonic and epiblast stem cells.

    Science.gov (United States)

    Hutchins, Andrew Paul; Choo, Siew Hua; Mistri, Tapan Kumar; Rahmani, Mehran; Woon, Chow Thai; Ng, Calista Keow Leng; Jauch, Ralf; Robson, Paul

    2013-02-01

    Transcription factors (TF) often bind in heterodimeric complexes with each TF recognizing a specific neighboring cis element in the regulatory region of the genome. Comprehension of this DNA motif grammar is opaque, yet recent developments have allowed the interrogation of genome-wide TF binding sites. We reasoned that within this data novel motif grammars could be identified that controlled distinct biological programs. For this purpose, we developed a novel motif-discovery tool termed fexcom that systematically interrogates ChIP-seq data to discover spatially constrained TF-TF composite motifs occurring over short DNA distances. We applied this to the extensive ChIP-seq data available from mouse embryonic stem cells (ESCs). In addition to the well-known and most prevalent sox-oct motif, we also discovered a novel constrained spacer motif for Esrrb and Sox2 with a gap of between 2 and 8 bps that Essrb and Sox2 cobind in a selective fashion. Through the use of knockdown experiments, we argue that the Esrrb-Sox2 complex is an arbiter of gene expression differences between ESCs and epiblast stem cells (EpiSC). A number of genes downregulated upon dual Esrrb/Sox2 knockdown (e.g., Klf4, Klf5, Jam2, Pecam1) are similarly downregulated in the ESC to EpiSC transition and contain the esrrb-sox motif. The prototypical Esrrb-Sox2 target gene, containing an esrrb-sox element conserved throughout eutherian and metatherian mammals, is Nr0b1. Through positive regulation of this transcriptional repressor, we argue the Esrrb-Sox2 complex promotes the ESC state through inhibition of the EpiSC transcriptional program and the same trio may also function to maintain trophoblast stem cells.

  4. Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

    Science.gov (United States)

    Hernández-Vásquez, Magda Nohemí; Adame-García, Sendi Rafael; Hamoud, Noumeira; Chidiac, Rony; Reyes-Cruz, Guadalupe; Gratton, Jean Philippe; Côté, Jean-François; Vázquez-Prado, José

    2017-07-21

    Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses. © 2017 by The American Society for

  5. Acquired pMHC I Complexes Greatly Enhance CD4+ Th Cell's Stimulatory Effect on CD8+ T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

    Institute of Scientific and Technical Information of China (English)

    Khawaja Ashfaque Ahmed; Yufeng Xie; Xueshu Zhang; Jim Xiang

    2008-01-01

    CD4+ helper T (Th) cells play pivotal roles in induction of CD8+ CTL immunity. However, the mechanism of CD4+ T cell help delivery to CD8+ T cells in vivo is still elusive. In this study, we used ovalbumin (OVA)-pulsed dendritic cells (DCovA) to activate OT-Ⅱ mouse CD4+ T cells, and then studied the help effect of these CD4+ T cells on CD8+ cytotoxic T lymphocyte (CTL) responses. We also examined CTL mediated islet β cell destruction which leaded to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter (RIP)-mOVA mice expressing β cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex (pMHC) I acquired from DCovA by DCovA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination with TCR transgenic OT-I mouse CD8+ T cells, the tolerogenic dosage of CD4+ Th cells with acquired pMHC I, but not CD4+ (Kb-/-) Th cells without acquired pMHC I were able to cause diabetes in 8/10 (80%) RIP-mOVA mice.This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4+ T cell-mediated help in CD8+ CTL induction. Cellular & Molecular Immunology. 2008;5(6):407-415.

  6. Technology-Use Mediation

    DEFF Research Database (Denmark)

    Bansler, Jørgen P.; Havn, Erling C.

    2004-01-01

    Implementation of new computer-mediated communication (CMC) systems in organizations is a complex socio-technical endeavour, involving the mutual adaptation of technology and organization over time. Drawing on the analytic concept of sensemaking, this paper provides a theoretical perspective...... that deepens our understanding of how organizations appropriate new electronic communication media. The paper analyzes how a group of mediators in a large, multinational company adapted a new web-based CMC technology (a virtual workspace) to the local organizational context (and vice versa) by modifying...... features of the technology, providing ongoing support for users, and promoting appropriate conventions of use. We found that these mediators exerted considerable influence on how the technology was established and used in the organization. The mediators were not neutral facilitators of a well...

  7. Effect of water molecules on the fluorescence enhancement of Aflatoxin B1 mediated by Aflatoxin B1:beta-cyclodextrin complexes. A theoretical study.

    Science.gov (United States)

    Ramírez-Galicia, Guillermo; Garduño-Juárez, Ramón; Gabriela Vargas, M

    2007-01-01

    In order to explain the observed fluorescence enhancement of Aflatoxin B1 (AFB1) when forming AFB1:beta-cyclodextrin (AFB1:beta-CD) inclusion complexes, we have performed a theoretical (quantum chemistry calculations) study of AFB1 and AFB1:beta-CD in vacuum and in the presence of aqueous solvent. The AM1 method was used to calculate the absorption and emission wavelengths of these molecules. With the help of density functional theory (DFT) and time-dependent DFT (TDDFT) vibrational frequencies and related excitation energies of AFB1 and AFB1.(H2O)m = 4,5,6,11 were calculated. On the basis of these calculations we propose a plausible mechanism for the fluorescence enhancement of AFB1 in the presence of beta-CD: (1) before photoexcitation of AFB1 to its S1 excited state, there is a vibrational coupling between the vibrational modes involving the AFB1 carbonyl groups and the bending modes of the nearby water molecules (CG + WM); (2) these interactions allow a thermal relaxation of the excited AFB1 molecules that results in fluorescence quenching; (3) when the AFB1 molecules form inclusion complexes with beta-CD the CG + WM interaction decreases; and (4) this gives rise to a fluorescence enhancement.

  8. A novel zinc finger protein Zfp277 mediates transcriptional repression of the Ink4a/arf locus through polycomb repressive complex 1

    DEFF Research Database (Denmark)

    Negishi, Masamitsu; Saraya, Atsunori; Mochizuki, Shinobu

    2010-01-01

    . METHODOLOGY/PRINCIPAL FINDINGS: We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through......BACKGROUND: Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood...... direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated...

  9. The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

    Science.gov (United States)

    Chen, Yi-An; Peng, Yi-Jheng; Hu, Meng-Chun; Huang, Jing-Jia; Chien, Yun-Chia; Wu, June-Tai; Chen, Tsung-Yu; Tang, Chih-Yung

    2015-05-29

    Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

  10. The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3-H4 tetramers and Asf1-H3-H4 complexes

    DEFF Research Database (Denmark)

    Hammond, Colin M; Sundaramoorthy, Ramasubramanian; Larance, Mark

    2016-01-01

    Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers...... catalysed histone H3 K9 acetylation. In the absence of Asf1 this model can be used to generate a complex consisting of a reconfigured Vps75 tetramer bound to a H3-H4 tetramer. This provides a structural explanation for many of the complexes detected biochemically and illustrates the ability of Vps75....... In the presence of equimolar H3-H4 and Vps75, the major species is a reconfigured Vps75 tetramer bound to a histone H3-H4 tetramer. However, in the presence of excess histones, a Vps75 dimer bound to a histone H3-H4 tetramer predominates. We show the Vps75-H3-H4 interaction is compatible with the histone...

  11. Kicking against the PRCs - A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2.

    Directory of Open Access Journals (Sweden)

    Shih Chieh Liang

    2015-12-01

    Full Text Available The Polycomb group (PcG and trithorax group (trxG genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1 gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1. Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2, a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF, we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1, a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1

  12. A sustained deficiency of mitochondrial respiratory complex III induces an apoptotic cell death through the p53-mediated inhibition of pro-survival activities of the activating transcription factor 4.

    Science.gov (United States)

    Evstafieva, A G; Garaeva, A A; Khutornenko, A A; Klepikova, A V; Logacheva, M D; Penin, A A; Novakovsky, G E; Kovaleva, I E; Chumakov, P M

    2014-11-06

    Generation of energy in mitochondria is subjected to physiological regulation at many levels, and its malfunction may result in mitochondrial diseases. Mitochondrial dysfunction is associated with different environmental influences or certain genetic conditions, and can be artificially induced by inhibitors acting at different steps of the mitochondrial electron transport chain (ETC). We found that a short-term (5 h) inhibition of ETC complex III with myxothiazol results in the phosphorylation of translation initiation factor eIF2α and upregulation of mRNA for the activating transcription factor 4 (ATF4) and several ATF4-regulated genes. The changes are characteristic for the adaptive integrated stress response (ISR), which is known to be triggered by unfolded proteins, nutrient and metabolic deficiency, and mitochondrial dysfunctions. However, after a prolonged incubation with myxothiazol (13-17 h), levels of ATF4 mRNA and ATF4-regulated transcripts were found substantially suppressed. The suppression was dependent on the p53 response, which is triggered by the impairment of the complex III-dependent de novo biosynthesis of pyrimidines by mitochondrial dihydroorotate dehydrogenase. The initial adaptive induction of ATF4/ISR acted to promote viability of cells by attenuating apoptosis. In contrast, the induction of p53 upon a sustained inhibition of ETC complex III produced a pro-apoptotic effect, which was additionally stimulated by the p53-mediated abrogation of the pro-survival activities of the ISR. Interestingly, a sustained inhibition of ETC complex I by piericidine did not induce the p53 response and stably maintained the pro-survival activation of ATF4/ISR. We conclude that a downregulation of mitochondrial ETC generally induces adaptive pro-survival responses, which are specifically abrogated by the suicidal p53 response triggered by the genetic risks of the pyrimidine nucleotide deficiency.

  13. Branched-chain amino acids enhance premature senescence through mammalian target of rapamycin complex I-mediated upregulation of p21 protein.

    Directory of Open Access Journals (Sweden)

    Masayuki Nakano

    Full Text Available Branched-chain amino acids (BCAAs have been applied as an oral supplementation to patients with liver cirrhosis. BCAAs not only improve nutritional status of patients but also decrease the incidence of liver cancer. Mammalian target of rapamycin (mTOR links cellular metabolism with growth and proliferation in response to nutrients, energy, and growth factors. BCAAs, especially leucine, have been shown to regulate protein synthesis through mTOR activities. On the other hand, cellular senescence is suggested to function as tumor suppressor mechanisms, and induced by a variety of stimuli including DNA damage-inducing drugs. However, it is not clear how BCAA supplementation prevents the incidence of liver cancer in patients with cirrhosis. Here we showed that human cancer cells, HepG2 and U2OS, cultured in medium containing BCAAs with Fischer's ratio about 3, which was shown to have highest activities to synthesize and secrete of albumin, had higher activities to induce premature senescence and elevate mTORC1 activities. Furthermore, BCAAs themselves enhanced the execution of premature senescence induced by DNA damage-inducing drugs, which was effectively prevented by rapamycin. These results strongly suggested the contribution of the mTORC1 pathway to the regulation of premature senescence. Interestingly, the protein levels of p21, a p53 target and well-known gene essential for the execution of cellular senescence, were upregulated in the presence of BCAAs. These results suggested that BCAAs possibly contribute to tumor suppression by enhancing cellular senescence mediated through the mTOR signalling pathway.

  14. ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex

    Directory of Open Access Journals (Sweden)

    Richard T. Timms

    2016-10-01

    Full Text Available The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus.

  15. ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex.

    Science.gov (United States)

    Timms, Richard T; Tchasovnikarova, Iva A; Antrobus, Robin; Dougan, Gordon; Lehner, Paul J

    2016-10-11

    The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus.

  16. Striatal adenosine A2A and cannabinoid CB1 receptors form functional heteromeric complexes that mediate the motor effects of cannabinoids.

    Science.gov (United States)

    Carriba, Paulina; Ortiz, Oskar; Patkar, Kshitij; Justinova, Zuzana; Stroik, Jessica; Themann, Andrea; Müller, Christa; Woods, Anima S; Hope, Bruce T; Ciruela, Francisco; Casadó, Vicent; Canela, Enric I; Lluis, Carme; Goldberg, Steven R; Moratalla, Rosario; Franco, Rafael; Ferré, Sergi

    2007-11-01

    The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.

  17. Resolution of complex fluorescence spectra of lipids and nicotinic acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid microenvironment

    CERN Document Server

    Wenz, Jorge J; 10.1186/1757-5036-1-6

    2009-01-01

    Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Forster resonance energy transfer (FRET) was observed between the protein and pyrene-PC and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to ...

  18. Polycomb repressive complex 2 (PRC2) protein ESC regulates insect developmental timing by mediating H3K27me3 and activating prothoracicotropic hormone gene expression.

    Science.gov (United States)

    Lu, Yu-Xuan; Denlinger, David L; Xu, Wei-Hua

    2013-08-09

    The decision made by insects to develop into adults or halt development (enter diapause and prolong lifespan) is commonly based on environmental signals that provide reliable predictors of future seasons of adversity. For example, the short day lengths of early autumn accurately foretell the advent of winter, but little is known about the molecular mechanisms that preside over the hormonal events dictating whether the insect proceeds with development or enters diapause. In Helicoverpa armigera we show that day length affects H3K27me3 by affecting polycomb repressive complex 2 (PRC2) protein extra sex comb (ESC) and regulates the prothoracicotropic hormone (PTTH) gene, thus directly influencing developmental timing. ESC expression in brains of developing (nondiapause) pupae is higher than in brains from diapausing pupae. High ESC expression is localized in two pairs of PTTH neurosecretory cells, and H3K27me3 recruits on the PTTH promoter. Double strand ESC and PRC2 inhibitor (DzNep) treatment in vitro show that ESC triggers PTTH promoter activity, which in turn depends on PRC2 methyltransferase activity. Injection of DzNep into pupae programmed for development reduces the H3K27me3 mark and PTTH gene expression, thereby delaying development. Although ESC is best known as a transcriptional repressor, our results show that ESC prompts development and metamorphosis. We believe this is the first report showing that the PRC2 complex functions as an activator and that a low level of H3K27me3 can prolong lifespan (i.e. induce diapause) by controlling PTTH gene expression in insects.

  19. Examination of nanoformulated crosslinked polymers complexed with copper/zinc superoxide dismutase as a therapeutic strategy for angiotensin II-mediated hypertension

    Science.gov (United States)

    Savalia, Krupa

    Excessive generation of superoxide (O2·-) has been extensively implicated as a signaling molecule in cardiovascular pathologies, including hypertension. As a major risk factor for myocardial infarction, stroke, and heart failure, the morbidity and mortality associated with hypertension is a worldwide epidemic. Although there are several standard therapies that effectively lower blood pressure, many hypertensive patients have uncontrolled blood pressure despite taking available medications. Thus, there is a necessity to develop new pharmacotherapies that target novel molecular effectors (e.g. O2·-) that have been implicated to be integral in the pathogenesis of hypertension. To overcome the failed therapeutic impact of currently available antioxidants in cardiovascular disease, we developed a nanomedicine-based delivery system for the O2 ·- scavenging enzyme, copper/zinc superoxide dismutase (CuZnSOD), in which CuZnSOD protein is electrostatically bound to poly-L-lysine (PLL 50)-polyethylene glycol (PEG) block co-polymer to form CuZnSOD nanozyme. Different formulations of CuZnSOD nanozyme are covalently stabilized by either reducible or non-reducible crosslinked bonds between the PLL50-PEG polymers. Herein, we tested the overall hypothesis that PLL50-PEG CuZnSOD nanozyme delivers active CuZnSOD protein to neurons and decreases blood pressure in a model of Angll-dependent hypertension. As determined by electron paramagnetic resonance (EPR) spectroscopy, nanozymes retain full SOD enzymatic activity. Furthermore, non-reducible crosslinked nanozyme delivers active CuZnSOD protein to central neurons in culture (CATH.a neurons) without inducing significant neuronal toxicity. In vivo studies conducted in Angll-mediated hypertensive adult male C57BL/6 mice demonstrate that the non-reducible crosslinked nanozyme significantly attenuates blood pressure when given directly into the brain and prevents the further increase in hypertension when intravenously (IV) administered

  20. Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

    Directory of Open Access Journals (Sweden)

    Gidda Satinder K

    2008-09-01

    Full Text Available Abstract Background Carnation Italian ringspot virus (CIRV is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36, are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. Results Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM, but not with the sorting and assembly machinery (SAM. Conclusion Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer

  1. Mediatized Humanitarianism

    DEFF Research Database (Denmark)

    Vestergaard, Anne

    2014-01-01

    The article investigates the implications of mediatization for the legitimation strategies of humanitarian organizations. Based on a (full population) corpus of ~400 pages of brochure material from 1970 to 2007, the micro-textual processes involved in humanitarian organizations' efforts to legiti......The article investigates the implications of mediatization for the legitimation strategies of humanitarian organizations. Based on a (full population) corpus of ~400 pages of brochure material from 1970 to 2007, the micro-textual processes involved in humanitarian organizations' efforts...... legitimation by accountancy, legitimation by institutionalization, and legitimation by compensation. The analysis relates these changes to a problem of trust associated with mediatization through processes of mediation....

  2. DELLA signaling mediates stress-induced cell differentiation in Arabidopsis leaves through modulation of anaphase-promoting complex/cyclosome activity.

    Science.gov (United States)

    Claeys, Hannes; Skirycz, Aleksandra; Maleux, Katrien; Inzé, Dirk

    2012-06-01

    Drought is responsible for considerable yield losses in agriculture due to its detrimental effects on growth. Drought responses have been extensively studied, but mostly on the level of complete plants or mature tissues. However, stress responses were shown to be highly tissue and developmental stage specific, and dividing tissues have developed unique mechanisms to respond to stress. Previously, we studied the effects of osmotic stress on dividing leaf cells in Arabidopsis (Arabidopsis thaliana) and found that stress causes early mitotic exit, in which cells end their mitotic division and start endoreduplication earlier. In this study, we analyzed this phenomenon in more detail. Osmotic stress induces changes in gibberellin metabolism, resulting in the stabilization of DELLAs, which are responsible for mitotic exit and earlier onset of endoreduplication. Consequently, this response is absent in mutants with altered gibberellin levels or DELLA activity. Mitotic exit and onset of endoreduplication do not correlate with an up-regulation of known cell cycle inhibitors but are the result of reduced levels of DP-E2F-LIKE1/E2Fe and UV-B-INSENSITIVE4, both inhibitors of the developmental transition from mitosis to endoreduplication by modulating anaphase-promoting complex/cyclosome activity, which are down-regulated rapidly after DELLA stabilization. This work fits into an emerging view of DELLAs as regulators of cell division by regulating the transition to endoreduplication and differentiation.

  3. Production of Omega-3 Fatty Acid Ethyl Esters from Menhaden Oil Using Proteus vulgaris Lipase-Mediated One-Step Transesterification and Urea Complexation.

    Science.gov (United States)

    Kim, Soo-Jin; Kim, Hyung Kwoun

    2016-05-01

    An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (ω-3 PUFA EEs). First, the lyophilized recombinant lipase K80 (LyoK80) was used to perform the transesterification reaction of menhaden oil and ethanol. LyoK80 produced the ω-3 PUFA EEs with a conversion yield of 82 % in the presence of 20 % water content via a three-step ethanol-feeding process; however, in a non-aqueous condition, LyoK80 produced only a slight amount of the ω-3 PUFA EEs. To enhance its reaction properties, the lipase K80 was immobilized on a hydrophobic bead to derive ImmK80; the biochemical properties and substrate specificity of ImmK80 are similar to those of LyoK80. ImmK80 was then used to produce ω-3 PUFA EEs in accordance with the same transesterification reaction. Unlike LyoK80, ImmK80 achieved a high ω-3 PUFA EE conversion yield of 86 % under a non-aqueous system via a one-step ethanol-feeding reaction. The ω-3 PUFA EEs were purified up to 92 % using a urea complexation method.

  4. β-Caryophyllene/Hydroxypropyl-β-Cyclodextrin Inclusion Complex Improves Cognitive Deficits in Rats with Vascular Dementia through the Cannabinoid Receptor Type 2 -Mediated Pathway

    Science.gov (United States)

    Lou, Jie; Teng, Zhipeng; Zhang, Liangke; Yang, Jiadan; Ma, Lianju; Wang, Fang; Tian, Xiaocui; An, Ruidi; Yang, Mei; Zhang, Qian; Xu, Lu; Dong, Zhi

    2017-01-01

    This work was conducted to prepare β-caryophyllene-hydroxypropyl-β-cyclodextrin inclusion complex (HPβCD/BCP) and investigate its effects and mechanisms on cognitive deficits in vascular dementia (VD) rats. First, HPβCD/BCP was prepared, optimized, characterized, and evaluated. HPβCD/BCP and AM630 were then administered to VD rats to upregulate and downregulate the cannabinoid receptor type 2 (CB2). Results showed that HPβCD/BCP can significantly increase the bioavailability of BCP. Through the Morris water maze test, HPβCD/BCP can attenuate learning and memory deficits in rats. Cerebral blood flow (CBF) monitoring results indicated that HPβCD/BCP can promote the recovery of CBF. Moreover, molecular biology experiments showed that HPβCD/BCP can increase the expression levels of CB2 in brain tissues, particularly the hippocampus and white matter tissues, as well as the expression levels of PI3K and Akt. Overall, the findings demonstrated the protective effects of HPβCD/BCP against cognitive deficits induced by chronic cerebral ischemia and suggested the potential of HPβCD/BCP in the therapy of vascular dementia in the future. PMID:28154534

  5. Fallen angels or risen apes? A tale of the intricate complexities of imbalanced immune responses in the pathogenesis and progression of immune-mediated and viral cancers

    Directory of Open Access Journals (Sweden)

    Beatrice Omusiro Ondondo

    2014-03-01

    Full Text Available Excessive immune responses directed against foreign pathogens, self-antigens or commensal microflora can cause cancer establishment and progression if the execution of tight immuno-regulatory mechanisms fails. On the other hand, induction of potent tumour antigen-specific immune responses together with stimulation of the innate immune system is a pre-requisite for effective antitumour immunity, and if suppressed by the strong immuno-regulatory mechanisms can lead to cancer progression. Therefore, it is crucial that the inevitable co-existence of these fundamental, yet conflicting roles of immune-regulatory cells is carefully streamlined as imbalances can be detrimental to the host. Infection with chronic persistent viruses is characterised by severe immune dysfunction resulting in T cell exhaustion and sometimes deletion of antigen-specific T cells. More often this is due to increased immuno-regulatory processes which are triggered to down-regulate immune responses and limit immunopathology. However, such heightened levels of immune disruption cause a concomitant loss of tumour immune-surveillance and create a permissive microenvironment for cancer establishment and progression, as demonstrated by increased incidences of cancer in immunosuppressed hosts. Paradoxically, while some cancers arise as a consequence of increased immuno-regulatory mechanisms that inhibit protective immune responses and impinge on tumour surveillance, other cancers arise due to impaired immuno-regulatory mechanisms and failure to limit pathogenic inflammatory responses. This intricate complexity, where immuno-regulatory cells can be beneficial in certain immune settings but detrimental in other settings underscores the need for carefully formulated interventions to equilibrate the balance between immuno-stimulatory and immuno-regulatory processes.

  6. Involuntary switching of attention mediates differences in event-related responses to complex tones between early and late Spanish-English bilinguals.

    Science.gov (United States)

    Ortiz-Mantilla, Silvia; Choudhury, Naseem; Alvarez, Barbara; Benasich, April A

    2010-11-29

    Most research with bilinguals has used speech stimuli to demonstrate differences in auditory processing abilities. Two main factors have been identified as modulators of such differences: proficiency and age of acquisition of the second language (L2). However, whether the bilingual brain differs from the monolingual in the efficient processing of non-verbal auditory events (known to be critical to the acoustic analysis of the speech stream) remains unclear. In this EEG/ERP study, using the mismatch negativity (MMN), P3a, and late negativity (LN), we examined differences in discrimination, involuntary switching of attention and reorienting of attention between monolinguals and bilinguals as they processed complex tones. Further, we examined the role that age of acquisition plays in modulating such responses. A group of English monolinguals and a group of proficient Spanish-English bilinguals were presented with a multiple-deviant oddball paradigm with four deviant conditions (duration, frequency, silent gap, and frequency modulation). Late bilinguals, who learned English after age 10, exhibited larger MMN and P3a responses than early bilinguals, across all deviant conditions. Significant associations were found between amplitude of the responses and both age of L2 acquisition and years of L2 experience. Individuals who acquired English at later ages and had fewer years of L2 experience had larger MMN, P3a, and LN responses than those who learned it earlier. These findings demonstrate that age of L2 acquisition is an important modulator of auditory responses in bilinguals even when processing non-speech signals. Involuntary attention switching is suggested as the main factor driving these differences.

  7. N-Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5β1 to α6β4.

    Science.gov (United States)

    Hang, Qinglei; Isaji, Tomoya; Hou, Sicong; Zhou, Ying; Fukuda, Tomohiko; Gu, Jianguo

    2016-09-19

    N-Glycosylation of integrin α5β1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3-5,10-14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3-5,10-14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5β1 or α6β4; i.e., the loss of site-11 switched EGFR-α5β1 to EGFR-α6β4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5β1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR.

  8. Down regulation of the TCR complex CD3 ζ-chain on CD3+ T cells: a potential mechanism for helminth mediated immune modulation

    Directory of Open Access Journals (Sweden)

    Laura Jane Appleby

    2015-02-01

    Full Text Available The CD3ζ forms part of the T cell receptor (TCR where it plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways leading to T cell effector functions. Down regulation of CD3ζ leads to impairment of immune responses including reduced cell proliferation and cytokine production. In experimental models helminth parasites have been shown to modulate immune responses directed against them and unrelated antigens, so called bystander antigens, but there is a lack of studies validating these observations in humans. This study focused on investigated the relationship between expression levels of the TCR CD3ζ chain with lymphocyte cell proliferation during human infection with the helminth parasite, Schistosoma haematobium which causes uro-genital schistosomiasis. Using flow cytometry, peripheral blood mononuclear cells (PBMCs from individuals naturally exposed to S. haematobium in rural Zimbabwe were phenotyped, and expression levels of CD3ζ on T cells were related to intensity of infection. In this population, parasite infection intensity was inversely related to CD3ζ expression levels (p<0.05, consistent with down-regulation of CD3ζ expression during helminth infection. Furthermore, PBMC proliferation was positively related to expression levels of CD3ζ (p<0.05 after allowing for confounding variables (host age, sex, infection level. CD3ζ expression levels had a differing relationship between immune correlates of susceptibility and immunity, measured by antibody responses, indicating a complex relationship between immune activation status and immunity. The relationships between the CD3ζ chain of the TCR and schistosome infection, PBMC proliferation and schistosome-specific antibody responses have not previously been reported, and these results may indicate a mechanism for the impaired T cell proliferative responses observed during human schistosome infection.

  9. Stabilization of the Low-Spin State in a Mononuclear Iron(II) Complex and High-Temperature Cooperative Spin Crossover Mediated by Hydrogen Bonding.

    Science.gov (United States)

    Zheng, Sipeng; Reintjens, Niels R M; Siegler, Maxime A; Roubeau, Olivier; Bouwman, Elisabeth; Rudavskyi, Andrii; Havenith, Remco W A; Bonnet, Sylvestre

    2016-01-04

    The tetrapyridyl ligand bbpya (bbpya=N,N-bis(2,2'-bipyrid-6-yl)amine) and its mononuclear coordination compound [Fe(bbpya)(NCS)2 ] (1) were prepared. According to magnetic susceptibility, differential scanning calorimetry fitted to Sorai's domain model, and powder X-ray diffraction measurements, 1 is low-spin at room temperature, and it exhibits spin crossover (SCO) at an exceptionally high transition temperature of T1/2 =418 K. Although the SCO of compound 1 spans a temperature range of more than 150 K, it is characterized by a wide (21 K) and dissymmetric hysteresis cycle, which suggests cooperativity. The crystal structure of the LS phase of compound 1 shows strong NH⋅⋅⋅S intermolecular H-bonding interactions that explain, at least in part, the cooperative SCO behavior observed for complex 1. DFT and CASPT2 calculations under vacuum demonstrate that the bbpya ligand generates a stronger ligand field around the iron(II) core than its analogue bapbpy (N,N'-di(pyrid-2-yl)-2,2'-bipyridine-6,6'-diamine); this stabilizes the LS state and destabilizes the HS state in 1 compared with [Fe(bapbpy)(NCS)2 ] (2). Periodic DFT calculations suggest that crystal-packing effects are significant for compound 2, in which they destabilize the HS state by about 1500 cm(-1) . The much lower transition temperature found for the SCO of 2 compared to 1 appears to be due to the combined effects of the different ligand field strengths and crystal packing.

  10. Biological Significance of Photoreceptor Photocycle Length: VIVID Photocycle Governs the Dynamic VIVID-White Collar Complex Pool Mediating Photo-adaptation and Response to Changes in Light Intensity.

    Directory of Open Access Journals (Sweden)

    Arko Dasgupta

    2015-05-01

    Full Text Available Most organisms on earth sense light through the use of chromophore-bearing photoreceptive proteins with distinct and characteristic photocycle lengths, yet the biological significance of this adduct decay length is neither understood nor has been tested. In the filamentous fungus Neurospora crassa VIVID (VVD is a critical player in the process of photoadaptation, the attenuation of light-induced responses and the ability to maintain photosensitivity in response to changing light intensities. Detailed in vitro analysis of the photochemistry of the blue light sensing, FAD binding, LOV domain of VVD has revealed residues around the site of photo-adduct formation that influence the stability of the adduct state (light state, that is, altering the photocycle length. We have examined the biological significance of VVD photocycle length to photoadaptation and report that a double substitution mutant (vvdI74VI85V, previously shown to have a very fast light to dark state reversion in vitro, shows significantly reduced interaction with the White Collar Complex (WCC resulting in a substantial photoadaptation defect. This reduced interaction impacts photoreceptor transcription factor WHITE COLLAR-1 (WC-1 protein stability when N. crassa is exposed to light: The fast-reverting mutant VVD is unable to form a dynamic VVD-WCC pool of the size required for photoadaptation as assayed both by attenuation of gene expression and the ability to respond to increasing light intensity. Additionally, transcription of the clock gene frequency (frq is sensitive to changing light intensity in a wild-type strain but not in the fast photo-reversion mutant indicating that the establishment of this dynamic VVD-WCC pool is essential in general photobiology and circadian biology. Thus, VVD photocycle length appears sculpted to establish a VVD-WCC reservoir of sufficient size to sustain photoadaptation while maintaining sensitivity to changing light intensity. The great diversity

  11. Molecular dynamics of sialic acid analogues complex with cholera toxin and DFT optimization of ethylene glycol-mediated zinc nanocluster conjugation.

    Science.gov (United States)

    Sharmila, D Jeya Sundara; Jino Blessy, J

    2017-01-01

    Cholera is an infectious disease caused by cholera toxin (CT) protein of bacterium Vibrio cholerae. A sequence of sialic acid (N-acetylneuraminic acid, NeuNAc or Neu5Ac) analogues modified in its C-5 position is modelled using molecular modelling techniques and docked against the CT followed by molecular dynamics simulations. Docking results suggest better binding affinity of NeuNAc analogue towards the binding site of CT. The NeuNAc analogues interact with the active site residues GLU:11, TYR:12, HIS:13, GLY:33, LYS:34, GLU:51, GLN:56, HIE:57, ILE:58, GLN:61, TRP:88, ASN:90 and LYS:91 through intermolecular hydrogen bonding. Analogues N-glycolyl-NeuNAc, N-Pentanoyl-NeuNAc and N-Propanoyl-NeuNAc show the least XPGscore (docking score) of -9.90, -9.16, and -8.91, respectively, and glide energy of -45.99, -42.14 and -41.66 kcal/mol, respectively. Stable nature of CT-N-glycolyl-NeuNAc, CT-N-Pentanoyl-NeuNAc and CT-N-Propanoyl-NeuNAc complexes was verified through molecular dynamics simulations, each for 40 ns using the software Desmond. All the nine NeuNAc analogues show better score for drug-like properties, so could be considered as suitable candidates for drug development for cholera infection. To improve the enhanced binding mode of NeuNAc analogues towards CT, the nine NeuNAc analogues are conjugated with Zn nanoclusters through ethylene glycol (EG) as carriers. The NeuNAc analogues conjugated with EG-Zn nanoclusters show better binding energy towards CT than the unconjugated nine NeuNAc analogues. The electronic structural optimization of EG-Zn nanoclusters was carried out for optimizing their performance as better delivery vehicles for NeuNAc analogues through density functional theory calculations. These sialic acid analogues may be considered as novel leads for the design of drug against cholera and the EG-Zn nanocluster may be a suitable carrier for sialic acid analogues.

  12. β7 Integrin controls mast cell recruitment, whereas αE integrin modulates the number and function of CD8+ T cells in immune complex-mediated tissue injury.

    Science.gov (United States)

    Yamada, Daisuke; Kadono, Takafumi; Masui, Yuri; Yanaba, Koichi; Sato, Shinichi

    2014-05-01

    Immune complex (IC) deposition causes significant tissue injury associated with various autoimmune diseases such as vasculitis. In the cascade of inflammation, cell-to-cell and cell-to-matrix adhesion via adhesion molecules are essential. To assess the role of αE and β7 integrin in IC-mediated tissue injury, peritoneal and cutaneous reverse-passive Arthus reaction was examined in mice lacking αE integrin (αE(-/-)) or β7 integrin (β7(-/-)). Both αE(-/-) and β7(-/-) mice exhibited significantly attenuated neutrophil infiltration in the peritoneal and cutaneous Arthus reaction. β7 integrin deficiency, not αE integrin deficiency, significantly reduced the number of mast cells in the peritoneal cavity, which was consistent with the result that mast cells expressed only α4β7 integrin, not αEβ7 integrin. αE(-/-) mice instead revealed the reduction of CD8(+) T cells in the peritoneal cavity, and nearly half of them in wild-type mice expressed αE integrin. These αE(+)CD8(+) T cells produced more proinflammatory cytokines than αE(-)CD8(+) T cells, and adoptive transfer of αE(+)CD8(+) T cell into αE(-/-) recipients restored cutaneous and peritoneal Arthus reaction. These results suggest that in the peritoneal and cutaneous reverse-passive Arthus reaction, α4β7 integrin is involved in the migration of mast cells for initial IC recognition. αEβ7 integrin, in contrast, contributes by recruiting αE(+)CD8(+) T cells, which produce more proinflammatory cytokines than αE(-)CD8(+) T cells and amplify IC-mediated inflammation.

  13. Fashion, Mediations & Method Assemblages

    DEFF Research Database (Denmark)

    Sommerlund, Julie; Jespersen, Astrid Pernille

    , respectively. The paper thus takes on aesthetics and the social in a manner closely related to a core argument of STS - namely that the scientific fact, and the social processes of constructing, distributing, and using that fact, are co-constructed (Callon, 1986; Latour, 1993). The paper thus contributes......, it is an important ambition of this paper to go into a methodological discussion of how "that which effectively happens" can be approached. To this end, the paper will combine Hennion's term of the "mediator" with John Laws methodological term of "method assemblages". Method assemblages is a suggested as a way...... of handling multiple, fluid realities with multiple, fluid methods. Empirically, the paper works with mediation in fashion - that is efforts the active shaping of relations between producer and consumer through communication, marketing and PR. Fashion mediation is by no means simple, but organise complex...

  14. Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by H-2K and H-2D serological regions of the murine major histocompatibility complex.

    Science.gov (United States)

    Shearer, G M; Rehn, T G; Garbarino, C A

    1975-06-01

    Splenic lymphocytes from four C57BL/10 congenic resistant mouse strains were sensitized in vitro with trinitrophenyl (TNP)-modified autologous spleen cellsmthe effector cells generated were incubated with 51-Cr-labeled unmodified or TNP-modified spleen or tumor target cells, and the percentage of specific lympholysis determined. The results obtained using syngeneic-, congenic-, recombinante, and allogeneic-modified target cells indicated that TNP modification of the target cells was a necessary but insufficient requirement for lympholysis. Intra-H-2 homology either between modified stimulating cells and modified target cells or between responding lymphocytes and modified target cells was also important in the specificity for lysis. Homology at the K serological region or at K plus I-A in the B10.A and B10BR strains, and at either the D serological region or at some other region (possibly K) in the B10.D2 and C57BL/10 strains were shown to be necessary in order to detect lympholysis. Experiments using (B10itimes C57BL/10)F1 responding lymphocytes sensitized and assayed with TNP-modified parental cells indicated that the homology required for lympholysis was between modified stimulating and modified target cellsmthe possibility is raised that histocompatibility antigens may serve in the autologous system as cell surface components which are modified by viruses or autoimmune complexes to form cell-bound modified-self antigens, which are particularly suited for cell-mediated immune reactions. Evidence is presented suggesting that H-2-linked Ir genes are expressed in the TNP-modified autologous cytotoxic system. These findings imply that the major histocompatibility complex can be functionally involved both in the response potential to and in the formation of new antigenic determinants involving modified-self components.

  15. Complex segmental duplications mediate a recurrent dup(X)(p11.22-p11.23) associated with mental retardation, speech delay, and EEG anomalies in males and females.

    Science.gov (United States)

    Giorda, Roberto; Bonaglia, M Clara; Beri, Silvana; Fichera, Marco; Novara, Francesca; Magini, Pamela; Urquhart, Jill; Sharkey, Freddie H; Zucca, Claudio; Grasso, Rita; Marelli, Susan; Castiglia, Lucia; Di Benedetto, Daniela; Musumeci, Sebastiano A; Vitello, Girolamo A; Failla, Pinella; Reitano, Santina; Avola, Emanuela; Bisulli, Francesca; Tinuper, Paolo; Mastrangelo, Massimo; Fiocchi, Isabella; Spaccini, Luigina; Torniero, Claudia; Fontana, Elena; Lynch, Sally Ann; Clayton-Smith, Jill; Black, Graeme; Jonveaux, Philippe; Leheup, Bruno; Seri, Marco; Romano, Corrado; dalla Bernardina, Bernardo; Zuffardi, Orsetta

    2009-09-01

    Submicroscopic copy-number variations make a considerable contribution to the genetic etiology of human disease. We have analyzed subjects with idiopathic mental retardation (MR) by using whole-genome oligonucleotide-based array comparative genomic hybridization (aCGH) and identified familial and de novo recurrent Xp11.22-p11.23 duplications in males and females with MR, speech delay, and a peculiar electroencephalographic (EEG) pattern in childhood. The size of the duplications ranges from 0.8-9.2 Mb. Most affected females show preferential activation of the duplicated X chromosome. Carriers of the smallest duplication show X-linked recessive inheritance. All other affected individuals present dominant expression and comparable clinical phenotypes irrespective of sex, duplication size, and X-inactivation pattern. The majority of the rearrangements are mediated by recombination between flanking complex segmental duplications. The identification of common clinical features, including the typical EEG pattern, predisposing genomic structure, and peculiar X-inactivation pattern, suggests that duplication of Xp11.22-p11.23 constitutes a previously undescribed syndrome.

  16. A computational insight into a metal mediated pathway for the ring-opening polymerization (ROP) of lactides by an ionic {(NHC)2Ag}(+)X(-) (X = halide) type N-heterocyclic carbene (NHC) complex.

    Science.gov (United States)

    Stephen, Raji; Sunoj, Raghavan B; Ghosh, Prasenjit

    2011-10-21

    A metal mediated coordination-insertion pathway for the ring-opening polymerization (ROP) of L-lactide by an ionic {(NHC)(2)Ag}(+)X(-) (X = halide) type silver complex of N-heterocyclic carbene (NHC) has been investigated using the density functional theory (DFT) method. A clear insight into the lactide insertion process could be obtained by modeling two consecutive monomer addition steps with the first one mimicking chain initiation with the second representing a propagation step. In particular, in each of the cycles, the reaction initiates with the formation of a lactide coordinated species, [1+LL] and [2+LL] that transforms into a metal bound cyclic lactide intermediate, I([1+LL]→2) and I([2+LL]→3), which subsequently ring opens to give the lactide inserted products, 2 and 3. The estimated overall activation barrier for the initiation step is 42.0 kcal mol(-1) while the same for the propagation step is 31.5 kcal mol(-1). Studies on higher monomer insertions showed a decrease in the relative product energies as anticipated for an addition polymerization pathway.

  17. MoDnm1 Dynamin Mediating Peroxisomal and Mitochondrial Fission in Complex with MoFis1 and MoMdv1 Is Important for Development of Functional Appressorium in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Kaili Zhong

    2016-08-01

    Full Text Available Dynamins are large superfamily GTPase proteins that are involved in various cellular processes including budding of transport vesicles, division of organelles, cytokinesis, and pathogen resistance. Here, we characterized several dynamin-related proteins from the rice blast fungus Magnaporthe oryzae and found that MoDnm1 is required for normal functions, including vegetative growth, conidiogenesis, and full pathogenicity. In addition, we found that MoDnm1 co-localizes with peroxisomes and mitochondria, which is consistent with the conserved role of dynamin proteins. Importantly, MoDnm1-dependent peroxisomal and mitochondrial fission involves functions of mitochondrial fission protein MoFis1 and WD-40 repeat protein MoMdv1. These two proteins display similar cellular functions and subcellular localizations as MoDnm1, and are also required for full pathogenicity. Further studies showed that MoDnm1, MoFis1 and MoMdv1 are in complex to regulate not only peroxisomal and mitochondrial fission, pexophagy and mitophagy progression, but also appressorium function and host penetration. In summary, our studies provide new insights into how MoDnm1 interacts with its partner proteins to mediate peroxisomal and mitochondrial functions and how such regulatory events may link to differentiation and pathogenicity in the rice blast fungus.

  18. Axion Mediation

    CERN Document Server

    Baryakhtar, Masha; March-Russell, John

    2013-01-01

    We explore the possibility that supersymmetry breaking is mediated to the Standard Model sector through the interactions of a generalized axion multiplet that gains a F-term expectation value. Using an effective field theory framework we enumerate the most general possible set of axion couplings and compute the Standard Model sector soft-supersymmetry-breaking terms. Unusual, non-minimal spectra, such as those of both natural and split supersymmetry are easily implemented. We discuss example models and low-energy spectra, as well as implications of the particularly minimal case of mediation via the QCD axion multiplet. We argue that if the Peccei-Quinn solution to the strong-CP problem is realized in string theory then such axion-mediation is generic, while in a field theory model it is a natural possibility in both DFSZ- and KSVZ-like regimes. Axion mediation can parametrically dominate gravity-mediation and is also cosmologically beneficial as the constraints arising from axino and gravitino overproduction ...

  19. Mediatized Parenthood

    DEFF Research Database (Denmark)

    Sonne Damkjær, Maja

    2017-01-01

    to parenthood? The dissertation explores this question on the basis of a synchronous study within an overall mediatization perspective. The first part of the dissertation focuses on a conceptualization of the relationship between digital media and parenting as well as an exploration of theoretical perspectives...... and methods that make it possible to study the interactions between the two. Concretely, the dissertation builds on a number of key studies within audience research, which have contributed knowledge about the media’s role in the family and the home. This is done by including three approaches to mediatization......) a family-oriented, b) a peer-oriented, c) an oppositional, and d) non-use. Secondary contribution: Based on qualitative audience research and mediatization theory, the dissertation contributes a conceptualization of the relationship between media and parenthood. This is carried out in a study design...

  20. Mediating Business

    DEFF Research Database (Denmark)

    "Mediating Business" is a study of the expansion of business journalism. Building on evidence from Denmark, Finland, Norway and Sweden, "Mediating Business" is a comparative and multidisciplinary study of one of the major transformations of the mass media and the realm of business - nationally...... and globally. The book explores the history of key innovations and innovators in the business press. It analyzes changes in the discourse of business journalism associated with the growth in business news and the development of new ways of framing business issues and events. Finally, it examines...... the organizational implications of the increased media visibility of business and, in particular, the development of corporate governance and media relations....

  1. Mediatized play

    DEFF Research Database (Denmark)

    Johansen, Stine Liv

    Children’s play must nowadays be understood as a mediatized field in society and culture. Media – understood in a very broad sense - holds severe explanatory power in describing and understanding the practice of play, since play happens both with, through and inspired by media of different sorts....... In this presentation the case of ‘playing soccer’ will be outlined through its different mediated manifestations, including soccer games and programs on TV, computer games, magazines, books, YouTube videos and soccer trading cards....

  2. The cellular RNA export receptor TAP/NXF1 is required for ICP27-mediated export of herpes simplex virus 1 RNA, but the TREX complex adaptor protein Aly/REF appears to be dispensable.

    Science.gov (United States)

    Johnson, Lisa A; Li, Ling; Sandri-Goldin, Rozanne M

    2009-07-01

    Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)(+) RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)(+) RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

  3. The Arabidopsis KINβγ Subunit of the SnRK1 Complex Regulates Pollen Hydration on the Stigma by Mediating the Level of Reactive Oxygen Species in Pollen.

    Science.gov (United States)

    Gao, Xin-Qi; Liu, Chang Zhen; Li, Dan Dan; Zhao, Ting Ting; Li, Fei; Jia, Xiao Na; Zhao, Xin-Ying; Zhang, Xian Sheng

    2016-07-01

    Pollen-stigma interactions are essential for pollen germination. The highly regulated process of pollen germination includes pollen adhesion, hydration, and germination on the stigma. However, the internal signaling of pollen that regulates pollen-stigma interactions is poorly understood. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex which plays important roles in the regulation of plant development. Here, we showed that KINβγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinβγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinβγ mutant pollen on the stigma was compromised. However, adding water to the stigma promoted the germination of the mutant pollen in vivo, suggesting that the compromised hydration of the mutant pollen led to its defective germination. In kinβγ mutant pollen, the structure of the mitochondria and peroxisomes was destroyed, and their numbers were significantly reduced compared with those in the wild type. Furthermore, we found that the kinβγ mutant exhibited reduced levels of reactive oxygen species (ROS) in pollen. The addition of H2O2 in vitro partially compensated for the reduced water absorption of the mutant pollen, and reducing ROS levels in pollen by overexpressing Arabidopsis CATALASE 3 resulted in compromised hydration of pollen on the stigma. These results indicate that Arabidopsis KINβγ is critical for the regulation of ROS levels by mediating the biogenesis of mitochondria and peroxisomes in pollen, which is required for pollen-stigma interactions during pollination.

  4. Carney Complex

    Science.gov (United States)

    ... Types of Cancer > Carney Complex Request Permissions Carney Complex Approved by the Cancer.Net Editorial Board , 11/2015 What is Carney complex? Carney complex is a hereditary condition associated with: ...

  5. A systemic study of stepwise chlorination-chemical vapor transport characteristics of pure rare earth oxides from Sc{sub 2}O{sub 3} to Lu{sub 2}O{sub 3} mediated by alkaline chlorides as complex former

    Energy Technology Data Exchange (ETDEWEB)

    Sun Yanhui [School of Chemistry and Environment, South China Normal University, Guangzhou 510631 (China)]. E-mail: sunyanhui0102@163.com; He Peng [Department of Ecology, School of Metallurgical and Ecological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Chen Huani [School of Chemistry and Environment, South China Normal University, Guangzhou 510631 (China)

    2007-08-30

    A systematic study has been carried out for the stepwise chlorination-chemical vapor transport (SC-CVT) characteristics of pure rare earth oxides from Sc{sub 2}O{sub 3} to Lu{sub 2}O{sub 3} mediated by the vapor complexes KLnCl{sub 4} and NaLnCl{sub 4} (Ln = Sc, Y and La-Lu) used NaCl and KCl as complex former, respectively. The results showed that the SC-CVT characteristics are similarly for NaCl and KCl as complex former, the main deposition temperature of the rare earth chlorides LnCl{sub 3} is in the increasing order ScCl{sub 3} < YCl{sub 3} < LaCl{sub 3}, and then with a systematically decreasing trend from the early lanthanide chlorides to the end one. The results also showed that the total transported amount of the produced chlorides is YCl{sub 3} > ScCl{sub 3}, and they are much higher than that of most lanthanoid chlorides. For lanthanoids, the total transported amount of chloride increases systematically from the early lanthanoid chlorides to the end one except for EuCl{sub 3} and GdCl{sub 3} mediated by KCl and NaCl as complex former, respectively, which showed the divergence effect of Gd in the total transport efficiency. But there are some differences in SC-CVT characteristics of pure rare earth oxide mediated by KCl and NaCl as complex former, that is the main deposition temperature region for the same rare earth element was lower for KCl than that for NaCl as complex former except for LaCl{sub 3}, CeCl{sub 3}, YbCl{sub 3} and LuCl{sub 3}, while the total transport amount of rare earth chloride for KCl as complex former is higher than that for NaCl except for LaCl{sub 3} and EuCl{sub 3}. More over, the discussion was carried out for Sc and Y on the one hand and the lanthanides contain 4f electron as another hand based on the 4f electron hybridization assumption. Further more, the transport characteristics of rare earth oxides with alkaline chlorides as complex former in this study were compared to that with AlCl{sub 3} as complex former.

  6. Complex Beauty

    OpenAIRE

    Franceschet, Massimo

    2014-01-01

    Complex systems and their underlying convoluted networks are ubiquitous, all we need is an eye for them. They pose problems of organized complexity which cannot be approached with a reductionist method. Complexity science and its emergent sister network science both come to grips with the inherent complexity of complex systems with an holistic strategy. The relevance of complexity, however, transcends the sciences. Complex systems and networks are the focal point of a philosophical, cultural ...

  7. Syntheses and structures of closely related copper(I) complexes of tridentate (2-pyridylmethyl)imine and (2-pyridylmethyl)amine ligands and their use in mediating atom transfer radical polymerizations.

    Science.gov (United States)

    Turner, Sara A; Remillard, Zachary D; Gijima, Desire T; Gao, Emily; Pike, Robert D; Goh, Christopher

    2012-10-15

    A series of five copper(I) bromide complexes of tridentate (N,N,L) pyridine-imine and pyridine-amine ligands with a third amine, ether, or thioether neutral donor was synthesized and utilized in the atom transfer radical polymerization of styrene. The ligand design illustrated a systematic approach to the development of copper complexes for use in ATRP. Variations in the nature of the ligand impacted the solid state structures of the complexes. A mononuclear [CuBr(L)] complex was observed for L = pyridine-amine-amine, whereas complexes of L = pyridine-imine-amine and -thioether formed dinuclear [CuBr(L)](2) structures with a central 10-membered ring. A doubly-bromide-bridged dimer was revealed for the [CuBr(L)] complex of L = pyridine-imine-ether and a polymeric species for [CuBr(L)], where L = pyridine-imine-amine and the imine-amine spacer was extended from two to three carbon atoms. In the application of these complexes to the ATRP of styrene, the redox potentials of the complexes were found to be one indicator of ATRP efficiency. Of the series presented, two complexes in particular provided fast polymerization rates and good to excellent molecular weight control. In both of these complexes, the ligand contained all nitrogen-based donor moieties.

  8. C-terminal functional unit of Rapana thomasiana (marine snail, gastropod) hemocyanin isoform RtH1: isolation and characterization.

    Science.gov (United States)

    Parvanova, Katja; Idakieva, Krassimira; Todinova, Svetla; Genov, Nicolay

    2003-09-23

    Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin (Hc) isoforms termed RtH1 and RtH2. Both subunit types are built up of eight functional units (FUs). The C-terminal functional unit (RtH1-h) of the Rapana Hc subunit 1 has been isolated by limited trypsinolysis of the subunit polypeptide chain. The oxy- and apo-forms of the unit are characterized by fluorescence spectroscopy. Upon excitation of RtH1-h at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein globule determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the indole emission of the oxy-RtH1-h. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-RtH1-h. The thermal stability of the apo-RtH1-h is characterized fluorimetrically by the "melting" temperature T(m) (65 degrees C) and by the transition temperature T(m) (83 degrees C) obtained by differential scanning calorimetry for oxy-RtH1-h. The results confirm the role of the copper-dioxygen complex for the stabilization of the Hc structure in solution.

  9. Family education and television mediation

    Directory of Open Access Journals (Sweden)

    Paz CÁNOVAS LEONHARDT

    2010-07-01

    Full Text Available This article try to deal with the complex influence of television viewing in the process of socialization of children and adolescents, focusing our attention on the importance of the family as the mediator-educator agency of particular relevance. Once analyzed the basic theoretical assumptions, we deepened in reality under study by providing data about how the studied population lives television and what extent parental mediation influences and affects the process. The article concludes with some reflections and pedagogical suggestions which trying to help to the optimization of the educational reality.

  10. Complexity explained

    CERN Document Server

    Erdi, Peter

    2008-01-01

    This book explains why complex systems research is important in understanding the structure, function and dynamics of complex natural and social phenomena. Readers will learn the basic concepts and methods of complex system research.

  11. Three tasks for mediatization research

    DEFF Research Database (Denmark)

    Ekstrøm, Mats; Fornäs, Johan; Jansson, André;

    2016-01-01

    Based on the interdisciplinary experience of a Swedish research committee, this article discusses critical conceptual issues raised by the current debate on mediatization – a concept that holds great potential to constitute a space for synthesized understandings of media-related social...... that mediatization researchers have sometimes formulated too grand claims as to mediatization’s status as a unitary approach, a meta-theory or a paradigm. Such claims have led to problematic confusions around the concept and should be abandoned in favour of a more open agenda. In line with such a call for openness...... transformations. In contrast to other, more metaphorical constructions, mediatization can be studied empirically in systematic ways through various sub-processes that together provide a complex picture of how culture and everyday life evolve in times of media saturation. The first part of this article argues...

  12. Mediation in Legal English Teaching

    Directory of Open Access Journals (Sweden)

    Chovancová Barbora

    2016-06-01

    Full Text Available Mediation is a language activity that has been unjustly neglected when preparing law students for their future professional careers. When trained in a professional context, students need to develop and improve complex communicative skills. These include not only the traditional language skills such as reading, writing, listening and speaking, but also more advanced skills such as summarizing, providing definitions, changing registers etc. All these are involved in the students’ acquisition of ‘soft skills’ that are particularly important for students of law since much of their future work involves interpersonal lawyer-client interaction. This article argues that mediation is a crucial (though previously underestimated skill and that law-oriented ESP instruction should provide training aimed at developing this skill. Showing a practical application of this approach, the paper demonstrates that mediation can be successfully integrated in the legal English syllabus and make the learning of legal English more effective.

  13. Nem tudo é midiatização: como entender, ver e analisar a complexidade dos processos comunicacionais sem banalizar (Not everything is mediatization: how to understand, regard and analyze the complexity of the communication processes without trivializing them

    Directory of Open Access Journals (Sweden)

    Adriana Domingues Garcia

    2011-01-01

    Full Text Available Resumo: O artigo apresenta reflexões e inferências sobre os estudos em midiatização. São discutidos a formação, as lógicas de funcionamento e os métodos de pesquisa sobre o fenômeno da midiatização para aplicação no trabalho de dissertação sobre as convergências de processos e práticas para a interação da sociedade em midiatização. O eixo argumentativo desdobra-se sobre as precauções mobilizadas para que as processualidades não sejam reduzidas à ampla abrangência e ao determinismo do pensamento único. Encaminha-se um raciocínio para que a midiatização seja visualizada como um jogo de relações de sentidos transversais, diferidos, difusos, heterogêneos e fragmentados, que produzem interações e vínculos por meio de práticas e processos em circulação na sociedade.Abstract: This paper presents reflections and insights on the mediatization studies. It aims at discussing the formation, the operating logic, and the methods of research on the mediatization phenomenon. This analysis intends to aid a dissertation about the convergence of processes and practices regarding the interaction of the mediatized society. The argumentation is centered on the precautions against the reduction of the media processes to the all-encompassing view and the determinism of the pensée unique. It is also argued that the mediatization should be viewed as a set of transversal relationships, which are deferred, diffused, heterogeneous and fragmented, and produce interactions and linkages through the existing practices and processes of the society.

  14. Bucolic Complexes

    CERN Document Server

    Brešar, Bostjan; Chepoi, Victor; Gologranc, Tanja; Osajda, Damian

    2012-01-01

    In this article, we introduce and investigate bucolic complexes, a common generalization of systolic complexes and of CAT(0) cubical complexes. This class of complexes is closed under Cartesian products and amalgamations over some convex subcomplexes. We study various approaches to bucolic complexes: from graph-theoretic and topological viewpoints, as well as from the point of view of geometric group theory. Bucolic complexes can be defined as locally-finite simply connected prism complexes satisfying some local combinatorial conditions. We show that bucolic complexes are contractible, and satisfy some nonpositive-curvature-like properties. In particular, we prove a version of the Cartan-Hadamard theorem, the fixed point theorem for finite group actions, and establish some results on groups acting geometrically on such complexes. We also characterize the 1-skeletons (which we call bucolic graphs) and the 2-skeletons of bucolic complexes. In particular, we prove that bucolic graphs are precisely retracts of Ca...

  15. [Co(η5-P5){η2-P2H(mes)}]2-: a phospha-organometallic complex obtained by the transition-metal-mediated activation of the heptaphosphide trianion.

    Science.gov (United States)

    Knapp, Caroline M; Westcott, Bethan H; Raybould, Melissa A C; McGrady, John E; Goicoechea, Jose M

    2012-09-03

    A carbon copy: The chemical activation of the heptaphosphide trianion with [Co(PEt(2)Ph)(2)(mes)(2)] (see picture; 1) yields the novel phospha-organometallic complex [Co(η(5)-P(5)){η(2)-P(2)H(mes)}](2-) (2). The reaction product maintains the nuclearity of the parent cluster, but extensive cage fragmentation takes place to yield a diamagnetic "inorganometallic" cobalt complex.

  16. Bioethics mediation: the role and importance of nursing advocacy.

    Science.gov (United States)

    Schlairet, Maura C

    2009-01-01

    Ethics consultations are utilized in health care to identify and manage conflict, difficult decision-making, and ethical issues. In bioethics mediation, a more updated approach using interpersonal, mediative, conflict management, and dispute resolution skills is merged with ethical principles to manage dilemmas arising in healthcare settings. This article argues, based on a professional obligation to advocate for the good of the client, that nurses must assume leadership roles in mediation processes. Nurses can initiate and fully participate in formal bioethics mediation and other mediative interventions. Nurse administrators can work to evolve existing ethics consult models to mediation models. Nonetheless, mediative efforts of individual nurses must be grounded in realization of the multifactorial nature of conflict and dilemma in healthcare settings. Multidisciplinary mediative interventions, framed by sound institutional policies, may best serve the complex needs of ethically vulnerable clients. To best advocate for these at-risk clients, nurses must assume various leadership roles in mediation processes.

  17. Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

    Science.gov (United States)

    Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

    2016-06-07

    Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

  18. Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy

    Science.gov (United States)

    Wang, Li; Li, Xueying; Shen, Hongchun; Mao, Nan; Wang, Honglian; Cui, Luke; Cheng, Yuan; Fan, Junming

    2016-01-01

    Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN. PMID:27485391

  19. In vivo expansion of regulatory T cells with IL-2/IL-2 mAb complexes prevents anti-factor VIII immune responses in hemophilia A mice treated with factor VIII plasmid-mediated gene therapy.

    Science.gov (United States)

    Liu, Chao-Lien; Ye, Peiqing; Yen, Benjamin C; Miao, Carol H

    2011-08-01

    Generation of transgene-specific immune responses can constitute a major complication following gene therapy treatment. An in vivo approach to inducing selective expansion of Regulatory T (Treg) cells by injecting interleukin-2 (IL-2) mixed with a specific IL-2 monoclonal antibody (JES6-1) was adopted to modulate anti-factor VIII (anti-FVIII) immune responses. Three consecutive IL-2 complexes treatments combined with FVIII plasmid injection prevented anti-FVIII formation and achieved persistent, therapeutic-level of FVIII expression in hemophilia A (HemA) mice. The IL-2 complexes treatment expanded CD4(+)CD25(+)Foxp3(+) Treg cells five- to sevenfold on peak day, and they gradually returned to normal levels within 7-14 days without changing other lymphocyte populations. The transiently expanded Treg cells are highly activated and display suppressive function in vitro. Adoptive transfer of the expanded Treg cells protected recipient mice from generation of high-titer antibodies following FVIII plasmid challenge. Repeated plasmid transfer is applicable in tolerized mice without eliciting immune responses. Mice treated with IL-2 complexes mounted immune responses against both T-dependent and T-independent neoantigens, indicating that IL-2 complexes did not hamper the immune system for long. These results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses and the potential of developing Treg cell expansion therapies that induce long-term tolerance to FVIII.

  20. A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation.

    Science.gov (United States)

    Hong, Eun-Jin Erica; Villén, Judit; Gerace, Erica L; Gygi, Steven P; Moazed, Danesh

    2005-01-01

    The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4(+)), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4(+), cmc1(+), cmc2(+) and rad24(+) results in a complete loss of silencing of a ura4(+) reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

  1. On the Dehydrocoupling of Alkenylacetylenes Mediated by Various Samarocene Complexes: A Charming Story of Metal Cooperativity Revealing a Novel Dual Metal σ-Bond Metathesis Type of Mechanism (DM|σ-BM

    Directory of Open Access Journals (Sweden)

    Christos E. Kefalidis

    2015-12-01

    Full Text Available The prevailing reductive chemistry of Sm(II has been accessed and explored mostly by the use of samarocene precursors. The highly reducing character of these congeners, along with their Lewis acidity and predominantly ionic bonding, allows for the relatively facile activation of C–H bonds, as well as peculiar transformations of unsaturated substrates (e.g., C–C couplings. Among other important C–C coupling reactions, the reaction of phenylacetylene with different mono- or bimetallic samarocene complexes affords trienediyl complexes of the type {[(C5Me52Sm]2(µ-η2:η2-PhC4Ph}. In contrast, when t-butylacetylene is used, uncoupled monomers of the type (C5Me52Sm(C≡C–tBu were obtained. Although this type of reactivity may appear to be simple, the mechanism underlying these transformations is complex. This conclusion is drawn from the density functional theory (DFT mechanistic studies presented herein. The operating mechanistic paths consist of: (i the oxidation of each samarium center and the concomitant double reduction of the alkyne to afford a binuclear intermediate; (ii the C–H scission of the acetylinic bond that lies in between the two metals; (iii a dual metal σ-bond metathesis (DM|σ-SBM process that releases H2; and eventually (iv the C–C coupling of the two bridged μ-alkynides to give the final bimetallic trienediyl complexes. For the latter mechanistic route, the experimentally used phenylacetylene was considered first as well as the aliphatic hex-1-yne. More interestingly, we shed light into the formation of the mono(alkynide complex, being the final experimental product of the reaction with t-butylacetylene.

  2. Communication complexity and information complexity

    Science.gov (United States)

    Pankratov, Denis

    Information complexity enables the use of information-theoretic tools in communication complexity theory. Prior to the results presented in this thesis, information complexity was mainly used for proving lower bounds and direct-sum theorems in the setting of communication complexity. We present three results that demonstrate new connections between information complexity and communication complexity. In the first contribution we thoroughly study the information complexity of the smallest nontrivial two-party function: the AND function. While computing the communication complexity of AND is trivial, computing its exact information complexity presents a major technical challenge. In overcoming this challenge, we reveal that information complexity gives rise to rich geometrical structures. Our analysis of information complexity relies on new analytic techniques and new characterizations of communication protocols. We also uncover a connection of information complexity to the theory of elliptic partial differential equations. Once we compute the exact information complexity of AND, we can compute exact communication complexity of several related functions on n-bit inputs with some additional technical work. Previous combinatorial and algebraic techniques could only prove bounds of the form theta( n). Interestingly, this level of precision is typical in the area of information theory, so our result demonstrates that this meta-property of precise bounds carries over to information complexity and in certain cases even to communication complexity. Our result does not only strengthen the lower bound on communication complexity of disjointness by making it more exact, but it also shows that information complexity provides the exact upper bound on communication complexity. In fact, this result is more general and applies to a whole class of communication problems. In the second contribution, we use self-reduction methods to prove strong lower bounds on the information

  3. Electrocatalytic reduction of organohalides mediated by the dihalo-molybdenum phosphinic complexes trans-[MoX(2)(Ph(2)PCH(2)CH(2)PPh(2))(2)] (X = I, Br)-A mechanistic study by cyclic voltammetry digital simulation.

    Science.gov (United States)

    Martins, Natércia C T; Guedes da Silva, M Fátima C; Wanke, Riccardo; Pombeiro, Armando J L

    2009-06-28

    trans-[MoX(2)(dppe)(2)] (X = I, Br) act as inner-sphere electron-transfer mediators for the electrocatalytic reduction of organohalides RX to R + X(-), at both Mo(II)--> Mo(I) and Mo(I)--> Mo(0) reduction processes, each of them involving a cathodically induced heterolytic metal-halide bond cleavage with liberation of X(-) that is followed by addition of RX to the metal. Digital simulation of cyclic voltammetry at a wide range of scan rates allowed to estimate the rate constants of the various chemical steps for both electrocatalytic cycles, which were compared in terms of Mo-X bond dissociation energies, electronic and stereochemical effects.

  4. Three tasks for mediatization research

    DEFF Research Database (Denmark)

    Ekstrøm, Mats; Fornäs, Johan; Jansson, André

    2016-01-01

    Based on the interdisciplinary experience of a Swedish research committee, this article discusses critical conceptual issues raised by the current debate on mediatization – a concept that holds great potential to constitute a space for synthesized understandings of media-related social transforma......Based on the interdisciplinary experience of a Swedish research committee, this article discusses critical conceptual issues raised by the current debate on mediatization – a concept that holds great potential to constitute a space for synthesized understandings of media-related social...... transformations. In contrast to other, more metaphorical constructions, mediatization can be studied empirically in systematic ways through various sub-processes that together provide a complex picture of how culture and everyday life evolve in times of media saturation. The first part of this article argues...... that mediatization researchers have sometimes formulated too grand claims as to mediatization’s status as a unitary approach, a meta-theory or a paradigm. Such claims have led to problematic confusions around the concept and should be abandoned in favour of a more open agenda. In line with such a call for openness...

  5. Complexity Plots

    KAUST Repository

    Thiyagalingam, Jeyarajan

    2013-06-01

    In this paper, we present a novel visualization technique for assisting the observation and analysis of algorithmic complexity. In comparison with conventional line graphs, this new technique is not sensitive to the units of measurement, allowing multivariate data series of different physical qualities (e.g., time, space and energy) to be juxtaposed together conveniently and consistently. It supports multivariate visualization as well as uncertainty visualization. It enables users to focus on algorithm categorization by complexity classes, while reducing visual impact caused by constants and algorithmic components that are insignificant to complexity analysis. It provides an effective means for observing the algorithmic complexity of programs with a mixture of algorithms and black-box software through visualization. Through two case studies, we demonstrate the effectiveness of complexity plots in complexity analysis in research, education and application. © 2013 The Author(s) Computer Graphics Forum © 2013 The Eurographics Association and Blackwell Publishing Ltd.

  6. mTOR Hyperactivation by Ablation of Tuberous Sclerosis Complex 2 in the Mouse Heart Induces Cardiac Dysfunction with the Increased Number of Small Mitochondria Mediated through the Down-Regulation of Autophagy.

    Science.gov (United States)

    Taneike, Manabu; Nishida, Kazuhiko; Omiya, Shigemiki; Zarrinpashneh, Elham; Misaka, Tomofumi; Kitazume-Taneike, Rika; Austin, Ruth; Takaoka, Minoru; Yamaguchi, Osamu; Gambello, Michael J; Shah, Ajay M; Otsu, Kinya

    2016-01-01

    Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth, proliferation and metabolism. mTORC1 regulates protein synthesis positively and autophagy negatively. Autophagy is a major system to manage bulk degradation and recycling of cytoplasmic components and organelles. Tuberous sclerosis complex (TSC) 1 and 2 form a heterodimeric complex and inactivate Ras homolog enriched in brain, resulting in inhibition of mTORC1. Here, we investigated the effects of hyperactivation of mTORC1 on cardiac function and structure using cardiac-specific TSC2-deficient (TSC2-/-) mice. TSC2-/- mice were born normally at the expected Mendelian ratio. However, the median life span of TSC2-/- mice was approximately 10 months and significantly shorter than that of control mice. TSC2-/- mice showed cardiac dysfunction and cardiomyocyte hypertrophy without considerable fibrosis, cell infiltration or apoptotic cardiomyocyte death. Ultrastructural analysis of TSC2-/- hearts revealed misalignment, aggregation and a decrease in the size and an increase in the number of mitochondria, but the mitochondrial function was maintained. Autophagic flux was inhibited, while the phosphorylation level of S6 or eukaryotic initiation factor 4E -binding protein 1, downstream of mTORC1, was increased. The upregulation of autophagic flux by trehalose treatment attenuated the cardiac phenotypes such as cardiac dysfunction and structural abnormalities of mitochondria in TSC2-/- hearts. The results suggest that autophagy via the TSC2-mTORC1 signaling pathway plays an important role in maintenance of cardiac function and mitochondrial quantity and size in the heart and could be a therapeutic target to maintain mitochondrial homeostasis in failing hearts.

  7. The Anti-Tumor Activity of Succinyl Macrolactin A Is Mediated through the β-Catenin Destruction Complex via the Suppression of Tankyrase and PI3K/Akt.

    Directory of Open Access Journals (Sweden)

    Sushil C Regmi

    Full Text Available Accumulated gene mutations in cancer suggest that multi-targeted suppression of affected signaling networks is a promising strategy for cancer treatment. In the present study, we report that 7-O-succinyl macrolactin A (SMA suppresses tumor growth by stabilizing the β-catenin destruction complex, which was achieved through inhibition of regulatory components associated with the complex. SMA significantly reduced the activities of PI3K/Akt, which corresponded with a decrease in GSK3β phosphorylation, an increase in β-catenin phosphorylation, and a reduction in nuclear β-catenin content in HT29 human colon cancer cells. At the same time, the activity of tankyrase, which inhibits the β-catenin destruction complex by destabilizing the axin level, was suppressed by SMA. Despite the low potency of SMA against tankyrase activity (IC50 of 50.1 μM and 15.5 μM for tankyrase 1 and 2, respectively compared to XAV939 (IC50 of 11 nM for tankyrase 1, a selective and potent tankyrase inhibitor, SMA had strong inhibitory effects on β-catenin-dependent TCF/LEF1 transcriptional activity (IC50 of 39.8 nM, which were similar to that of XAV939 (IC50 of 28.1 nM. In addition to suppressing the colony forming ability of colon cancer cells in vitro, SMA significantly inhibited tumor growth in CT26 syngenic and HT29 xenograft mouse tumor models. Furthermore, treating mice with SMA in combination with 5-FU in a colon cancer xenograft model or with cisplatin in an A549 lung cancer xenograft model resulted in greater anti-tumor activity than did treatment with the drugs alone. In the xenograft tumor tissues, SMA dose-dependently inhibited nuclear β-catenin along with reductions in GSK3β phosphorylation and increases in axin levels. These results suggest that SMA is a possible candidate as an effective anti-cancer agent alone or in combination with cytotoxic chemotherapeutic drugs, such as 5-FU and cisplatin, and that the mode of action for SMA involves stabilization

  8. Metal complexes with 2,3-bis(diphenylphosphino)-1,4-diazadiene ligands: synthesis, structures, and an intramolecular metal-mediated [4 + 2] cycloaddition employing a benzene ring as a dienophile.

    Science.gov (United States)

    Walther, Dirk; Liesicke, Stefan; Böttcher, Lars; Fischer, Reinald; Görls, Helmar; Vaughan, Gavin

    2003-01-27

    2,3-Bis(diphenylphosphino)-1,4-diazadienes RN=C(PPh2)-C(PPh2)=NR (1a, R = 4-tolyl; 1b, R = 4-tert-butylphenyl; 1c, R = mesityl) were used as novel ligands for transition metals. The metal complexes [(1c)Mo(CO)4] (2a), [(1c)[Mo(CO)4]2] (2b), [(1a)Cu(Cl)(PPh3)] (3), and [(1b)[(NiBr2(THF))]2] (4) were characterized by elemental analysis, MS, and 31P[1H], 1H, and 13C NMR spectra (except the paramagnetic complex 4). Additionally, the molecular structure of the complexes in the solid state was determined by single-crystal X-ray diffraction. In 2a and 2b the chelating ligand coordinates via the N,P donor set, whereas in 3 the chelating ligand coordinates via the two P atoms. 4 contains a square-planar (P,P)NiBr2 moiety on the one side of the bridging ligand 1b. On the opposite side the 1,2-dimine unit bonds to another Ni center having octahedral geometry. The bulkier ligand 1c reacts to form the mononuclear compound 5. X-ray diffraction analysis of single crystals shows that 5 contains a quinoxaline derivative with a cyclohexa-1,3-diene ring in the peripheral position. Furthermore, it contains a bis(diphenylphosphino)-ethylene unit coordinating the NiBr2. This arrangement is the result of an intramolecular [4 + 2] cycloaddition between the 1,2-diimine unit (as diheterodiene) and the benzene ring of the 4-tolyl-N substituent (as dieneophile). The same type of ring-closing reaction followed by a tautomerization reaction to form the mononuclear compound 6 occurred by dissolution of the binuclear complex 4 in methanol. This reaction can be used as a simple method for the synthesis of novel 1,2-bis(diarylphosphanyl)ethylenes containing a quinoxaline backbone.

  9. Engaging complexity

    Directory of Open Access Journals (Sweden)

    Gys M. Loubser

    2014-01-01

    Full Text Available In this article, I discuss studies in complexity and its epistemological implications for systematic and practical theology. I argue that engagement with complexity does not necessarily assurea non-reductionist approach. However, if complexity is engaged transversally, it becomes possible to transcend reductionist approaches. Moreover, systematic and practical the ologians can draw on complexity in developing new ways of understanding and, therefore, new ways of describing the focus, epistemic scope and heuristic structures of systematic and practical theology. Firstly, Edgar Morin draws a distinction between restricted and general complexity based on the epistemology drawn upon in studies in complexity. Moving away from foundationalist approaches to epistemology, Morin argues for a paradigm of systems. Secondly,I discuss Kees van Kooten Niekerk�s distinction between epistemology, methodology andontology in studies in complexity and offer an example of a theological argument that drawson complexity. Thirdly, I argue for the importance of transversality in engaging complexity by drawing on the work of Wentzel van Huyssteen and Paul Cilliers. In conclusion, I argue that theologians have to be conscious of the epistemic foundations of each study in complexity, and these studies illuminate the heart of Reformed theology.Intradisciplinary and/or interdisciplinary implications: Therefore, this article has both intradisciplinary and interdisciplinary implications. When theologians engage studies incomplexity, the epistemological roots of these studies need to be considered seeing thatresearchers in complexity draw on different epistemologies. Drawing on transversality wouldenhance such considerations. Furthermore, Edgar Morin�s and Paul Cilliers� approach tocomplexity will inform practical and theoretical considerations in church polity and unity.

  10. mediation: R Package for Causal Mediation Analysis

    Directory of Open Access Journals (Sweden)

    Dustin Tingley

    2014-09-01

    Full Text Available In this paper, we describe the R package mediation for conducting causal mediation analysis in applied empirical research. In many scientific disciplines, the goal of researchers is not only estimating causal effects of a treatment but also understanding the process in which the treatment causally affects the outcome. Causal mediation analysis is frequently used to assess potential causal mechanisms. The mediation package implements a comprehensive suite of statistical tools for conducting such an analysis. The package is organized into two distinct approaches. Using the model-based approach, researchers can estimate causal mediation effects and conduct sensitivity analysis under the standard research design. Furthermore, the design-based approach provides several analysis tools that are applicable under different experimental designs. This approach requires weaker assumptions than the model-based approach. We also implement a statistical method for dealing with multiple (causally dependent mediators, which are often encountered in practice. Finally, the package also offers a methodology for assessing causal mediation in the presence of treatment noncompliance, a common problem in randomized trials.

  11. CD4 T cells mediate both positive and negative regulation of the immune response to HIV infection: complex role of T follicular helper cells and Regulatory T cells in pathogenesis

    Directory of Open Access Journals (Sweden)

    Chansavath ePhetsouphanh

    2015-01-01

    Full Text Available HIV-1 infection results in chronic activation of cells in lymphoid tissue, including T cells, B cells and myeloid lineage cells. The resulting characteristic hyperplasia is an amalgam of proliferating host immune cells in the adaptive response, increased concentrations of innate response mediators due to viral and bacterial products, and homeostatic responses to inflammation. While it is generally thought that CD4 T cells are greatly depleted, in fact, two types of CD4 T cells appear to be increased, namely regulatory T cells (Tregs and T follicular helper cells (Tfh. These cells have opposing roles, but may both be important in the pathogenic process. Whether Tregs are failing in their role to limit lymphocyte activation is unclear, but there is no doubt now that Tfh are associated with B cell hyperplasia and increased germinal centre activity. Antiretroviral therapy (ART may reduce the lymphocyte activation, but not completely, and therefore there is a need for interventions that selectively enhance normal CD4 function without exacerbating Tfh, B cell or Treg dysfunction.

  12. MEDIATION AGREEMENTS LEGAL MODEL

    Directory of Open Access Journals (Sweden)

    Alexander Ponomarev

    2015-07-01

    Full Text Available This article focuses on the legal model of mediation agreements in Russian and international legislation. The authors consider the main provisions of the mediation agreements in civil matters, in particular, is defined by such features of the legal model as the requirements for this type of agreements. In addition, the article discusses the problematic issues of implementation of mediation agreements.

  13. Mediation as Signal

    NARCIS (Netherlands)

    Holler, M.J.; Lindner, I.

    2004-01-01

    This paper analyzes mediation as a signal. Starting from a stylized case, a game theoretical model of one-sided incomplete information, taken from Cho and Kreps (1987), is applied to discuss strategic effects of mediation. It turns out that to reject mediation can be interpreted as a ”negative signa

  14. Mediation in Special Education.

    Science.gov (United States)

    Fielding, Pamela S.

    1990-01-01

    This article presents a perspective for viewing mediation in resolving conflicts between parents and school personnel about the education of handicapped students. The appropriateness of mediation as well as its limitations are discussed, as are current uses of mediation in special education and legal problems and issues arising from its use.…

  15. Bayesian Mediation Analysis

    Science.gov (United States)

    Yuan, Ying; MacKinnon, David P.

    2009-01-01

    In this article, we propose Bayesian analysis of mediation effects. Compared with conventional frequentist mediation analysis, the Bayesian approach has several advantages. First, it allows researchers to incorporate prior information into the mediation analysis, thus potentially improving the efficiency of estimates. Second, under the Bayesian…

  16. Computational Complexity

    Directory of Open Access Journals (Sweden)

    J. A. Tenreiro Machado

    2017-02-01

    Full Text Available Complex systems (CS involve many elements that interact at different scales in time and space. The challenges in modeling CS led to the development of novel computational tools with applications in a wide range of scientific areas. The computational problems posed by CS exhibit intrinsic difficulties that are a major concern in Computational Complexity Theory. [...

  17. Complex narratives

    NARCIS (Netherlands)

    Simons, J.

    2008-01-01

    This paper brings together narratology, game theory, and complexity theory to untangle the intricate nature of complex narratives in contemporary cinema. It interrogates the different terms - forking-path narratives, mind-game films, modular narratives, multiple-draft films, database narratives, puz

  18. Complex odontoma.

    Science.gov (United States)

    Preetha, A; Balikai, Bharati S; Sujatha, D; Pai, Anuradha; Ganapathy, K S

    2010-01-01

    Odontomas are hamartomatous lesions or malformations composed of mature enamel, dentin, and pulp. They may be compound or complex, depending on the extent of morphodifferentiation or their resemblance to normal teeth. The etiology of odontoma is unknown, although several theories have been proposed. This article describes a case of a large infected complex odontoma in the residual mandibular ridge, resulting in considerable mandibular expansion.

  19. Simplifying complexity

    NARCIS (Netherlands)

    Leemput, van de I.A.

    2016-01-01

    In this thesis I use mathematical models to explore the properties of complex systems ranging from microbial nitrogen pathways and coral reefs to the human state of mind. All are examples of complex systems, defined as systems composed of a number of interconnected parts, where the systemic behavior

  20. Catalytic mechanisms of direct pyrrole synthesis via dehydrogenative coupling mediated by PNP-Ir or PNN-Ru pincer complexes: Crucial role of proton-transfer shuttles in the PNP-Ir system

    KAUST Repository

    Qu, Shuanglin

    2014-04-02

    Kempe et al. and Milstein et al. have recently advanced the dehydrogenative coupling methodology to synthesize pyrroles from secondary alcohols (e.g., 3) and β-amino alcohols (e.g., 4), using PNP-Ir (1) and PNN-Ru (2) pincer complexes, respectively. We herein present a DFT study to characterize the catalytic mechanism of these reactions. After precatalyst activation to give active 1A/2A, the transformation proceeds via four stages: 1A/2A-catalyzed alcohol (3) dehydrogenation to give ketone (11), base-facilitated C-N coupling of 11 and 4 to form an imine-alcohol intermediate (18), base-promoted cyclization of 18, and catalyst regeneration via H2 release from 1R/2R. For alcohol dehydrogenations, the bifunctional double hydrogen-transfer pathway is more favorable than that via β-hydride elimination. Generally, proton-transfer (H-transfer) shuttles facilitate various H-transfer processes in both systems. Notwithstanding, H-transfer shuttles play a much more crucial role in the PNP-Ir system than in the PNN-Ru system. Without H-transfer shuttles, the key barriers up to 45.9 kcal/mol in PNP-Ir system are too high to be accessible, while the corresponding barriers (<32.0 kcal/mol) in PNN-Ru system are not unreachable. Another significant difference between the two systems is that the addition of alcohol to 1A giving an alkoxo complex is endergonic by 8.1 kcal/mol, whereas the addition to 2A is exergonic by 8.9 kcal/mol. The thermodynamic difference could be the main reason for PNP-Ir system requiring lower catalyst loading than the PNN-Ru system. We discuss how the differences are resulted in terms of electronic and geometric structures of the catalysts and how to use the features in catalyst development. © 2014 American Chemical Society.

  1. Dynamic interaction of hTRPC6 with the Orai1-STIM1 complex or hTRPC3 mediates its role in capacitative or non-capacitative Ca(2+) entry pathways.

    Science.gov (United States)

    Jardin, Isaac; Gómez, Luis J; Salido, Gines M; Rosado, Juan A

    2009-05-13

    TRPC (canonical transient receptor potential) channel subunits have been shown to assemble into homo- or hetero-meric channel complexes, including different Ca2+-handling proteins, required for the activation of CCE (capacitative Ca2+ entry) or NCCE (non-CCE) pathways. In the present study we found evidence for the dynamic interaction between endogenously expressed hTRPC6 (human TRPC6) with either both Orai1 and STIM1 (stromal interaction molecule 1) or hTRPC3 to participate in CCE or NCCE. Electrotransjection of cells with an anti-hTRPC6 antibody, directed towards the C-terminal region, reduces CCE induced by TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine], which reduces the intraluminal free Ca2+ concentration. Cell stimulation with thrombin or extensive Ca2+-store depletion by TG (thapsigargin)+ionomycin enhanced the interaction between hTRPC6 and the CCE proteins Orai1 and STIM1. In contrast, stimulation with the diacylglycerol analogue OAG (1-oleoyl-2-acetyl-sn-glycerol) displaces hTRPC6 from Orai1 and STIM1 and enhances the association between hTRPC6 and hTRPC3. The interaction between hTRPC6 and hTRPC3 was abolished by dimethyl-BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid] loading, which indicates that this phenomenon is Ca2+-dependent. These findings support the hypothesis that hTRPC6 participates both in CCE and NCCE through its interaction with the Orai1-STIM1 complex or hTRPC3 respectively.

  2. Complex variables

    CERN Document Server

    Fisher, Stephen D

    1999-01-01

    The most important topics in the theory and application of complex variables receive a thorough, coherent treatment in this introductory text. Intended for undergraduates or graduate students in science, mathematics, and engineering, this volume features hundreds of solved examples, exercises, and applications designed to foster a complete understanding of complex variables as well as an appreciation of their mathematical beauty and elegance. Prerequisites are minimal; a three-semester course in calculus will suffice to prepare students for discussions of these topics: the complex plane, basic

  3. Managing Complexity

    DEFF Research Database (Denmark)

    Maylath, Bruce; Vandepitte, Sonia; Minacori, Patricia

    2013-01-01

    This article discusses the largest and most complex international learning-by-doing project to date- a project involving translation from Danish and Dutch into English and editing into American English alongside a project involving writing, usability testing, and translation from English into Dut...... and into French. The complexity of the undertaking proved to be a central element in the students' learning, as the collaboration closely resembles the complexity of international documentation workplaces of language service providers. © Association of Teachers of Technical Writing....

  4. Lecithin Complex

    African Journals Online (AJOL)

    yellow power was collected as polydatin-lecithin complex. ... performed on an Agilent 1260 HPLC system. The injection volume .... rabbits. Biomed. Pharmacother 2009; 63: 457-462. 4. Liu B, Du J, Zeng J, Chen C, Niu S. Characterization and.

  5. Oxidation of guanine by carbonate radicals derived from photolysis of carbonatotetramminecobalt(III) complexes and the pH dependence of intrastrand DNA cross-links mediated by guanine radical reactions.

    Science.gov (United States)

    Crean, Conor; Lee, Young Ae; Yun, Byeong Hwa; Geacintov, Nicholas E; Shafirovich, Vladimir

    2008-08-11

    The carbonate radical anion CO(3)(*-) is a decomposition product of nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, an important biological byproduct of the inflammatory response. The selective oxidation of guanine in DNA by CO(3)(*-) radicals is known to yield spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) products, and also a novel intrastrand cross-linked product: 5'-d(CCATCG*CT*ACC), featuring a linkage between guanine C8 (G*) and thymine N3 (T*) atoms in the oligonucleotide (Crean et al., Nucleic Acids Res. 2008, 36, 742-755). Involvement of the T-N3 (pK(a) of N3-H is 9.67) suggests that the formation of 5'-d(CCATCG*CT*ACC) might be pH-dependent. This hypothesis was tested by generating CO(3)(*-) radicals through the photodissociation of carbonatotetramminecobalt(III) complexes by steady-state UV irradiation, which allowed for studies of product yields in the pH 5.0-10.0 range. The yield of 5'-d(CCATCG*CT*ACC) at pH 10.0 is approximately 45 times greater than at pH 5.0; this is consistent with the proposed mechanism, which requires N3(H) thymine proton dissociation followed by nucleophilic addition to the C8 guanine radical.

  6. Oxoaporphine Metal Complexes (CoII, NiII, ZnII) with High Antitumor Activity by Inducing Mitochondria-Mediated Apoptosis and S-phase Arrest in HepG2

    Science.gov (United States)

    Qin, Jiao-Lan; Shen, Wen-Ying; Chen, Zhen-Feng; Zhao, Li-Fang; Qin, Qi-Pin; Yu, Yan-Cheng; Liang, Hong

    2017-01-01

    Three new oxoaporphine Co(II), Ni(II) and Zn(II) complexes 1–3 have been synthesized and fully characterized. 1–3 have similar mononuclear structures with the metal and ligand ratio of 1:2. 1–3 exhibited higher cytotoxicity than the OD ligand and cisplatin against HepG2, T-24, BEL-7404, MGC80–3 and SK-OV-3/DDP cells, with IC50 value of 0.23−4.31 μM. Interestingly, 0.5 μM 1–3 significantly caused HepG2 arrest at S-phase, which was associated with the up-regulation of p53, p21, p27, Chk1 and Chk2 proteins, and decrease in cyclin A, CDK2, Cdc25A, PCNA proteins. In addition, 1–3 induced HepG2 apoptosis via a caspase-dependent mitochondrion pathway as evidenced by p53 activation, ROS production, Bax up-regulation and Bcl-2 down-regulation, mitochondrial dysfunction, cytochrome c release, caspase activation and PARP cleavage. Furthermore, 3 inhibited tumor growth in HepG2 xenograft model, and displayed more safety profile in vivo than cisplatin. PMID:28436418

  7. Water mediated ligand functional group cooperativity: the contribution of a methyl group to binding affinity is enhanced by a COO(-) group through changes in the structure and thermodynamics of the hydration waters of ligand-thermolysin complexes.

    Science.gov (United States)

    Nasief, Nader N; Tan, Hongwei; Kong, Jing; Hangauer, David

    2012-10-11

    Ligand functional groups can modulate the contributions of one another to the ligand-protein binding thermodynamics, producing either positive or negative cooperativity. Data presented for four thermolysin phosphonamidate inhibitors demonstrate that the differential binding free energy and enthalpy caused by replacement of a H with a Me group, which binds in the well-hydrated S2' pocket, are more favorable in presence of a ligand carboxylate. The differential entropy is however less favorable. Dissection of these differential thermodynamic parameters, X-ray crystallography, and density-functional theory calculations suggest that these cooperativities are caused by variations in the thermodynamics of the complex hydration shell changes accompanying the H→Me replacement. Specifically, the COO(-) reduces both the enthalpic penalty and the entropic advantage of displacing water molecules from the S2' pocket and causes a subsequent acquisition of a more enthalpically, less entropically, favorable water network. This study contributes to understanding the important role water plays in ligand-protein binding.

  8. Mediators of homologous DNA pairing.

    Science.gov (United States)

    Zelensky, Alex; Kanaar, Roland; Wyman, Claire

    2014-10-09

    Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  9. Complex analysis

    CERN Document Server

    Freitag, Eberhard

    2005-01-01

    The guiding principle of this presentation of ``Classical Complex Analysis'' is to proceed as quickly as possible to the central results while using a small number of notions and concepts from other fields. Thus the prerequisites for understanding this book are minimal; only elementary facts of calculus and algebra are required. The first four chapters cover the essential core of complex analysis: - differentiation in C (including elementary facts about conformal mappings) - integration in C (including complex line integrals, Cauchy's Integral Theorem, and the Integral Formulas) - sequences and series of analytic functions, (isolated) singularities, Laurent series, calculus of residues - construction of analytic functions: the gamma function, Weierstrass' Factorization Theorem, Mittag-Leffler Partial Fraction Decomposition, and -as a particular highlight- the Riemann Mapping Theorem, which characterizes the simply connected domains in C. Further topics included are: - the theory of elliptic functions based on...

  10. Complex Networks

    CERN Document Server

    Evsukoff, Alexandre; González, Marta

    2013-01-01

    In the last decade we have seen the emergence of a new inter-disciplinary field focusing on the understanding of networks which are dynamic, large, open, and have a structure sometimes called random-biased. The field of Complex Networks is helping us better understand many complex phenomena such as the spread of  deseases, protein interactions, social relationships, to name but a few. Studies in Complex Networks are gaining attention due to some major scientific breakthroughs proposed by network scientists helping us understand and model interactions contained in large datasets. In fact, if we could point to one event leading to the widespread use of complex network analysis is the availability of online databases. Theories of Random Graphs from Erdös and Rényi from the late 1950s led us to believe that most networks had random characteristics. The work on large online datasets told us otherwise. Starting with the work of Barabási and Albert as well as Watts and Strogatz in the late 1990s, we now know th...

  11. Let's talk conflict: using mediation in healthcare security disputes.

    Science.gov (United States)

    Armstrong, Brad

    2013-01-01

    Healthcare security administrators face weekly, if not daily challenges and conflict. This article considers that security leaders must anticipate disputes in highly complex healthcare systems. When disputes cannot be resolved by organizational efforts, security administrators may be in a position to recommend or participate in mediation. Here the concept of mediation is introduced to healthcare security leaders as a viable means to resolve disputes with patients, visitors, and the community. This includes a description of the facilitative versus evaluative mediation processes, as well as pragmatic guidance when preparing for mediation.

  12. Complex chemistry with complex compounds

    Directory of Open Access Journals (Sweden)

    Eichler Robert

    2016-01-01

    Full Text Available In recent years gas-phase chemical studies assisted by physical pre-separation allowed for the investigation of fragile single molecular species by gas-phase chromatography. The latest success with the heaviest group 6 transactinide seaborgium is highlighted. The formation of a very volatile hexacarbonyl compound Sg(CO6 was observed similarly to its lighter homologues molybdenum and tungsten. The interactions of these gaseous carbonyl complex compounds with quartz surfaces were investigated by thermochromatography. Second-generation experiments are under way to investigate the intramolecular bond between the central metal atom of the complexes and the ligands addressing the influence of relativistic effects in the heaviest compounds. Our contribution comprises some aspects of the ongoing challenging experiments as well as an outlook towards other interesting compounds related to volatile complex compounds in the gas phase.

  13. Ruimte voor mediation

    NARCIS (Netherlands)

    Combrink-Kuiters, L.; Niemeijer, E.; Voert, M. ter

    2003-01-01

    Dit onderzoek is samen met ADR en mediation Rechterlijke macht gepubliceerd. Het doel was enerzijds na te gaan of, en onder welke condities, mediation in de Nederlandse context een effectief en efficiënt alternatief is voor de gerechtelijke geschillenbeslechting. Anderzijds inzicht te verwerven in d

  14. Teaching Mediated Public Relations.

    Science.gov (United States)

    Kent, Michael L.

    2001-01-01

    Discusses approaches to teaching a mediated public relations course, emphasizing the World Wide Web. Outlines five course objectives, assignments and activities, evaluation, texts, and lecture topics. Argues that students mastering these course objectives will understand ethical issues relating to media use, using mediated technology in public…

  15. Dynamic public service mediation

    NARCIS (Netherlands)

    Hofman, W.; Staalduinen, M. van

    2010-01-01

    This paper presents an approach to dynamic public service mediation. It is based on a conceptual model and the use of search and ranking algorithms. The conceptual model is based on Abstract State Machine theory. Requirements for dynamic service mediation were derived from a real-world case. The con

  16. PIC Activation through Functional Interplay between Mediator and TFIIH.

    Science.gov (United States)

    Malik, Sohail; Molina, Henrik; Xue, Zhu

    2017-01-06

    The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process.

  17. Managing Complexity

    Energy Technology Data Exchange (ETDEWEB)

    Chassin, David P.; Posse, Christian; Malard, Joel M.

    2004-08-01

    Physical analogs have shown considerable promise for understanding the behavior of complex adaptive systems, including macroeconomics, biological systems, social networks, and electric power markets. Many of today’s most challenging technical and policy questions can be reduced to a distributed economic control problem. Indeed, economically-based control of large-scale systems is founded on the conjecture that the price-based regulation (e.g., auctions, markets) results in an optimal allocation of resources and emergent optimal system control. This paper explores the state of the art in the use physical analogs for understanding the behavior of some econophysical systems and to deriving stable and robust control strategies for them. In particular we review and discussion applications of some analytic methods based on the thermodynamic metaphor according to which the interplay between system entropy and conservation laws gives rise to intuitive and governing global properties of complex systems that cannot be otherwise understood.

  18. [Mediation in health].

    Science.gov (United States)

    Decastello, Alice

    2008-02-10

    The author presents mediation as an alternative dispute resolution method. Mediation is a process where the parties are ready to settle their dispute out of court, by way of negotiation and with the involvement of an independent third person as mediator. In the mediation process the mediator shall not decide the dispute, nor examine the default or give legal advice or express his/her opinion - the mediator's duty is to help the parties bring their positions closer and come to a settlement agreement within a short time (120 days). The author gives a summary of the applications of the Hungarian Act on Mediation in Public Health and draws conclusions from the practical experience since entry into force of the legislation and illustrates the advantages of mediation over the court procedure (which may drag on for years). The primary advantages of mediation are that both the mediators and the parties are bound by the obligation of secrecy, the procedure is cheaper than the court proceedings, and the parties can "save their faces" because in mediation the winner-winner formula asserts itself - against lawsuits where the winner-loser positions are confronted. The author also analyses the specific data and information available so far. As for the future, the legislation needs to be amended at several points. It is particularly expedient to regulate the legal relationship between the insurance companies and the health service providers because the liability insurance may not cover the damages the courts adjudicate. And so some of the service providers may go bankrupt as the difference in excess of the upper limit of coverage - it might as well be up to HUF 5 million per case - shall be paid from own budget, to the charge of the upkeep costs. It is also required to review and amend the regulations on expert activities, just as it is inevitable to make data supply compulsory - otherwise it will be impossible to monitor the number of mediation procedures in health. At present

  19. General resonance mediation

    Energy Technology Data Exchange (ETDEWEB)

    McGarrie, Moritz

    2012-07-15

    We extend the framework of general gauge mediation to cases where the mediating fields have a nontrivial spectral function, as might arise from strong dynamics. We demonstrate through examples that this setup describes a broad class of possible models of gauge mediated supersymmetry breaking. A main emphasis is to give general formulas for cross sections for {sigma}(visible {yields} hidden) in these resonance models. We will also give formulas for soft masses, A-terms and demonstrate the framework with a holographic setup.

  20. Sensemaking in Technology-Use Mediation

    DEFF Research Database (Denmark)

    Bansler, Jørgen P.; Havn, Erling C.

    2006-01-01

    of advanced CSCW technologies is basically a problem of sensemaking. We analyze how a group of “technology-use mediators” (Orlikowski et al. Org. Sci. (1995) 6(4), 423) in a large, multinational company adapted a groupware technology (a “virtual workspace”) to the local organizational context (and vice versa......) by modifying features of the technology, providing ongoing support for users, and promoting appropriate conventions of use. Our findings corroborate earlier research on technology-use mediation, which suggests that such mediators can exert considerable influence on how a particular technology...... will be established and used in an organization. However, we also find that the process of technology-use mediation is much more complex and indeterminate than prior research suggests. The reason being, we argue, that new, advanced CSCW technologies, such as “virtual workspaces” and other groupware applications...

  1. Complex variables

    CERN Document Server

    Taylor, Joseph L

    2011-01-01

    The text covers a broad spectrum between basic and advanced complex variables on the one hand and between theoretical and applied or computational material on the other hand. With careful selection of the emphasis put on the various sections, examples, and exercises, the book can be used in a one- or two-semester course for undergraduate mathematics majors, a one-semester course for engineering or physics majors, or a one-semester course for first-year mathematics graduate students. It has been tested in all three settings at the University of Utah. The exposition is clear, concise, and lively

  2. Complex variables

    CERN Document Server

    Flanigan, Francis J

    2010-01-01

    A caution to mathematics professors: Complex Variables does not follow conventional outlines of course material. One reviewer noting its originality wrote: ""A standard text is often preferred [to a superior text like this] because the professor knows the order of topics and the problems, and doesn't really have to pay attention to the text. He can go to class without preparation."" Not so here-Dr. Flanigan treats this most important field of contemporary mathematics in a most unusual way. While all the material for an advanced undergraduate or first-year graduate course is covered, discussion

  3. Natural generalized mirage mediation

    CERN Document Server

    Baer, Howard; Serce, Hasan; Tata, Xerxes

    2016-01-01

    In the supersymmetric scenario known as mirage mediation (MM), the soft SUSY breaking terms receive comparable anomaly-mediation and moduli-mediation contributions leading to the phenomenon of mirage unification. The simplest MM SUSY breaking models which are consistent with the measured Higgs mass and sparticle mass constraints are strongly disfavoured by fine-tuning considerations. However, while MM makes robust predictions for gaugino masses, the scalar sector is quite sensitive to specific mechanisms for moduli stabilization and potential uplifting. We suggest here a broader setup of generalized mirage mediation (GMM), where heretofore discrete parameters are allowed as continuous to better parametrize these other schemes. We find that natural SUSY spectra consistent with both the measured value of m(h). as well as LHC lower bounds on superpartner masses are then possible. We explicitly show that models generated from natural GMM may be beyond the reach of even high-luminosity LHC searches. In such a case...

  4. The Maieutic Force of Mediating Instruments

    DEFF Research Database (Denmark)

    Revellino, Silvana; Mouritsen, Jan

    This paper investigates how compromising plays a role when framing, developing and appraising capital budgeting projects. Motivated by Miller & O’Leary’s (1997, 2005a, 2005b, 2007) call for further research on capital budgeting to include not only valuation practices but also practices for managing...... and coordinating complex investments in inter-organizational spaces, the paper explores the role of the Chronoprogram in managing compromises and directing attention to concerns in relation to the development of an Italian piece of motorway(the Variante di Valico).The Chronoprogram acts as a mediating instrument...... transform actions in unexpected directions which reconfigure the capital budgeting complex investment project and also extend the motorway object. The paper thus further develops the notion of mediating instruments by highlighting their maieutic ability to address the dialectics between matters of fact...

  5. Mechanisms Mediating the Perception of Complex Acoustic Patterns

    Science.gov (United States)

    1990-11-09

    Wisconsin, 53201. 2. This study was supported in part by grants from the National Institute on Deafness and Other Communication Disorders (DC 00208) and...verbal form (mostly nonsense). However, the syllables reported always followed the phonological rules of English. In addition to the form, there was

  6. Gold nanoparticle-fluorophore complex for conditionally fluorescing signal mediator

    Energy Technology Data Exchange (ETDEWEB)

    Wang Jianting [Department of Chemical Engineering University of, Louisville, KY 40292 (United States); Achilefu, Samuel [Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Nantz, Michael [Department of Chemistry, University of Louisville, Louisville, KY 40292 (United States); Kang, Kyung A., E-mail: kyung.kang@louisville.edu [Department of Chemical Engineering University of, Louisville, KY 40292 (United States)

    2011-06-10

    Fluorescent contrast agents with high specificity and sensitivity are valuable for accurate disease detection and diagnosis. Spherical gold nanoparticles (GNPs) can be smartly utilized for developing highly effective agents. The strong electromagnetic (plasmon) field on their surface can be very effective in influencing the electrons of fluorophores and, thus, manipulating the fluorescence output (i.e., either quenching or enhancement). Fluorescence quenching can be used for negative sensing, or for conditional de-quenching to increase the specificity. Fluorescence enhancement allows sensing to be more sensitive. The level of fluorescence alteration depends on the GNP size, the excitation and emission wavelengths and quantum yield of the fluorophore, and the distance between the GNP and the fluorophore. To understand the mechanisms of the fluorescence change by GNP, we have theoretically analyzed the parameters involved in the fluorescence alteration for commonly used fluorophores, with an emphasis on quenching. The results showed that the fluorescence of fluorophores with the excitation (Ex) and emission (Ex) wavelengths close to the GNP resonance peak tended to be significantly quenched by GNPs. For those fluorophores emitting fluorescence in red or near infrared, to achieve quenching, the distance between GNP and the fluorophore was required to be very short. In general, a shorter distance resulted in more quenching. Bigger GNPs require a shorter distance to achieve the same level of quenching. The fluorescence of a fluorophore with a lower quantum yield (especially the one with emission in far-red or near-infrared) is more difficult to be quenched by GNPs (requires very short distance). Instead, it can be enhanced. Based on the theoretical study, we have developed a near-infrared contrast agent, i.e., Cypate conjugated GNP via a short peptide spacer. Normally the fluorescence of Cypate was quenched. The spacer has a motif of a substrate for urokinase type plasminogen activator (uPA; cancer-secreting enzyme). This contrast agent emits fluorescence only in the presence of uPA, where the uPA cleaves the spacer. This design can be used in characterization of the cancer type and also in diagnosing other diseases with signature enzymes.

  7. Complex dynamics

    CERN Document Server

    Carleson, Lennart

    1993-01-01

    Complex dynamics is today very much a focus of interest. Though several fine expository articles were available, by P. Blanchard and by M. Yu. Lyubich in particular, until recently there was no single source where students could find the material with proofs. For anyone in our position, gathering and organizing the material required a great deal of work going through preprints and papers and in some cases even finding a proof. We hope that the results of our efforts will be of help to others who plan to learn about complex dynamics and perhaps even lecture. Meanwhile books in the field a. re beginning to appear. The Stony Brook course notes of J. Milnor were particularly welcome and useful. Still we hope that our special emphasis on the analytic side will satisfy a need. This book is a revised and expanded version of notes based on lectures of the first author at UCLA over several \\Vinter Quarters, particularly 1986 and 1990. We owe Chris Bishop a great deal of gratitude for supervising the production of cour...

  8. DREDed Anomaly Mediation

    CERN Document Server

    Boyda, E; Pierce, A T; Boyda, Ed; Murayama, Hitoshi; Pierce, Aaron

    2002-01-01

    We offer a guide to dimensional reduction (DRED) in theories with anomaly mediated supersymmetry breaking. Evanescent operators proportional to epsilon arise in the bare Lagrangian when it is reduced from d=4 to d= (4-2 epsilon) dimensions. In the course of a detailed diagrammatic calculation, we show that inclusion of these operators is crucial. The evanescent operators conspire to drive the supersymmetry-breaking parameters along anomaly-mediation trajectories across heavy particle thresholds, guaranteeing the ultraviolet insensitivity.

  9. Green oxidations with laccase-mediator systems.

    Science.gov (United States)

    Wells, A; Teria, M; Eve, T

    2006-04-01

    Laccases are oxidase enzymes produced by 'white rot' fungi as part of a complex armoury of redox enzymes used to break down lignin--part of the carbon cycle of nature. Laccases alone or in combination with redox co-catalysts have been shown to oxidize xenobiotic compounds under conditions that can be described as 'green'. This paper describes some novel oxidations using the laccase-mediator method and some current limitations to the use of this technology.

  10. Cosmic Complexity

    Science.gov (United States)

    Mather, John C.

    2012-01-01

    What explains the extraordinary complexity of the observed universe, on all scales from quarks to the accelerating universe? My favorite explanation (which I certainty did not invent) ls that the fundamental laws of physics produce natural instability, energy flows, and chaos. Some call the result the Life Force, some note that the Earth is a living system itself (Gaia, a "tough bitch" according to Margulis), and some conclude that the observed complexity requires a supernatural explanation (of which we have many). But my dad was a statistician (of dairy cows) and he told me about cells and genes and evolution and chance when I was very small. So a scientist must look for me explanation of how nature's laws and statistics brought us into conscious existence. And how is that seemll"!gly Improbable events are actually happening a!1 the time? Well, the physicists have countless examples of natural instability, in which energy is released to power change from simplicity to complexity. One of the most common to see is that cooling water vapor below the freezing point produces snowflakes, no two alike, and all complex and beautiful. We see it often so we are not amazed. But physlc!sts have observed so many kinds of these changes from one structure to another (we call them phase transitions) that the Nobel Prize in 1992 could be awarded for understanding the mathematics of their common features. Now for a few examples of how the laws of nature produce the instabilities that lead to our own existence. First, the Big Bang (what an insufficient name!) apparently came from an instability, in which the "false vacuum" eventually decayed into the ordinary vacuum we have today, plus the most fundamental particles we know, the quarks and leptons. So the universe as a whole started with an instability. Then, a great expansion and cooling happened, and the loose quarks, finding themselves unstable too, bound themselves together into today's less elementary particles like protons and

  11. Cosmic Complexity

    Science.gov (United States)

    Mather, John C.

    2012-01-01

    What explains the extraordinary complexity of the observed universe, on all scales from quarks to the accelerating universe? My favorite explanation (which I certainty did not invent) ls that the fundamental laws of physics produce natural instability, energy flows, and chaos. Some call the result the Life Force, some note that the Earth is a living system itself (Gaia, a "tough bitch" according to Margulis), and some conclude that the observed complexity requires a supernatural explanation (of which we have many). But my dad was a statistician (of dairy cows) and he told me about cells and genes and evolution and chance when I was very small. So a scientist must look for me explanation of how nature's laws and statistics brought us into conscious existence. And how is that seemll"!gly Improbable events are actually happening a!1 the time? Well, the physicists have countless examples of natural instability, in which energy is released to power change from simplicity to complexity. One of the most common to see is that cooling water vapor below the freezing point produces snowflakes, no two alike, and all complex and beautiful. We see it often so we are not amazed. But physlc!sts have observed so many kinds of these changes from one structure to another (we call them phase transitions) that the Nobel Prize in 1992 could be awarded for understanding the mathematics of their common features. Now for a few examples of how the laws of nature produce the instabilities that lead to our own existence. First, the Big Bang (what an insufficient name!) apparently came from an instability, in which the "false vacuum" eventually decayed into the ordinary vacuum we have today, plus the most fundamental particles we know, the quarks and leptons. So the universe as a whole started with an instability. Then, a great expansion and cooling happened, and the loose quarks, finding themselves unstable too, bound themselves together into today's less elementary particles like protons and

  12. Fluorescence properties and conformational stability of the beta-hemocyanin of Helix pomatia.

    Science.gov (United States)

    Idakieva, Krassimira; Siddiqui, Nurul I; Parvanova, Katja; Nikolov, Peter; Gielens, Constant

    2006-04-01

    The beta-hemocyanin (beta-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-beta-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the beta-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-beta-HpH is characterized by a transition temperature (Tm) of 84 degrees C and 63 degrees C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper-dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule.

  13. Metadata based mediator generation

    Energy Technology Data Exchange (ETDEWEB)

    Critchlow, T

    1998-03-01

    Mediators are a critical component of any data warehouse, particularly one utilizing partially materialized views; they transform data from its source format to the warehouse representation while resolving semantic and syntactic conflicts. The close relationship between mediators and databases, requires a mediator to be updated whenever an associated schema is modified. This maintenance may be a significant undertaking if a warehouse integrates several dynamic data sources. However, failure to quickly perform these updates significantly reduces the reliability of the warehouse because queries do not have access to the m current data. This may result in incorrect or misleading responses, and reduce user confidence in the warehouse. This paper describes a metadata framework, and associated software designed to automate a significant portion of the mediator generation task and thereby reduce the effort involved in adapting the schema changes. By allowing the DBA to concentrate on identifying the modifications at a high level, instead of reprogramming the mediator, turnaround time is reduced and warehouse reliability is improved.

  14. Promoter-mediated transcriptional dynamics.

    Science.gov (United States)

    Zhang, Jiajun; Zhou, Tianshou

    2014-01-21

    Genes in eukaryotic cells are typically regulated by complex promoters containing multiple binding sites for a variety of transcription factors, but how promoter dynamics affect transcriptional dynamics has remained poorly understood. In this study, we analyze gene models at the transcriptional regulation level, which incorporate the complexity of promoter structure (PS) defined as transcriptional exits (i.e., ON states of the promoter) and the transition pattern (described by a matrix consisting of transition rates among promoter activity states). We show that multiple exits of transcription are the essential origin of generating multimodal distributions of mRNA, but promoters with the same transition pattern can lead to multimodality of different modes, depending on the regulation of transcriptional factors. In turn, for similar mRNA distributions in the models, the mean ON or OFF time distributions may exhibit different characteristics, thus providing the supplemental information on PS. In addition, we demonstrate that the transcriptional noise can be characterized by a nonlinear function of mean ON and OFF times. These results not only reveal essential characteristics of promoter-mediated transcriptional dynamics but also provide signatures useful for inferring PS based on characteristics of transcriptional outputs. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. The Strategic Mediator

    DEFF Research Database (Denmark)

    Rossignoli, Cecilia; Carugati, Andrea; Mola, Lapo

    2009-01-01

    The last 10 years have witnessed the emergence of electronic marketplaces as players that leverage new technologies to facilitate B2B internet-mediated collaborative business. Nowadays these players are augmenting their services from simple intermediation to include new inter-organizational relat......The last 10 years have witnessed the emergence of electronic marketplaces as players that leverage new technologies to facilitate B2B internet-mediated collaborative business. Nowadays these players are augmenting their services from simple intermediation to include new inter......-marketplace assumes the paradoxical role of strategic mediator: an agent who upholds and heightens the fences of the transactions instead of leveling them. The results have implication in shaping how we see the role of technology as strategic or commoditized....

  16. [Immune-mediated neuropathies].

    Science.gov (United States)

    Stoll, G; Reiners, K

    2016-08-01

    The Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are the most common immune-mediated polyneuropathies, which can show variable clinical and electrophysiological manifestations. Rarer immune-mediated neuropathies encompass paraproteinemic neuropathies (PPN), multifocal motor neuropathy (MMN) and vasculitic neuropathies. The diagnosis usually relies on the history of symptom evolution, distribution of nerve dysfunction and particularly on characteristic features in nerve conduction studies, aided by cerebrospinal fluid (CSF) examination and nerve biopsy findings. The therapeutic toolbox encompasses corticosteroids, immunoglobulins and plasmapheresis often accompanied by long-term immunosuppression. It is important to note that immune-mediated neuropathies selectively respond to treatment and contraindications need to be considered. Despite treatment a considerable number of patients suffer from permanent neurological deficits.

  17. Antibody-Mediated Immunity against Tuberculosis: Implications for Vaccine Development

    OpenAIRE

    Achkar, Jacqueline M; Casadevall, Arturo

    2013-01-01

    There is an urgent need for new and better vaccines against tuberculosis (TB). Current vaccine design strategies are generally focused on the enhancement of cell-mediated immunity. Antibody-based approaches are not being considered, mostly due to the paradigm that humoral immunity plays little role in the protection against intracellular pathogens. Here, we reappraise and update the increasing evidence for antibody-mediated immunity against Mycobacterium tuberculosis, discuss the complexity o...

  18. Mediated intimacy in families

    DEFF Research Database (Denmark)

    Stougaard, Malthe Kirkhoff

    2006-01-01

    Mediating intimacy between children and their parents is still limited investigated and at the same time, we find that, emerging technologies are about to change and affect the way we interact with each other. In this paper, we report from an empirical study where we investigated the social...... with other types of intimate relations such as strong-tie intimacy (couples cohabiting). However, we also identified several issues of intimacy unique to the special relation between children and their parents. These unique acts of intimacy propose challenges when designing technologies for mediated intimacy...

  19. Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.

    Directory of Open Access Journals (Sweden)

    Yumiko Kurokawa

    2008-04-01

    Full Text Available In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog-mediated homologous recombination (HR and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA, which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.

  20. Membrane tethering complexes in the endosomal system

    Directory of Open Access Journals (Sweden)

    Anne eSpang

    2016-05-01

    Full Text Available Vesicles that are generated by endocytic events at the plasma membrane are destined to early endosomes. A prerequisite for proper fusion is the tethering of two membrane entities. Tethering of vesicles to early endosomes is mediated by the CORVET complex, while fusion of late endosomes with lysosomes depends on the HOPS complex. Recycling through the TGN and to the plasma membrane is facilitated by the GARP and EARP complexes, respectively. However, there are other tethering functions in the endosomal system as there are multiple pathways through which proteins can be delivered from endosomes to either the TGN or the plasma membrane. Furthermore, complexes that may be part of novel tethering complexes have been recently identified. Thus it is likely that more tethering factors exist. In this review, I will provide an overview of different tethering complexes of the endosomal system and discuss how they may provide specificity in membrane traffic.

  1. Fungal mediator tail subunits contain classical transcriptional activation domains.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

  2. Mediation and Automatization.

    Science.gov (United States)

    Hutchins, Edwin

    This paper discusses the relationship between the mediation of task performance by some structure that is not inherent in the task domain itself and the phenomenon of automatization, in which skilled performance becomes effortless or phenomenologically "automatic" after extensive practice. The use of a common simple explicit mediating…

  3. Thermally favourable gauge mediation

    Energy Technology Data Exchange (ETDEWEB)

    Dalianis, Ioannis, E-mail: Ioannis.Dalianis@fuw.edu.p [Institute of Theoretical Physics, Faculty of Physics, University of Warsaw, ul. Hoza 69, Warsaw (Poland); Lalak, Zygmunt, E-mail: Zygmunt.Lalak@fuw.edu.p [Institute of Theoretical Physics, Faculty of Physics, University of Warsaw, ul. Hoza 69, Warsaw (Poland)

    2011-03-14

    We discuss the thermal evolution of the spurion and messenger fields of ordinary gauge mediation models taking into account the Standard Model degrees of freedom. It is shown that for thermalized messengers the metastable susy breaking vacuum becomes thermally selected provided that the susy breaking sector is sufficiently weakly coupled to messengers or to any other observable field.

  4. Teachers as mediators

    DEFF Research Database (Denmark)

    Dorf, Hans; Kelly, Peter; Hohmann, Ulrike

    2012-01-01

    Within the context of lower secondary English teaching in South West England, this study identifies in broad terms the competing goals between which English teachers mediate and the explicit and hidden tensions that result. To understand the interactions of competing goals, teachers’ goal...

  5. Mediatization and Government Communication

    DEFF Research Database (Denmark)

    Laursen, Bo; Valentini, Chiara

    2015-01-01

    in the light of mediatization and government communication theories. Without one pan-European public sphere, the European Parliament, like the other European Union (EU) institutions, competes with national actors for the news media’s attention in the EU’s twenty-eight national public spheres, where EU affairs...

  6. Expanding mediation theory

    NARCIS (Netherlands)

    Verbeek, P.P.C.C.

    2012-01-01

    In his article In Between Us, Yoni van den Eede expands existing theories of mediation into the realm of the social and the political, focusing on the notions of opacity and transparency. His approach is rich and promising, but two pitfalls should be avoided. First, his concept of ‘in-between’ runs

  7. String Mediated Supersymmetry Breaking

    Energy Technology Data Exchange (ETDEWEB)

    Brodie, John H

    2001-07-25

    We consider the 3+1 visible sector to live on a Hanany-Witten D-brane construction in type IIA string theory. The messenger sector consists of stretched strings from the visible brane to a hidden D6-brane in the extra spatial dimensions. In the open string channel supersymmetry is broken by gauge mediation while in the closed string channel supersymmetry is broken by gravity mediation. Hence, we call this kind of mediation ''string mediation''. We propose an extension of the Dimopoulos-Georgi theorem to brane models: only detached probe branes can break supersymmetry without generating a tachyon. Fermion masses are generated at one loop if the branes break a sufficient amount of the ten dimensional Lorentz group while scalar potentials are generated if there is a force between the visible brane and the hidden brane. Scalars can be lifted at two loops through a combination of brane bending and brane forces. We find a large class of stable non-supersymmetric brane configurations of ten dimensional string theory.

  8. Natural generalized mirage mediation

    Science.gov (United States)

    Baer, Howard; Barger, Vernon; Serce, Hasan; Tata, Xerxes

    2016-12-01

    In the supersymmetric scenario known as mirage mediation (MM), the soft supersymmetry (SUSY) breaking terms receive comparable anomaly-mediation and moduli-mediation contributions leading to the phenomenon of mirage unification. The simplest MM SUSY breaking models which are consistent with the measured Higgs mass and sparticle mass constraints are strongly disfavored by fine-tuning considerations. However, while MM makes robust predictions for gaugino masses, the scalar sector is quite sensitive to specific mechanisms for moduli stabilization and potential uplifting. We suggest here a broader setup of generalized mirage mediation (GMM), where heretofore discrete parameters are allowed as continuous to better parametrize these other schemes. We find that natural SUSY spectra consistent with both the measured value of mh as well as LHC lower bounds on superpartner masses are then possible. We explicitly show that models generated from natural GMM may be beyond the reach of even high-luminosity LHC searches. In such a case, the proposed International Linear e+e- Collider will be required for natural SUSY discovery via higgsino pair production reactions. We also outline prospects for detection of higgsino-like WIMPs from natural GMM.

  9. Nutritionally Mediated Oxidative Stress and Inflammation

    Directory of Open Access Journals (Sweden)

    Alexandra Muñoz

    2013-01-01

    Full Text Available There are many sources of nutritionally mediated oxidative stress that trigger inflammatory cascades along short and long time frames. These events are primarily mediated via NFκB. On the short-term scale postprandial inflammation is characterized by an increase in circulating levels of IL-6 and TNF-α and is mirrored on the long-term by proinflammatory gene expression changes in the adipocytes and peripheral blood mononuclear cells (PBMCs of obese individuals. Specifically the upregulation of CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CXCL2/MIP-2α, and CXCL3/MIP-2β is noted because these changes have been observed in both adipocytes and PBMC of obese humans. In comparing numerous human intervention studies it is clear that pro-inflammatory and anti-inflammatory consumption choices mediate gene expression in humans adipocytes and peripheral blood mononuclear cells. Arachidonic acid and saturated fatty acids (SFAs both demonstrate an ability to increase pro-inflammatory IL-8 along with numerous other inflammatory factors including IL-6, TNFα, IL-1β, and CXCL1 for arachidonic acid and IGB2 and CTSS for SFA. Antioxidant rich foods including olive oil, fruits, and vegetables all demonstrate an ability to lower levels of IL-6 in PBMCs. Thus, dietary choices play a complex role in the mediation of unavoidable oxidative stress and can serve to exacerbate or dampen the level of inflammation.

  10. Fluoroquinolone-Gyrase-DNA Complexes

    Science.gov (United States)

    Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M.; Hiasa, Hiroshi; Marks, Kevin R.; Kerns, Robert J.; Berger, James M.; Drlica, Karl

    2014-01-01

    DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly81 and GyrB-Glu466 residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases. PMID:24497635

  11. The mediatization of journalism

    Directory of Open Access Journals (Sweden)

    Aske Kammer

    2014-06-01

    Full Text Available Proposing an explanation of current macro-sociological changes and institutional transformations in journalism, this article argues that journalism is currently undergoing a process of mediatization. Drawing upon the international research literature as well as statements from interviews with news workers working on Danish news websites, the article examines four current trends in journalism that are closely connected to the rise of news on the web, namely the use of the affordances of news websites, radical commercialization, increased audience participation in news production, and the increased multi-skilling and simultaneous de-skilling of journalists. Taken together, these trends reflect a process through which journalism increasingly subsumes itself to the logic of the media, suggesting mediatization as an adequate explanatory framework. One implication of such a process is that journalism seems to be transforming from an occupational profession into an organizational one.

  12. Dissociation in mediation

    Directory of Open Access Journals (Sweden)

    Daniela Muraru

    2008-01-01

    Full Text Available This paper approaches several texts that are part of the so-called discourse of mediation, adopting a pragma-dialectical perspective of the theory of dissociation. It is an attempt to identify the uses of dissociative patterns, with special emphasis on the indicators of dissociation. The paper investigates the various uses of the concept of dissociation as a discursive technique in the argumentation on the different aspects that are involved in international conflict, such as the discussion of the notion of peace. The purpose is to identify the role of dissociation, as a device strategically used by the mediator to help the parties minimize the disagreement space, and come to a conflict resolution.

  13. Nanofluids mediating surface forces.

    Science.gov (United States)

    Pilkington, Georgia A; Briscoe, Wuge H

    2012-11-01

    Fluids containing nanostructures, known as nanofluids, are increasingly found in a wide array of applications due to their unique physical properties as compared with their base fluids and larger colloidal suspensions. With several tuneable parameters such as the size, shape and surface chemistry of nanostructures, as well as numerous base fluids available, nanofluids also offer a new paradigm for mediating surface forces. Other properties such as local surface plasmon resonance and size dependent magnetism of nanostructures also present novel mechanisms for imparting tuneable surface interactions. However, our fundamental understanding, experimentally and theoretically, of how these parameters might affect surface forces remains incomplete. Here we review recent results on equilibrium and dynamic surface forces between macroscopic surfaces in nanofluids, highlighting the overriding trends in the correlation between the physical parameters that characterise nanofluids and the surface forces they mediate. We also discuss the challenges that confront existing surface force knowledge as a result of this new paradigm.

  14. When Memories are Mediated

    DEFF Research Database (Denmark)

    Frølunde, Lisbeth; Bjerregaard, Mette

    2013-01-01

    that are mediated through stories: told and retold as oral stories through generations, as myths or sagas, or remediated as contemporary documentary film accounts or more fictional film accounts. In these processes of retelling acts of violence, transformations of meanings across time, cultural, social......Acts of mass violence, including murder on civilians, genocide, oppression and wars, can mobilize memories of the involved persons and following generations in a certain historical situation. Acts of mass violence can also create a sort of looking glass of culturally dominant memories...... makes meaning about past events. In the discussion, we consider how mediated memories affect audiences, and the potential of achieving development of present political and cultural understandings of past acts of violence....

  15. Teachers as mediators

    DEFF Research Database (Denmark)

    Dorf, Hans; Kelly, Peter; Hohmann, Ulrike

    2012-01-01

    Within the context of lower secondary English teaching in South West England, this study identifies in broad terms the competing goals between which English teachers mediate and the explicit and hidden tensions that result. To understand the interactions of competing goals, teachers’ goal-oriente...... and cultural influences on practice. Yet the teachers observed moved smoothly between goal-oriented behaviours in a continuous and comfortable style, easily and without reflecting any tensions between them. Thus, this article elaborates an account of situated English teaching.......Within the context of lower secondary English teaching in South West England, this study identifies in broad terms the competing goals between which English teachers mediate and the explicit and hidden tensions that result. To understand the interactions of competing goals, teachers’ goal...

  16. Ran-dependent nuclear export mediators: a structural perspective.

    Science.gov (United States)

    Güttler, Thomas; Görlich, Dirk

    2011-08-31

    Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP-exportin-cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions.

  17. Surface Mediated Photocatalysis.

    Science.gov (United States)

    1987-12-01

    Bipolar Photoelectrodes," M.A. Fox, Nouv. J. Chim. 1987, II, 129. 4. "Effect of Cosolvent Additives on Relative Rates of Photooxidation on...Semiconductor Surfaces," D.D. Sackett, M.A. Fox, J. Phys. Org. Chem, 19_8, in press. 5. "Selectivity in the Semiconductor-Mediated Photooxidation of Polyols ...surfaced properties of the heteropolytungstates were varied. The conceptual mechanistic picture involved in the heteropolyoxo induced photooxidations

  18. [Healthcare mediation model for nerologists].

    Science.gov (United States)

    Ando, Tetsuo

    2011-11-01

    Mediation offers a process by which two parties work towards an agreement with the aid of a neutral third party. Physicians and nurses can apply healthcare mediation model to ordinary medical practice for preventing conflict. Communication using mediation skills improves patient-physician relationship, and prevents medical malpractice and conflict.

  19. SV40-mediated immortalization of human fibroblasts.

    Science.gov (United States)

    Ozer, H L; Banga, S S; Dasgupta, T; Houghton, J; Hubbard, K; Jha, K K; Kim, S H; Lenahan, M; Pang, Z; Pardinas, J R; Patsalis, P C

    1996-01-01

    We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

  20. Immune-Mediated Muscle Diseases of the Horse.

    Science.gov (United States)

    Durward-Akhurst, S A; Valberg, S J

    2017-01-01

    In horses, immune-mediated muscle disorders can arise from an overzealous immune response to concurrent infections or potentially from an inherent immune response to host muscle antigens. Streptococcus equi ss. equi infection or vaccination can result in infarctive purpura hemorrhagica (IPH) in which vascular deposition of IgA-streptococcal M protein complexes produces ischemia and complete focal infarction of skeletal muscle and internal organs. In Quarter Horse-related breeds with immune-mediated myositis, an apparent abnormal immune response to muscle antigens results in upregulation of major histocompatibility complex class (MHC) I and II on muscle cell membranes, lymphocytic infiltration of lumbar and gluteal myofibers, and subsequent gross muscle atrophy. Rarely, an inflammatory event results in myositis with subsequent systemic calcinosis characterized by a pathognomonic hyperphosphatemia and high fatality rate. This review presents an overview of these immune-mediated myopathies and highlights clinical and pathological features as well as the suspected pathophysiology.

  1. What carries a mediation process? Configural analysis of mediation.

    Science.gov (United States)

    von Eye, Alexander; Mun, Eun Young; Mair, Patrick

    2009-09-01

    Mediation is a process that links a predictor and a criterion via a mediator variable. Mediation can be full or partial. This well-established definition operates at the level of variables even if they are categorical. In this article, two new approaches to the analysis of mediation are proposed. Both of these approaches focus on the analysis of categorical variables. The first involves mediation analysis at the level of configurations instead of variables. Thus, mediation can be incorporated into the arsenal of methods of analysis for person-oriented research. Second, it is proposed that Configural Frequency Analysis (CFA) can be used for both exploration and confirmation of mediation relationships among categorical variables. The implications of using CFA are first that mediation hypotheses can be tested at the level of individual configurations instead of variables. Second, this approach leaves the door open for different types of mediation processes to exist within the same set. Using a data example, it is illustrated that aggregate-level analysis can overlook mediation processes that operate at the level of individual configurations.

  2. The human body on Exhibit: promoting socio-cultural mediations in a science museum

    OpenAIRE

    Silvania Sousa do Nascimento

    2008-01-01

    This paper discusses three mediation concept approaches and, consequently, three facets of mediator action. The approaches presented start with a bibliographical review of the concept of mediation present in education and scientific communication studies. These approaches serve as a basis for interpreting a semi-directive interview with the director of the Museum of Morphological Sciences of the Federal University of Minas Gerais (UFMG). They also help us reflect on the complexity of organ...

  3. Fc-mediated immune precipitation. III. Visualization by electron microscopy

    DEFF Research Database (Denmark)

    Møller, NPH; Christiansen, Gunna

    1983-01-01

    Fc-mediated interactions between immune complexes are of major importance for the precipitin reaction. In the present study these interactions were investigated by means of electron microscopy. Keyhole limpet haemocyanin (KLH) was adsorbed to a thin glow charged carbon supporting film and reacted...

  4. Mediatization: Theorizing the Interplay Between Media, Culture, and Society

    DEFF Research Database (Denmark)

    Hepp, Andreas; Hjarvard, Stig; Lundby, Knut

    2015-01-01

    with the complex relationship between changes in media and communication on the one hand and changes in various fields of culture and society on the other. We conclude that the emergence of the concept of mediatization is part of a paradigmatic shift within media and communication research....

  5. Design of a wearable research tool for warm mediated social touches

    NARCIS (Netherlands)

    Pfab, Isabel; Willemse, Christian J.A.M.

    2015-01-01

    Social touches are essential in interpersonal communication, for instance to show affect. Despite this importance, mediated interpersonal communication oftentimes lacks the possibility to touch. A human touch is a complex composition of several physical qualities and parameters, but different haptic

  6. Computer Mediated Communication

    Science.gov (United States)

    Fano, Robert M.

    1984-08-01

    The use of computers in organizations is discussed in terms of its present and potential role in facilitating and mediating communication between people. This approach clarifies the impact that computers may have on the operation of organizations and on the individuals comprising them. Communication, which is essential to collaborative activities, must be properly controlled to protect individual and group privacy, which is equally essential. Our understanding of the human and organizational aspects of controlling communication and access to information presently lags behind our technical ability to implement the controls that may be needed.

  7. Holographic Gauge Mediation

    Energy Technology Data Exchange (ETDEWEB)

    Benini, Francesco; /Princeton U.; Dymarsky, Anatoly; /Stanford U., ITP; Franco, Sebastian; /Santa Barbara, KITP; Kachru, Shamit; Simic, Dusan; /Stanford U., ITP /SLAC; Verlinde, Herman; /Princeton, Inst. Advanced Study

    2009-06-19

    We discuss gravitational backgrounds where supersymmetry is broken at the end of a warped throat, and the SUSY-breaking is transmitted to the Standard Model via gauginos which live in (part of) the bulk of the throat geometry. We find that the leading effect arises from splittings of certain 'messenger mesons,' which are adjoint KK-modes of the D-branes supporting the Standard Model gauge group. This picture is a gravity dual of a strongly coupled field theory where SUSY is broken in a hidden sector and transmitted to the Standard Model via a relative of semi-direct gauge mediation.

  8. Sociocultural mediators of remembering

    DEFF Research Database (Denmark)

    Wagoner, Brady; Gillespie, Alex

    2014-01-01

    , questioning and deferring contribute to the transformation and conventionalization of the material. These diverse sociocultural mediators are integrated into a partially coherent recollection by participants self-reflecting, or as Bartlett termed it, turning around upon their schemas. We demonstrate...... that this self-reflection is both a social and a psychological process, occurring because participants are responding to their own utterances in the same way that they respond to the utterances of other people. These empirical findings are used to make a case for using discursive data to look not only...

  9. DNA-Mediated Electrochemistry

    Science.gov (United States)

    Gorodetsky, Alon A.; Buzzeo, Marisa C.

    2009-01-01

    The base pair stack of DNA has been demonstrated as a medium for long range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry. PMID:18980370

  10. Mediatization and Government Communication

    DEFF Research Database (Denmark)

    Laursen, Bo; Valentini, Chiara

    2015-01-01

    in the light of mediatization and government communication theories. Without one pan-European public sphere, the European Parliament, like the other European Union (EU) institutions, competes with national actors for the news media’s attention in the EU’s twenty-eight national public spheres, where EU affairs......, and particularly into the thinking that guides the efforts of these European Parliament officials to increase European citizens’ awareness of, and support for, the European Parliament that is meant to voice the citizens’ concerns in political processes at the EU level....

  11. Surface-Mediated Photocatalysis

    Science.gov (United States)

    1992-02-04

    Photocatalysis 6. AUTHOR(S) DIMarye Anne Fox E -LECTE ’ / 7. PERFORMING ORGANIZATION NAME(S) AND AD ES). 8. PERFORMING ORGANIZATION REPORT NUMBER University of...Form 298 (Rev 2-89) Prescr d bv ANJ std Z39.1s 291-102 j Final Report on Surface-Mediated Photocatalysis "a,. . ARO Proposal No. 28298-CH u - Work from...Produced by Anodic Oxidation and by Photoelectrochemical Activation of TiO2 ," Marye Anne Fox and Karl L. Worthen, Chem. Mater. 1991, 3, 253. "Surface

  12. Tracing the Policy Mediation Process in the Implementation of a Change in the Life Sciences Curriculum

    Science.gov (United States)

    Singh-Pillay, Asheena; Alant, Busisiwe

    2015-01-01

    This paper accounts for the enacted realities of curriculum reform in South Africa, in particular the mediation of curriculum change. Curriculum implementation is viewed as a complex networked process of transforming or mediating policy into classroom practice. The fact that curriculum implementation is seen as problematic requires attention for…

  13. Current trends in inflammatory and immunomodulatory mediators in sepsis

    Science.gov (United States)

    Aziz, Monowar; Jacob, Asha; Yang, Weng-Lang; Matsuda, Akihisa; Wang, Ping

    2013-01-01

    Sepsis refers to severe systemic inflammation in response to invading pathogens. An overwhelming immune response, as mediated by the release of various inflammatory mediators, can lead to shock, multiple organ damage, and even death. Cytokines, proteases, lipid mediators, gaseous substances, vasoactive peptides, and cell stress markers play key roles in sepsis pathophysiology. Various adhesion molecules and chemokines sequester and activate neutrophils into the target organs, further augmenting inflammation and tissue damage. Although the anti-inflammatory substances counterbalance proinflammatory mediators, prolonged immune modulation may cause host susceptibility to concurrent infections, thus reflecting enormous challenge toward developing effective clinical therapy against sepsis. To understand the complex interplay between pro- and anti-inflammatory phenomenon in sepsis, there is still an unmet need to study newly characterized mediators. In addition, revealing the current trends of novel mediators will upgrade our understanding on their signal transduction, cross-talk, and synergistic and immunomodulating roles during sepsis. This review highlights the latest discoveries of the mediators in sepsis linking to innate and adaptive immune systems, which may lead to resolution of many unexplored queries. PMID:23136259

  14. 45 CFR 16.18 - Mediation.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Mediation. 16.18 Section 16.18 Public Welfare... BOARD § 16.18 Mediation. (a) In cases pending before the Board. If the Board decides that mediation... mediation techniques and will provide or assist in selecting a mediator. The mediator may take any...

  15. Mediation Revisited: The Interactive Organization of Mediation in Learning Environments

    OpenAIRE

    Pekarek Doehler, Simona

    2013-01-01

    This article is concerned with the social organization of mediation in learning environments. It seeks to further articulate the sociocultural notion of mediation in sociointeractional terms, combining insights from the sociocultural approach to cognition and the microinteractionist, especially ethnomethodological approach to social activities. A microanalysis of mediation in communicative 2nd-language classroom activities where the task at hand is the management of interaction itself is pres...

  16. Magnetically mediated thermoacoustic imaging

    Science.gov (United States)

    Feng, Xiaohua; Gao, Fei; Zheng, Yuanjin

    2014-03-01

    In this paper, alternating magnetic field is explored for inducing thermoacoustic effect on dielectric objects. Termed as magnetically mediated thermo-acoustic (MMTA) effect that provides a contrast in conductivity, this approach employs magnetic resonance for delivering energy to a desired location by applying a large transient current at radio frequency below 50MHz to a compact magnetically resonant coil. The alternating magnetic field induces large electric field inside conductive objects, which then undergoes joule heating and emanates acoustic signal thermo-elastically. The magnetic mediation approach with low radio frequency can potentially provide deeper penetration than microwave radiation due to the non-magnetic nature of human body and therefore extend thermoacoustic imaging to deep laid organs. Both incoherent time domain method that applies a pulsed radio frequency current and coherent frequency domain approach that employs a linear chirp signal to modulate the envelop of the current are discussed. Owing to the coherent processing nature, the latter approach is capable of achieving much better signal to noise ratio and therefore potential for portable imaging system. Phantom experiments are carried out to demonstrate the signal generation together with some preliminary imaging results. Ex-vivo tissue studies are also investigated.

  17. CCAN Assembly Configures Composite Binding Interfaces to Promote Cross-Linking of Ndc80 Complexes at the Kinetochore

    National Research Council Canada - National Science Library

    Pekgöz Altunkaya, Gülsah; Malvezzi, Francesca; Demianova, Zuzana; Zimniak, Tomasz; Litos, Gabriele; Weissmann, Florian; Mechtler, Karl; Herzog, Franz; Westermann, Stefan

    2016-01-01

    .... Kinetochores contain two supramolecular protein assemblies. The ten-protein KMN network harbors key microtubule-binding sites in the Ndc80 complex and mediates assembly of checkpoint complexes via the KNL-1/Spc105 protein [1, 2...

  18. Domain interactions within the Ski2/3/8 complex and between the Ski complex and Ski7p

    OpenAIRE

    Wang, Lingna; LEWIS, MARC S.; Johnson, Arlen W

    2005-01-01

    The Ski complex (composed of Ski3p, Ski8p, and the DEVH ATPase Ski2p) is a central component of the 3′–5′ cytoplasmic mRNA degradation pathway in yeast. Although the proteins of the complex interact with each other as well as with Ski7p to mediate degradation by exosome, a 3′-exonuclease complex, the nature of these interactions is not well understood. Here we explore interactions within the Ski complex and between the Ski complex and Ski7p using a directed two-hybrid approach combined with c...

  19. Workplace mediation: the participant experience

    OpenAIRE

    Saundry, Richard; Bennett, Anthony Joseph William; Wibberley, Gemma

    2013-01-01

    This paper reports on a qualitative study of the perceptions and experiences of participants in workplace mediation. In total, 25 individuals, from a variety of occupations and organisations, were interviewed by researchers. The project sought to: explore the trajectory of individual disputes and assess participants’ views of the effectiveness of mediation provision and sustainability of outcomes. Furthermore, the research attempted to examine the broader impact of participation in mediation ...

  20. Histone chaperone-mediated nucleosome assembly process.

    Science.gov (United States)

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

  1. Inflammatory mediators and intestinal injury.

    Science.gov (United States)

    Caplan, M S; MacKendrick, W

    1994-06-01

    Although the causes of necrotizing enterocolitis (NEC) are not well understood, there is compelling evidence to suggest that the inflammatory mediators play an important role in the pathophysiology of the disease. This article examines the role of platelet-activating factor (PAF) and other mediators on the development of NEC, and attempts to explain the association of the putative NEC risk factors with altered mediator production and subsequent intestinal injury. The authors hypothesize that PAF is a key mediator in the final common pathway leading to NEC.

  2. Mediating Trust in Terrorism Coverage

    DEFF Research Database (Denmark)

    Mogensen, Kirsten

    Mass mediated risk communication can contribute to perceptions of threats and fear of “others” and/or to perceptions of trust in fellow citizens and society to overcome problems. This paper outlines a cross-disciplinary holistic framework for research in mediated trust building during an acute...... crisis. While the framework is presented in the context of television coverage of a terror-related crisis situation, it can equally be used in connection with all other forms of mediated trust. Key words: National crisis, risk communication, crisis management, television coverage, mediated trust....

  3. Endogenous ion channel complexes: the NMDA receptor.

    Science.gov (United States)

    Frank, René A W

    2011-06-01

    Ionotropic receptors, including the NMDAR (N-methyl-D-aspartate receptor) mediate fast neurotransmission, neurodevelopment, neuronal excitability and learning. In the present article, the structure and function of the NMDAR is reviewed with the aim to condense our current understanding and highlight frontiers where important questions regarding the biology of this receptor remain unanswered. In the second part of the present review, new biochemical and genetic approaches for the investigation of ion channel receptor complexes will be discussed.

  4. Protein- mediated enamel mineralization

    Science.gov (United States)

    Moradian-Oldak, Janet

    2012-01-01

    Enamel is a hard nanocomposite bioceramic with significant resilience that protects the mammalian tooth from external physical and chemical damages. The remarkable mechanical properties of enamel are associated with its hierarchical structural organization and its thorough connection with underlying dentin. This dynamic mineralizing system offers scientists a wealth of information that allows the study of basic principals of organic matrix-mediated biomineralization and can potentially be utilized in the fields of material science and engineering for development and design of biomimetic materials. This chapter will provide a brief overview of enamel hierarchical structure and properties as well as the process and stages of amelogenesis. Particular emphasis is given to current knowledge of extracellular matrix protein and proteinases, and the structural chemistry of the matrix components and their putative functions. The chapter will conclude by discussing the potential of enamel for regrowth. PMID:22652761

  5. Mediated Cultural Memories

    DEFF Research Database (Denmark)

    Frølunde, Lisbeth; Bjerregaard, Mette

    2013-01-01

    (A revised, full paper will be published in the journal Mediekultur, spring 2014) This paper explores two examples of narratives representing memories of acts of mass violence: Gzim Rewind (Sweden, 2011, director Knutte Wester) about 1990’s Kosovo, and The Act of Killing (Denmark, 2012, director...... perspectives of semiosis (meaning-making) in relation to the films as redefining genres and what sorts of meanings different audiences create about the films. Acts of mass violence, including murder on civilians, genocide, and wars, can be seen as seeds for memories of the involved persons and following...... generations. Acts of mass violence also construct a sort of looking glass of culturally dominant memories that are mediated through stories: retold as oral stories through generations, as myths or sagas, or remediated in contemporary documentary or fiction films. In these processes of retelling acts...

  6. Neutrino assisted gauge mediation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyung Do; Mo, Doh Young; Seo, Min-Seok [Seoul National University, Department of Physics and Astronomy and Center for Theoretical Physics, Seoul (Korea, Republic of)

    2013-06-15

    Recent observation shows that the Higgs mass is at around 125 GeV while the prediction of the minimal supersymmetric standard model is below 120 GeV for stop mass lighter than 2 TeV unless the top squark has a maximal mixing. We consider the right-handed neutrino supermultiplets as messengers in addition to the usual gauge mediation to obtain sizeable trilinear soft parameters A{sub t} needed for the maximal stop mixing. Neutrino messengers can explain the observed Higgs mass for stop mass around 1 TeV. Neutrino assistance can also generate charged lepton flavor violation including {mu}{yields}e {gamma} as a possible signature of the neutrino messengers. We consider the S{sub 4} discrete flavor model and show the relation of the charged lepton flavor violation, {theta} {sub 13} of neutrino oscillation and the muon's g-2. (orig.)

  7. When Memories are Mediated

    DEFF Research Database (Denmark)

    Frølunde, Lisbeth; Bjerregaard, Mette

    2013-01-01

    and political contexts and media platforms take place and become contexts for audience reception. This paper explores two examples of narratives that construct memories of acts of mass violence: “Gzim Rewind” (Sweden, 2011, director Knutte Wester) about 1990’s Kosovo, and “The Act of Killing” (Denmark, 2012......Acts of mass violence, including murder on civilians, genocide, oppression and wars, can mobilize memories of the involved persons and following generations in a certain historical situation. Acts of mass violence can also create a sort of looking glass of culturally dominant memories...... that are mediated through stories: told and retold as oral stories through generations, as myths or sagas, or remediated as contemporary documentary film accounts or more fictional film accounts. In these processes of retelling acts of violence, transformations of meanings across time, cultural, social...

  8. The mediation procedure in Romania

    Directory of Open Access Journals (Sweden)

    Alexandrina Zaharia

    2009-06-01

    Full Text Available The mediation activity as an alternative way of solving conflicts occupies an important place in modernsociety. Currently, the mediation reached its maturity worldwide being adopted without reservations.The future of solving conflicts is undoubtedly closely related to mediation. XXth century is the century of solvingconflicts amiably outside the court room. In Romania and the mediation profession were regulated by the Law no.192/2006, on the basis of the idea that mediation is one of the major themes of the reform strategy of the judicialsystem 2005-2007. By adopting the mentioned law it was followed the idea of reducing the volume of activitycourts, and therefore, relieve them of as many cases, with the direct effect on the quality of justice. Mediation is avoluntary process in which the parties with a neutral and impartial third party, without power of decision - themediator - who is qualified to assist the parties to negotiate, facilitating the communication between them andhelping them to reach a unanimous effective and sustainable agreement. The parties may resort to mediation beforeor after triggering a trial. Mediation can be applied, in principle, on any type of conflict. However, theRomanian legislator has established special stipulations on conflict mediation in criminal, civil and familylaw. Although not expressly provided, the stipulations regarding the civil conflicts and also apply to commercialconflicts. Therefore, the mediation is applicable to most types of lawsuits, except those relating to personalrights. As a "win- win" principle, the mediation does not convert any of the parties defeated or victorious; allthose involved have gained by applying this procedure.

  9. Identifying Sources of Clinical Conflict: A Tool for Practice and Training in Bioethics Mediation.

    Science.gov (United States)

    Bergman, Edward J

    2015-01-01

    Bioethics mediators manage a wide range of clinical conflict emanating from diverse sources. Parties to clinical conflict are often not fully aware of, nor willing to express, the true nature and scope of their conflict. As such, a significant task of the bioethics mediator is to help define that conflict. The ability to assess and apply the tools necessary for an effective mediation process can be facilitated by each mediator's creation of a personal compendium of sources that generate clinical conflict, to provide an orientation for the successful management of complex dilemmatic cases.

  10. Hyper Space Complex Number

    OpenAIRE

    Tan, Shanguang

    2007-01-01

    A new kind of numbers called Hyper Space Complex Numbers and its algebras are defined and proved. It is with good properties as the classic Complex Numbers, such as expressed in coordinates, triangular and exponent forms and following the associative and commutative laws of addition and multiplication. So the classic Complex Number is developed from in complex plane with two dimensions to in complex space with N dimensions and the number system is enlarged also.

  11. Mesenchymal stem cell-mediated functional tooth regeneration in swine.

    Directory of Open Access Journals (Sweden)

    Wataru Sonoyama

    Full Text Available Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla. Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

  12. Mesenchymal stem cell-mediated functional tooth regeneration in swine.

    Science.gov (United States)

    Sonoyama, Wataru; Liu, Yi; Fang, Dianji; Yamaza, Takayoshi; Seo, Byoung-Moo; Zhang, Chunmei; Liu, He; Gronthos, Stan; Wang, Cun-Yu; Wang, Songlin; Shi, Songtao

    2006-12-20

    Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

  13. Anomaly mediation deformed by axion

    Energy Technology Data Exchange (ETDEWEB)

    Nakayama, Kazunori, E-mail: kazunori@hep-th.phys.s.u-tokyo.ac.jp [Department of Physics, University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Kavli Institute for the Physics and Mathematics of the Universe, University of Tokyo, Kashiwa 277-8583 (Japan); Yanagida, Tsutomu T. [Kavli Institute for the Physics and Mathematics of the Universe, University of Tokyo, Kashiwa 277-8583 (Japan)

    2013-05-13

    We show that in supersymmetric axion models the axion supermultiplet obtains a sizable F-term due to a non-supersymmetric dynamics and it generally gives the gaugino masses comparable to the anomaly mediation contribution. Thus the gaugino mass relation predicted by the anomaly mediation effect can be significantly modified in the presence of axion to solve the strong CP problem.

  14. Theorizing with/out "Mediators".

    Science.gov (United States)

    Roth, Wolff-Michael; Jornet, Alfredo

    2017-01-05

    Mediation is one of the most often cited concepts in current cultural-historical theory literature, in which cultural actions and artifacts are often characterized as mediators standing between situational stimuli and behavioral responses. Most often presented as a means to overcome Cartesian dualism between subject and object, and between individual and society, some scholars have nonetheless raised criticism suggesting that such mediators are problematic for a dialectical psychology that takes a unit analysis (monist) approach. In fact, Spinoza develops a monist theory of mind and body that goes without and even excludes every form of mediation. In this study, we follow up on the latter criticisms and explore what we consider to be problematic uses of the notion of mediation as an analytical construct in the literature. We elaborate an empirically grounded discussion on the ways the concept of mediation may lead to dualistic readings; and we offer an alternative account where the notion of mediator is not needed. We conclude discussing prospects for and implications of a cultural-historical theory where the notion of mediation no longer is invoked to account for human action and development.

  15. Complex networks analysis of language complexity

    CERN Document Server

    Amancio, Diego R; Oliveira, Osvaldo N; Costa, Luciano da F; 10.1209/0295-5075/100/58002

    2013-01-01

    Methods from statistical physics, such as those involving complex networks, have been increasingly used in quantitative analysis of linguistic phenomena. In this paper, we represented pieces of text with different levels of simplification in co-occurrence networks and found that topological regularity correlated negatively with textual complexity. Furthermore, in less complex texts the distance between concepts, represented as nodes, tended to decrease. The complex networks metrics were treated with multivariate pattern recognition techniques, which allowed us to distinguish between original texts and their simplified versions. For each original text, two simplified versions were generated manually with increasing number of simplification operations. As expected, distinction was easier for the strongly simplified versions, where the most relevant metrics were node strength, shortest paths and diversity. Also, the discrimination of complex texts was improved with higher hierarchical network metrics, thus point...

  16. Mediated Communities in the Age of Electronic Communication

    Directory of Open Access Journals (Sweden)

    Gábor Szécsi

    2012-01-01

    Full Text Available The electronically mediated communication has transformed our notionof the relation between place and community. With a greater proportionof our communicative acts taking place via electronic media, physical co-presence, the co-located interpersonal relations are diminishing as determinants of the nature of human interactions. This paper argues that the electronically mediated communication contributes to the construction of new, mediated forms of communities which are based on the interaction or operational synthesis of virtual and physical communities. The appearance of these new forms of communitiesleads to a new conceptualization of the relation between self and community. The aim of this paper is to show that the medium of the mediatization and new conceptualization of community is a specific pictorial language of electronically mediated communication, the semantic structure of which offers new opportunities to grasp and understand the complex notion of new mediated communities and to adopt the idea of a new global, community building language in local and national communities.

  17. Nacnac‐Cobalt‐Mediated P4 Transformations

    Science.gov (United States)

    Spitzer, Fabian; Graßl, Christian; Balázs, Gábor; Mädl, Eric; Keilwerth, Martin; Zolnhofer, Eva M.; Meyer, Karsten

    2017-01-01

    Abstract A comparison of P4 activations mediated by low‐valent β‐diketiminato (L) cobalt complexes is presented. The formal Co0 source [K2(L3Co)2(μ2:η1,η1‐N2)] (1) reacts with P4 to form a mixture of the monoanionic complexes [K(thf)6][(L3Co)2(μ2:η4,η4‐P4)] (2) and [K(thf)6][(L3Co)2(μ2:η3,η3‐P3)] (3). The analogue CoI precursor [L3Co(tol)] (4 a), however, selectively yields the corresponding neutral derivative [(L3Co)2(μ2:η4,η4‐P4)] (5 a). Compound 5 a undergoes thermal P atom loss to form the unprecedented complex [(L3Co)2(μ2:η3,η3‐P3)] (6). The products 2 and 3 can be obtained selectively by an one‐electron reduction of their neutral precursors 5 a and 6, respectively. The electrochemical behaviour of 2, 3, 5 a, and 6 is monitored by cyclic voltammetry and their magnetism is examined by SQUID measurements and the Evans method. The initial CoI‐mediated P4 activation is not influenced by applying the structurally different ligands L1 and L2, which is proven by the formation of the isostructural products [(LCo)2(μ2:η4,η4‐P4)] [L=L3 (5 a), L1 (5 b), L2 (5 c)]. PMID:28032678

  18. Practical Guide to Civil Mediation

    CERN Multimedia

    2006-01-01

    The Permanent Mission of Switzerland has informed CERN that the Département des Institutions of the Republic and Canton of Geneva and the Groupement suisse des Magistrats pour la médiation (GEMME) - Swiss Association of Magistrates for Mediation have published a multilingual Practical Guide to Civil Mediation (including English). In this context, the Swiss Mission has underlined the benefits of resorting to mediation, especially for the personnel of International Organizations, and which the Secretary-General of the GEMME has summarised as follows: it is a private process not requiring the waiver of the parties' immunities; the confidentiality of the mediation process is guaranteed both by the mediator and the parties to it; the search for an amicable settlement does not need to be determined by reference to law (provided that public order is respected); the process is faster (2 to 3 sessions), less costly and more flexible than civil or arbitration procedures; in order to reinforce the agreem...

  19. Practical Guide to Civil Mediation

    CERN Multimedia

    2006-01-01

    The Permanent Mission of Switzerland has informed CERN that the Département des Institutions of the Republic and Canton of Geneva and the Groupement suisse des Magistrats pour la médiation (GEMME) - Swiss Association of Magistrates for Mediation have published a multilingual Practical Guide to Civil Mediation (including English). In this context, the Swiss Mission has underlined the benefits of resorting to mediation, especially for the personnel of international organizations, and which the Secretary-General of the GEMME has summarised as follows: it is a private process not requiring the waiver of the parties' immunities; the confidentiality of the mediation process is guaranteed both by th