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Sample records for ptb promotes formation

  1. Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation

    PubMed Central

    Besse, Florence; López de Quinto, Sonia; Marchand, Virginie; Trucco, Alvar; Ephrussi, Anne

    2009-01-01

    Local translation of asymmetrically enriched mRNAs is a powerful mechanism for functional polarization of the cell. In Drosophila, exclusive accumulation of Oskar protein at the posterior pole of the oocyte is essential for development of the future embryo. This is achieved by the formation of a dynamic oskar ribonucleoprotein (RNP) complex regulating the transport of oskar mRNA, its translational repression while unlocalized, and its translational activation upon arrival at the posterior pole. We identified the nucleo–cytoplasmic shuttling protein PTB (polypyrimidine tract-binding protein)/hnRNP I as a new factor associating with the oskar RNP in vivo. While PTB function is largely dispensable for oskar mRNA transport, it is necessary for translational repression of the localizing mRNA. Unexpectedly, a cytoplasmic form of PTB can associate with oskar mRNA and repress its translation, suggesting that nuclear recruitment of PTB to oskar complexes is not required for its regulatory function. Furthermore, PTB binds directly to multiple sites along the oskar 3′ untranslated region and mediates assembly of high-order complexes containing multiple oskar RNA molecules in vivo. Thus, PTB is a key structural component of oskar RNP complexes that dually controls formation of high-order RNP particles and translational silencing. PMID:19131435

  2. Robot goniophotometry at PTB

    NASA Astrophysics Data System (ADS)

    Lindemann, M.; Maass, R.; Sauter, G.

    2015-04-01

    The total luminous flux of a light source is the complete integration of its spectral radiance distribution weighted with the photopic observer and taken over all parts of its surface and over the full solid angle of emittance. The spatial distributions are measured with various types of goniophotometers and the PTB robot goniophotometer is a new type with many unique features. It is built as an arrangement of three robots with arms of more than 6 m in length and with 7 degrees of freedom each. The extreme flexibility of the robots allows computer controlled tracks with variable radii and speeds up to 3 m and 1 m s-1, respectively. One robot aligns the light source and the two other robots move photometers and array spectrometers in their hemispheres simultaneously measuring planar illuminance and the related relative spectral distribution. The robot goniophotometer is optimized for the realisation of the luminous flux unit, the lumen and it is completely characterized in this report. The relevant properties and correction factors are explained, as well as the implementation of techniques for synchronisation and stabilisation of spatially resolved or integrated photometric and colorimetric quantities. Finally, all contributions are combined in the model of evaluation for the (total) luminous flux value and the measurement uncertainty associated with that value is evaluated in the presented uncertainty budget. The goniophotometric determination of the values for colorimetric quantities is explained for the total luminous flux and the spatially distributed radiant power.

  3. Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region.

    PubMed Central

    Tillmar, Linda; Welsh, Nils

    2002-01-01

    BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway. PMID:12359957

  4. Toward Efficient Thick Active PTB7 Photovoltaic Layers Using Diphenyl Ether as a Solvent Additive.

    PubMed

    Zheng, Yifan; Goh, Tenghooi; Fan, Pu; Shi, Wei; Yu, Junsheng; Taylor, André D

    2016-06-22

    The development of thick organic photovoltaics (OPV) could increase absorption in the active layer and ease manufacturing constraints in large-scale solar panel production. However, the efficiencies of most low-bandgap OPVs decrease substantially when the active layers exceed ∼100 nm in thickness (because of low crystallinity and a short exciton diffusion length). Herein, we report the use of solvent additive diphenyl ether (DPE) that facilitates the fabrication of thick (180 nm) active layers and triples the power conversion efficiency (PCE) of conventional thienothiophene-co-benzodithiophene polymer (PTB7)-based OPVs from 1.75 to 6.19%. These results demonstrate a PCE 20% higher than those of conventional (PTB7)-based OPV devices using 1,8-diiodooctane. Morphology studies reveal that DPE promotes the formation of nanofibrillar networks and ordered packing of PTB7 in the active layer that facilitate charge transport over longer distances. We further demonstrate that DPE improves the fill factor and photocurrent collection by enhancing the overall optical absorption, reducing the series resistance, and suppressing bimolecular recombination.

  5. INCOME INCONGRUITY, RACE AND PRETERM BIRTH (PTB)

    EPA Science Inventory

    Previous research using birth records has found income incongruity associated with adverse birth outcomes. The effects of negative income incongruity (reporting lower household income than the census tract median household income) on PTB (<37 weeks completed gestation) are examin...

  6. The origin of high PCE in PTB7 based photovoltaics: proper charge neutrality level and free energy of charge separation at PTB7/PC71BM interface

    PubMed Central

    Park, Soohyung; Jeong, Junkyeong; Hyun, Gyeongho; Kim, Minju; Lee, Hyunbok; Yi, Yeonjin

    2016-01-01

    The energy level alignments at donor/acceptor interfaces in organic photovoltaics (OPVs) play a decisive role in device performance. However, little is known about the interfacial energetics in polymer OPVs due to technical issues of the solution process. Here, the frontier ortbial line-ups at the donor/acceptor interface in high performance polymer OPVs, PTB7/PC71BM, were investigated using in situ UPS, XPS and IPES. The evolution of energy levels during PTB7/PC71BM interface formation was investigated using vacuum electrospray deposition, and was compared with that of P3HT/PC61BM. At the PTB7/PC71BM interface, the interface dipole and the band bending were absent due to their identical charge neutrality levels. In contrast, a large interfacial dipole was observed at the P3HT/PC61BM interface. The measured photovoltaic energy gap (EPVG) was 1.10 eV for PTB7/PC71BM and 0.90 eV for P3HT/PC61BM. This difference in the EPVG leads to a larger open-circuit voltage of PTB7/PC71BM than that of P3HT/PC61BM. PMID:27734957

  7. The origin of high PCE in PTB7 based photovoltaics: proper charge neutrality level and free energy of charge separation at PTB7/PC71BM interface.

    PubMed

    Park, Soohyung; Jeong, Junkyeong; Hyun, Gyeongho; Kim, Minju; Lee, Hyunbok; Yi, Yeonjin

    2016-10-13

    The energy level alignments at donor/acceptor interfaces in organic photovoltaics (OPVs) play a decisive role in device performance. However, little is known about the interfacial energetics in polymer OPVs due to technical issues of the solution process. Here, the frontier ortbial line-ups at the donor/acceptor interface in high performance polymer OPVs, PTB7/PC71BM, were investigated using in situ UPS, XPS and IPES. The evolution of energy levels during PTB7/PC71BM interface formation was investigated using vacuum electrospray deposition, and was compared with that of P3HT/PC61BM. At the PTB7/PC71BM interface, the interface dipole and the band bending were absent due to their identical charge neutrality levels. In contrast, a large interfacial dipole was observed at the P3HT/PC61BM interface. The measured photovoltaic energy gap (EPVG) was 1.10 eV for PTB7/PC71BM and 0.90 eV for P3HT/PC61BM. This difference in the EPVG leads to a larger open-circuit voltage of PTB7/PC71BM than that of P3HT/PC61BM.

  8. Promoting proximal formative assessment with relational discourse

    NASA Astrophysics Data System (ADS)

    Scherr, Rachel E.; Close, Hunter G.; McKagan, Sarah B.

    2012-02-01

    The practice of proximal formative assessment - the continual, responsive attention to students' developing understanding as it is expressed in real time - depends on students' sharing their ideas with instructors and on teachers' attending to them. Rogerian psychology presents an account of the conditions under which proximal formative assessment may be promoted or inhibited: (1) Normal classroom conditions, characterized by evaluation and attention to learning targets, may present threats to students' sense of their own competence and value, causing them to conceal their ideas and reducing the potential for proximal formative assessment. (2) In contrast, discourse patterns characterized by positive anticipation and attention to learner ideas increase the potential for proximal formative assessment and promote self-directed learning. We present an analysis methodology based on these principles and demonstrate its utility for understanding episodes of university physics instruction.

  9. Cu(II) promotes amyloid pore formation

    SciTech Connect

    Zhang, Hangyu; Rochet, Jean-Christophe; Stanciu, Lia A.

    2015-08-14

    The aggregation of α-synuclein is associated with dopamine neuron death in Parkinson's disease. There is controversy in the field over the question of which species of the aggregates, fibrils or protofibrils, are toxic. Moreover, compelling evidence suggested the exposure to heavy metals to be a risk of PD. Nevertheless, the mechanism of metal ions in promoting PD remains unclear. In this research, we investigated the structural basis of Cu(II) induced aggregation of α-synuclein. Using transmission electron microscopy experiments, Cu(II) was found to promote in vitro aggregation of α-synuclein by facilitating annular protofibril formation rather than fibril formation. Furthermore, neuroprotective baicalein disaggregated annular protofibrils accompanied by considerable decrease of β-sheet content. These results strongly support the hypothesis that annular protofibrils are the toxic species, rather than fibrils, thereby inspiring us to search novel therapeutic strategies for the suppression of the toxic annular protofibril formation. - Highlights: • Cu(II) promoted the annular protofibril formation of α-synuclein in vitro. • Cu(II) postponed the in vitro fibrillization of α-synuclein. • Neuroprotective baicalein disaggregated annular protofibrils.

  10. Extension of PTB's EUV metrology facilities

    NASA Astrophysics Data System (ADS)

    Laubis, Christian; Fischer, Andreas; Scholze, Frank

    2012-03-01

    After developing metrology with synchrotron radiation in its laboratories at the electron storage rings BESSY I and BESSY II for almost 30 years, PTB is extending its capabilities for EUV metrology with the EUV beamline at the Metrology Light Source. With the new instrumentation, PTB is prepared for the metrological challenges when EUV lithography changes over from R&D to pilot production. PTB's EUV reflectometer for large optical components, e.g. collector mirrors for LPP sources, will be transferred to this new dedicated EUV beamline. This allows us to offer services to customers independent of the operating schedule of BESSY - a basic research facility with regular shut-down times. The new beamline also provides much higher radiant power in the EUV spectral range up to 50 nm wavelength. This will particularly benefit the characterization of sensors regarding responsivity and stability, and the characterization of EUV components in the out-of-band spectral range. Reliable detector characterization is the basis for source power meters or tool-internal sensors. We present an updated overview of our new metrological capabilities with recent measurement examples.

  11. Structural and evolutionary division of phosphotyrosine binding (PTB) domains.

    PubMed

    Uhlik, Mark T; Temple, Brenda; Bencharit, Sompop; Kimple, Adam J; Siderovski, David P; Johnson, Gary L

    2005-01-07

    Proteins encoding phosphotyrosine binding (PTB) domains function as adaptors or scaffolds to organize the signaling complexes involved in wide-ranging physiological processes including neural development, immunity, tissue homeostasis and cell growth. There are more than 200 proteins in eukaryotes and nearly 60 human proteins having PTB domains. Six PTB domain encoded proteins have been found to have mutations that contribute to inherited human diseases including familial stroke, hypercholesteremia, coronary artery disease, Alzheimer's disease and diabetes, demonstrating the importance of PTB scaffold proteins in organizing critical signaling complexes. PTB domains bind both peptides and headgroups of phosphatidylinositides, utilizing two distinct binding motifs to mediate spatial organization and localization within cells. The structure of PTB domains confers specificity for binding peptides having a NPXY motif with differing requirements for phosphorylation of the tyrosine within this recognition sequence. In this review, we use structural, evolutionary and functional analysis to divide PTB domains into three groups represented by phosphotyrosine-dependent Shc-like, phosphotyrosine-dependent IRS-like and phosphotyrosine-independent Dab-like PTBs, with the Dab-like PTB domains representing nearly 75% of proteins encoding PTB domains. In addition, we further define the binding characteristics of the cognate ligands for each group of PTB domains. The signaling complexes organized by PTB domain encoded proteins are largely unknown and represents an important challenge in systems biology for the future.

  12. The spectral irradiance traceability chain at PTB

    SciTech Connect

    Sperfeld, P.; Pape, S.; Nevas, S.

    2013-05-10

    Spectral irradiance is a fundamental radiometric unit. Its application to measurement results requires qualified traceability to basic units of the international system of units (Systeme international d'unites, SI). The Physikalisch-Technische Bundesanstalt (PTB) is amongst other national metrological institutes (NMIs) responsible for the realization, maintenance and dissemination of various radiometric and photometric units based on and traceable to national standards. The unit of spectral irradiance is realized and represented by a blackbody-radiator as the national primary standard of the PTB. Based on Planck's radiation law, the irradiance is calculated and realized for any wavelength taking into account the exact knowledge of the radiation temperature and the geometrical parameters. Using a double-monochromator-based spectroradiometer system, secondary standard lamps can be calibrated by direct comparison to the blackbody-radiator (substitution method). These secondary standard lamps are then used at the PTB to calibrate standard lamps of customers. The customers themselves use these so-called transfer standards to calibrate their working standard lamps. These working standards are then used to calibrate own spectroradiometers or sources. This rather complex calibration chain is a common procedural method that for the customers generally leads to satisfying measurement results on site. Nevertheless, the standard lamps in use have to fulfill highest requirements concerning stability and reproducibility. Only this allows achieving comparably low transfer measurement uncertainties, which occur at each calibration step. Thus, the PTB is constantly investigating the improvement and further development of transfer standards and measurement methods for various spectral regions. The realization and dissemination of the spectral irradiance using the blackbody-radiator at the PTB is accomplished with worldwide approved minimized measurement uncertainties confirmed by

  13. The spectral irradiance traceability chain at PTB

    NASA Astrophysics Data System (ADS)

    Sperfeld, P.; Pape, S.; Nevas, S.

    2013-05-01

    Spectral irradiance is a fundamental radiometric unit. Its application to measurement results requires qualified traceability to basic units of the international system of units (Système international d'unités, SI). The Physikalisch-Technische Bundesanstalt (PTB) is amongst other national metrological institutes (NMIs) responsible for the realization, maintenance and dissemination of various radiometric and photometric units based on and traceable to national standards. The unit of spectral irradiance is realized and represented by a blackbody-radiator as the national primary standard of the PTB. Based on Planck's radiation law, the irradiance is calculated and realized for any wavelength taking into account the exact knowledge of the radiation temperature and the geometrical parameters. Using a double-monochromator-based spectroradiometer system, secondary standard lamps can be calibrated by direct comparison to the blackbody-radiator (substitution method). These secondary standard lamps are then used at the PTB to calibrate standard lamps of customers. The customers themselves use these so-called transfer standards to calibrate their working standard lamps. These working standards are then used to calibrate own spectroradiometers or sources. This rather complex calibration chain is a common procedural method that for the customers generally leads to satisfying measurement results on site. Nevertheless, the standard lamps in use have to fulfill highest requirements concerning stability and reproducibility. Only this allows achieving comparably low transfer measurement uncertainties, which occur at each calibration step. Thus, the PTB is constantly investigating the improvement and further development of transfer standards and measurement methods for various spectral regions. The realization and dissemination of the spectral irradiance using the blackbody-radiator at the PTB is accomplished with worldwide approved minimized measurement uncertainties confirmed by

  14. Rationale for a novel nutraceutical complex 'K-water': potassium taurine bicarbonate (PTB).

    PubMed

    McCarty, Mark F

    2006-01-01

    Potassium taurine bicarbonate (PTB), an equimolar blend of potassium bicarbonate and taurine, provides a convenient and feasible means of delivering physiologically significant doses of potassium, taurine, and organic base when dissolved in water ("K-water"). This brief essay reviews the versatile and complementary health benefits that likely would accrue in individuals making regular use of K-water; in particular, an adequate intake of PTB could be expected to aid blood pressure control, lessen risk for atherosclerosis and its thromboembolic complications (particularly stroke), promote maintenance of bone density, help to prevent calcium renal stones, and possibly reduce risk for weight gain and diabetes.

  15. PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

    PubMed Central

    Espinosa-Hernández, Wendy; Velez-Uriza, Dora; Valdés, Jesús; Vélez-Del Valle, Cristina; Salas-Benito, Juan; Martínez-Contreras, Rebeca; García-Espítia, Matilde; Salas-Benito, Mariana; Vega-Almeida, Tania; De Nova-Ocampo, Mónica

    2014-01-01

    The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication. PMID:25406089

  16. Modelling PTB's spatial angle autocollimator calibrator

    NASA Astrophysics Data System (ADS)

    Kranz, Oliver; Geckeler, Ralf D.; Just, Andreas; Krause, Michael

    2013-05-01

    The accurate and traceable form measurement of optical surfaces has been greatly advanced by a new generation of surface profilometers which are based on the reflection of light at the surface and the measurement of the reflection angle. For this application, high-resolution electronic autocollimators provide accurate and traceable angle metrology. In recent years, great progress has been made at the Physikalisch-Technische Bundesanstalt (PTB) in autocollimator calibration. For an advanced autocollimator characterisation, a novel calibration device has been built up at PTB: the Spatial Angle Autocollimator Calibrator (SAAC). The system makes use of an innovative Cartesian arrangement of three autocollimators (two reference autocollimators and the autocollimator to be calibrated), which allows a precise measurement of the angular orientation of a reflector cube. Each reference autocollimator is sensitive primarily to changes in one of the two relevant tilt angles, whereas the autocollimator to be calibrated is sensitive to both. The distance between the reflector cube and the autocollimator to be calibrated can be varied flexibly. In this contribution, we present the SAAC and aspects of the mathematical modelling of the system for deriving analytical expressions for the autocollimators' angle responses. These efforts will allow advancing the form measurement substantially with autocollimator-based profilometers and approaching fundamental measurement limits. Additionally, they will help manufacturers of autocollimators to improve their instruments and will provide improved angle measurement methods for precision engineering.

  17. MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3

    PubMed Central

    Gooding, Clare; Edge, Christopher; Lorenz, Mike; Coelho, Miguel B.; Winters, Mikael; Kaminski, Clemens F.; Cherny, Dmitry; Eperon, Ian C.; Smith, Christopher W.J.

    2013-01-01

    Exon 3 of the rat α-tropomyosin (Tpm1) gene is repressed in smooth muscle cells, allowing inclusion of the mutually exclusive partner exon 2. Two key types of elements affect repression of exon 3 splicing: binding sites for polypyrimidine tract-binding protein (PTB) and additional negative regulatory elements consisting of clusters of UGC or CUG motifs. Here, we show that the UGC clusters are bound by muscleblind-like proteins (MBNL), which act as repressors of Tpm1 exon 3. We show that the N-terminal region of MBNL1, containing its four CCCH zinc-finger domains, is sufficient to mediate repression. The same region of MBNL1 can make a direct protein-to-protein interaction with PTB, and RNA binding by MBNL promotes this interaction, apparently by inducing a conformational change in MBNL. Moreover, single molecule analysis showed that MBNL-binding sites increase the binding of PTB to its own sites. Our data suggest that the smooth muscle splicing of Tpm1 is mediated by allosteric assembly of an RNA–protein complex minimally comprising PTB, MBNL and their cognate RNA-binding sites. PMID:23511971

  18. Status of the nanometer comparator at PTB

    NASA Astrophysics Data System (ADS)

    Fluegge, Jens; Koening, Rainer G.

    2001-10-01

    Due to increasing demands on the photolithography of integrated circuits and the progress of interferometric linear encoders, length measurement systems with a reproducibility under 3 nm are used in industry today, whereas the connection to the unit of length exhibits an uncertainty of about 25 nm. To resolve this problem a new one dimensional length comparator, the nanometer comparator, was developed in a cooperation between the Physikalisch-Technische Bundesanstalt (PTB), the Dr. Johannes Heidenhain GmbH and Werth Me#technik GmbH. The nanometer comparator will be able to perform one dimensional calibrations of photo masks, line-graduation scales, incremental linear encoders and laser interferometers in one axis up to a maximum length of 610 mm. To ensure the highest level of measurement performance, the interferometer is completely located in vacuum using metal bellows, whilst the calibration objects can be mounted under atmospheric conditions. The interferometer set-up compensates the dilatation and the bending of the granite base and minimizes the measurement circle of the comparator. This will minimize the influence of thermal and mechanical distortions. The interferometer design can be used with a heterodyne or a homodyne signal detection electronics. Due to their high power dissipation, the laser is arranged far apart from the comparator and light is fed to the interferometers by means of glass fibers. The light source is a frequency-doubled Nd:YAG laser frequency stabilized by an iodine absorption line. Different measuring systems for the structure localization can be attached to an universal sensor carrier on a solid bridge above the measuring carriage. Incremental reading heads and two photoelectric microscopes are now available for this purpose.

  19. Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

    PubMed Central

    Stern, Michael Zeev; Gupta, Sachin Kumar; Salmon-Divon, Mali; Haham, Tomer; Barda, Omer; Levi, Sarit; Wachtel, Chaim; Nilsen, Timothy W.; Michaeli, Shulamit

    2009-01-01

    Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), but the function of most of these proteins in mRNA metabolism is currently unknown. Here, we report the function of two such proteins that we have named PTB1 and PTB2, which resemble the mammalian polypyrimidine tract binding proteins (PTB). RNAi silencing of these factors indicates that both are essential for life. PTB1 and PTB2 reside mostly in the nucleus, but are found in the cytoplasm, as well. Microarray analysis performed on PTB1 and PTB2 RNAi silenced cells indicates that each of these factors differentially affects the transcriptome, thus regulating a different subset of mRNAs. PTB1 and PTB2 substrates were categorized bioinformatically, based on the presence of PTB binding sites in their 5′ and 3′ flanking sequences. Both proteins were shown to regulate mRNA stability. Interestingly, PTB proteins are essential for trans-splicing of genes containing C-rich polypyrimidine tracts. PTB1, but not PTB2, also affects cis-splicing. The specificity of binding of PTB1 was established in vivo and in vitro using a model substrate. This study demonstrates for the first time that trans-splicing of only certain substrates requires specific factors such as PTB proteins for their splicing. The trypanosome PTB proteins, like their mammalian homologs, represent multivalent RNA binding proteins that regulate mRNAs from their synthesis to degradation. PMID:19218552

  20. Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.

    PubMed

    Simpson, Craig G; Lewandowska, Dominika; Liney, Michele; Davidson, Diane; Chapman, Sean; Fuller, John; McNicol, Jim; Shaw, Paul; Brown, John W S

    2014-07-01

    This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence.

  1. Glycerol metabolism promotes biofilm formation by Pseudomonas aeruginosa.

    PubMed

    Scoffield, Jessica; Silo-Suh, Laura

    2016-08-01

    Pseudomonas aeruginosa causes persistent infections in the airways of cystic fibrosis (CF) patients. Airway sputum contains various host-derived nutrients that can be utilized by P. aeruginosa, including phosphotidylcholine, a major component of host cell membranes. Phosphotidylcholine can be degraded by P. aeruginosa to glycerol and fatty acids to increase the availability of glycerol in the CF lung. In this study, we explored the role that glycerol metabolism plays in biofilm formation by P. aeruginosa. We report that glycerol metabolism promotes biofilm formation by both a chronic CF isolate (FRD1) and a wound isolate (PAO1) of P. aeruginosa. Moreover, loss of the GlpR regulator, which represses the expression of genes involved in glycerol metabolism, enhances biofilm formation in FRD1 through the upregulation of Pel polysaccharide. Taken together, our results suggest that glycerol metabolism may be a key factor that contributes to P. aeruginosa persistence by promoting biofilm formation.

  2. Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation.

    PubMed

    Hall, Megan P; Nagel, Roland J; Fagg, W Samuel; Shiue, Lily; Cline, Melissa S; Perriman, Rhonda J; Donohue, John Paul; Ares, Manuel

    2013-05-01

    Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near muscle-specific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3' UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two- to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB- and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

  3. Interferometry at the PTB Nanometer Comparator: design, status and development

    NASA Astrophysics Data System (ADS)

    Flügge, J.; Weichert, Ch.; Hu, H.; Köning, R.; Bosse, H.; Wiegmann, A.; Schulz, M.; Elster, C.; Geckeler, R. D.

    2008-10-01

    To minimize the measurement uncertainty of one dimensional length measurements on line scales, linear encoders and interferometers the PTB in cooperation with the Dr. Johannes Heidenhain GmbH had built up a new length comparator. The Nanometer Comparator [1,2] has already proven its performance during the measurements of incremental encoders and line scales with an expanded measurement uncertainty of below 5 nm [3,4,5]. Due to the introduction of double and multiple exposure in advanced lithography techniques the overlay and registration metrology requirements will drastically increase so that reference metrology tools need to be developed further to be able to follow the resulting decrease of the specifications. Therefore, the PTB further develops the new 1D vacuum comparator to add a measurement possibility for straightness and to reach a measurement accuracy in the sub nanometer range [6]. One key development will be the interferometric measurement of all six degrees of freedom of the measurement slide of the comparator. A new multi axis heterodyne interferometer electronics and optical interferometer designs minimizing nonlinearities by spatially separated beams are under development.

  4. Vanadium promotes hydroxyl radical formation by activated human neutrophils.

    PubMed

    Fickl, Heidi; Theron, Annette J; Grimmer, Heidi; Oommen, Joyce; Ramafi, Grace J; Steel, Helen C; Visser, Susanna S; Anderson, Ronald

    2006-01-01

    This study was undertaken to investigate the effects of vanadium in the +2, +3, +4, and +5 valence states on superoxide generation, myeloperoxidase (MPO) activity, and hydroxyl radical formation by activated human neutrophils in vitro, using lucigenin-enhanced chemiluminescence (LECL), autoiodination, and electron spin resonance with 5,5-dimethyl-l-pyrroline N-oxide as the spin trap, respectively. At concentrations of up to 25 microM, vanadium, in the four different valence states used, did not affect the LECL responses of neutrophils activated with either the chemoattractant, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), or the phorbol ester, phorbol 12-myristate 12-acetate (25 ng/ml). However, exposure to vanadium in the +2, +3, and +4, but not the +5, valence states was accompanied by significant augmentation of hydroxyl radical formation by activated neutrophils and attenuation of MPO-mediated iodination. With respect to hydroxyl radical formation, similar effects were observed using cell-free systems containing either hydrogen peroxide (100 microM) or xanthine/xanthine oxidase together with vanadium (+2, +3, +4), while the activity of purified MPO was inhibited by the metal in these valence states. These results demonstrate that vanadium in the +2, +3, and +4 valence states interacts prooxidatively with human neutrophils, competing effectively with MPO for hydrogen peroxide to promote formation of the highly toxic hydroxyl radical.

  5. Why stellar feedback promotes disc formation in simulated galaxies

    NASA Astrophysics Data System (ADS)

    Übler, Hannah; Naab, Thorsten; Oser, Ludwig; Aumer, Michael; Sales, Laura V.; White, Simon D. M.

    2014-09-01

    We study how feedback influences baryon infall on to galaxies using cosmological, zoom-in simulations of haloes with present mass Mvir = 6.9 × 1011 to 1.7 × 1012 M⊙. Starting at z = 4 from identical initial conditions, implementations of weak and strong stellar feedback produce bulge- and disc-dominated galaxies, respectively. Strong feedback favours disc formation: (1) because conversion of gas into stars is suppressed at early times, as required by abundance matching arguments, resulting in flat star formation histories and higher gas fractions; (2) because 50 per cent of the stars form in situ from recycled disc gas with angular momentum only weakly related to that of the z = 0 dark halo; (3) because late-time gas accretion is typically an order of magnitude stronger and has higher specific angular momentum, with recycled gas dominating over primordial infall; (4) because 25-30 per cent of the total accreted gas is ejected entirely before z ˜ 1, removing primarily low angular momentum material which enriches the nearby intergalactic medium. Most recycled gas roughly conserves its angular momentum, but material ejected for long times and to large radii can gain significant angular momentum before re-accretion. These processes lower galaxy formation efficiency in addition to promoting disc formation.

  6. Multi-protein Delivery by Nanodiamonds Promotes Bone Formation

    PubMed Central

    Moore, L.; Gatica, M.; Kim, H.; Osawa, E.; Ho, D.

    2013-01-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE® for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation. PMID:24045646

  7. Multi-protein delivery by nanodiamonds promotes bone formation.

    PubMed

    Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

    2013-11-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

  8. Dirt and diarrhoea: formative research in hygiene promotion programmes.

    PubMed

    Curtis, V; Kanki, B; Cousens, S; Sanou, A; Diallo, I; Mertens, T

    1997-06-01

    Investment in the promotion of better hygiene for the prevention of diarrhoeal diseases and as a component of water and sanitation programmes is increasing. Before designing programmes capable of sustainably modifying hygiene behaviour in large populations, valid answers to a number of basic questions concerning the site and the intended beneficiaries have to be obtained. Such questions include 'what practices favour the transmission of enteric pathogens?', 'what advantages will be perceived by those who adopt safe practices?' and 'what channels of communication are currently employed by the target population?' A study of hygiene and diarrhoea in Bobo-Dioulasso, Burkina Faso, used a mixture of methods to address such questions. This paper draws on that experience to propose a plan of preliminary research using a variety of techniques which could be implemented over a period of a few months by planners of hygiene promotion programmes. The techniques discussed include structured observation, focus group discussions and behavioural trials. Modest investment in such systematic formative research with clear and limited goals is likely to be repaid many times over in the increased effectiveness of hygiene promotion programmes.

  9. Biostratigraphy and event stratigraphy in Iran around the Permian Triassic Boundary (PTB): Implications for the causes of the PTB biotic crisis

    NASA Astrophysics Data System (ADS)

    Kozur, H. W.

    2007-01-01

    The conodont succession and stratigraphic events around the Permian-Triassic boundary (PTB) have been investigated in detail in the open sea deposits of Iran (Abadeh and Shahreza in central Iran, and Jolfa and Zal in northwestern Iran). This investigation produced a very detailed conodont zonation from the Clarkina nodosa Zone up to the Isarcicella isarcica Zone. All significant events have been accurately located and dated within this zonation, and the duration of most of these conodont zones has been calculated by cross-correlation with continental lake deposits that display obvious Milankovitch cyclicity. The unusually short duration of all conodont zones in the interval from the C. nodosa up to the Hindeodus parvus Zone indicates that there was persistent high ecological stress during this time interval. Most of the conodont zones can be accurately correlated with South China. In the interval from the C. hauschkei Zone to the H. parvus Zone, even correlation with the Arctic is possible. Within three thin stratigraphic intervals, the Changhsingian (Dorashamian) warm water conodont fauna of the C. subcarinata lineage is replaced by a cool water fauna with small H. typicalis, rare Merrillina sp., and cool water Clarkina that have very widely spaced denticles. The uppermost cool water fauna horizon comprises the lower C. zhangi Zone and can be accurately correlated with continental beds by recognition of a short reversed magnetozone below the long uppermost Permian-lowermost Triassic normal magnetozone. In Iran and Transcaucasia, this short reversed zone comprises the upper C. changxingensis- C. deflecta Zone and most of the C. zhangi Zone. Its top lies 50 cm below the top of the Paratirolites Limestone (s.s.) in the Dorasham 2 section, which is at the beginning of the upper quarter of the C. zhangi Zone. In the Germanic Basin, this short palaeomagnetic interval comprises the lower and the basal part of the upper Fulda Formation. On the Russian Platform, the

  10. New PTB thermal neutron calibration facility: first results.

    PubMed

    Luszik-Bhadra, M; Reginatto, M; Wershofen, H; Wiegel, B; Zimbal, A

    2014-10-01

    A new thermal neutron calibration facility based on a moderator assembly has been set up at PTB. It consists of 16 (241)Am-Be radionuclide sources mounted in a graphite block, 1.5 m wide, 1.5 m high and 1.8 m deep. The sources are distributed to eight different positions, at a mean distance of ∼1.25 m from the front face of the moderator. The neutron field at the reference position, 30 cm in front of the moderator device and 75 cm above the floor, has been characterised using calculations, Bonner sphere measurements and gold foil activation. First results are shown. The field is highly thermalised: 99 % in terms of fluence. It is quite homogenous within a 20 cm×20 cm area, but the absolute value of the thermal neutron fluence rate is small and yields an ambient dose equivalent rate of 3 µSv h(-1).

  11. Microglia promote learning-dependent synapse formation through BDNF

    PubMed Central

    Parkhurst, Christopher N.; Yang, Guang; Ninan, Ipe; Savas, Jeffrey N.; Yates, John R.; Lafaille, Juan J.; Hempstead, Barbara L.; Littman, Dan R.; Gan, Wen-Biao

    2014-01-01

    SUMMARY Microglia are the resident macrophages of the central nervous system and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and function, however, remain unclear. To address this question, we generated CX3CR1CreER mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function in microglia. Using CX3CR1CreER to drive diphtheria toxin receptor expression in microglia, we found that microglia could be specifically depleted from the brain upon diphtheria toxin administration. Mice depleted of microglia show deficits in multiple learning tasks and a significant reduction in motor learning-dependent synapse formation. Furthermore, Cre-dependent removal of brain-derived neurotrophic factor (BDNF) from microglia largely recapitulated the effects of microglia depletion. Microglial BDNF increases neuronal TrkB phosphorylation, a key mediator of synaptic plasticity. Together, our findings reveal important physiological functions of microglia in learning and memory by promoting learning-related synapse formation through BDNF signaling. PMID:24360280

  12. High throughput sequencing analysis of Trypanosoma brucei DRBD3/PTB1-bound mRNAs.

    PubMed

    Das, Anish; Bellofatto, Vivian; Rosenfeld, Jeffrey; Carrington, Mark; Romero-Zaliz, Rocío; del Val, Coral; Estévez, Antonio M

    2015-01-01

    Trypanosomes are early-branched eukaryotes that show an unusual dependence on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins are crucial in controlling mRNA fate in these organisms, but their RNA substrates remain largely unknown. Here we have analyzed on a global scale the mRNAs associated with the Trypanosoma brucei RNA-binding protein DRBD3/PTB1, by capturing ribonucleoprotein complexes using UV cross-linking and subsequent immunoprecipitation. DRBD3/PTB1 associates with many transcripts encoding ribosomal proteins and translation factors. Consequently, silencing of DRBD3/PTB1 expression altered the protein synthesis rate. DRBD3/PTB1 also binds to mRNAs encoding the enzymes required to obtain energy through the oxidation of proline to succinate. We hypothesize that DRBD3/PTB1 is a key player in RNA regulon-based gene control influencing protein synthesis in trypanosomes.

  13. Polypyrimidine tract binding protein-1 (PTB1) is a determinant of the tissue and host tropism of a human rhinovirus/poliovirus chimera PV1(RIPO).

    PubMed

    Jahan, Nusrat; Wimmer, Eckard; Mueller, Steffen

    2013-01-01

    The internal ribosomal entry site (IRES) of picornavirus genomes serves as the nucleation site of a highly structured ribonucleoprotein complex essential to the binding of the 40S ribosomal subunit and initiation of viral protein translation. The transition from naked RNA to a functional "IRESome" complex are poorly understood, involving the folding of secondary and tertiary RNA structure, facilitated by a tightly concerted binding of various host cell proteins that are commonly referred to as IRES trans-acting factors (ITAFs). Here we have investigated the influence of one ITAF, the polypyrimidine tract-binding protein 1 (PTB1), on the tropism of PV1(RIPO), a chimeric poliovirus in which translation of the poliovirus polyprotein is under the control of a human rhinovirus type 2 (HRV2) IRES element. We show that PV1(RIPO)'s growth defect in restrictive mouse cells is partly due to the inability of its IRES to interact with endogenous murine PTB. Over-expression of human PTB1 stimulated the HRV2 IRES-mediated translation, resulting in increased growth of PV1(RIPO) in murine cells and human neuronal SK-N-MC cells. Mutations within the PV1(RIPO) IRES, selected to grow in restrictive mouse cells, eliminated the human PTB1 supplementation requirement, by restoring the ability of the IRES to interact with endogenous murine PTB. In combination with our previous findings these results give a compelling insight into the thermodynamic behavior of IRES structures. We have uncovered three distinct thermodynamic aspects of IRES formation which may independently contribute to overcome the observed PV1(RIPO) IRES block by lowering the free energy δG of the IRESome formation, and stabilizing the correct and functional structure: 1) lowering the growth temperature, 2) modifying the complement of ITAFs in restricted cells, or 3) selection of adaptive mutations. All three mechanisms can conceivably modulate the thermodynamics of RNA folding, and thus facilitate and stabilize the

  14. Vibration acceleration promotes bone formation in rodent models

    PubMed Central

    Uchida, Ryohei; Nakata, Ken; Kawano, Fuminori; Yonetani, Yasukazu; Ogasawara, Issei; Nakai, Naoya; Mae, Tatsuo; Matsuo, Tomohiko; Tachibana, Yuta; Yokoi, Hiroyuki; Yoshikawa, Hideki

    2017-01-01

    centrifuge acceleration group had no significant difference compared those in control-CA group. Union rate and BV in the low-magnitude group of RFH model were also significantly higher than those in the other groups (Union rate: 60% v.s. 0% in the high-magnitude group and 10% in the control-VA group, BV: 0.69±0.30mm3 v.s. 0.15±0.09mm3 in high-magnitude group and 0.22±0.17mm3 in control-VA group). BV/TV in the low-magnitude group of RFH model was significantly higher than that in control-VA group (59.4±14.9% v.s. 35.8±13.5%). On the other hand, radiographic union rate (10% in centrifuge acceleration group v.s. 20% in control-CA group) and micro-CT parameters in RFH model were not significantly different between two groups in the constant acceleration studies. Radiographic images of non-union rib fractures showed cartilage at the fracture site and poor new bone formation, whereas union samples showed only new bone. In conclusion, low-magnitude vibration acceleration promoted bone formation at the trunk in both BMP-induced ectopic bone formation and rib fracture healing models. However, the micro-CT parameters were not similar between two models, which suggested that there might be difference in the mechanism of effect by vibration between two models. PMID:28264058

  15. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    PubMed Central

    Li, Shan; Shu, Bing; Zhang, Yanquan; Li, Jia; Guo, Junwei; Wang, Yinyin; Ren, Fangli; Xiao, Guozhi; Chang, Zhijie; Chen, Di

    2014-01-01

    Objective Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling. Methods The bone phenotype of Chip−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays. Results In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription. Conclusion CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases. PMID:24578159

  16. Conduit speed limit promotes formation of explosive `super slugs'

    NASA Astrophysics Data System (ADS)

    Llewellin, E. W.; Burton, M. R.; Mader, H. M.; Polacci, M.

    2014-12-01

    Strombolian activity - characterized by discrete, impulsive explosions - is common at basaltic volcanoes. The explosions are caused by the arrival, at the volcanic vent, of over-pressured `slugs' of magmatic gas, which have ascended the volcanic conduit. We present data from large-scale analogue experiments which reveal a previously-undescribed process which promotes the formation of large, highly over-pressured `super slugs'. We propose that these large slugs may drive the most violent Strombolian eruptions. Experiments were performed at the Large Analogue Volcano Apparatus at Durham University, UK, which comprises a 13m tall, 24cm diameter transparent conduit, surmounting a reservoir of analogue magma held at constant pressure. We simulate a vesiculation event deep in the sub-volcanic plumbing system by fluxing gas into the reservoir. Magma analogues with a range of viscosities are used, giving slug Reynolds numbers in the range 16 to 140,000. At moderate-to-high Reynolds number, we find that the gas rapidly self-organizes to form a conduit-filling lead slug; this slug ascends the column slowly, at a velocity limited by the flux of the falling film of liquid around it. Trailing bubbles, which ascend through the wake of the lead slug, rise much more rapidly. As they catch and coalesce with the lead slug, it grows and becomes increasingly over-pressured. This mechanism causes large slugs to form rapidly and we propose that it underpins the formation of the very large slugs that are responsible for the most explosive strombolian eruptions.

  17. Understanding low bandgap polymer PTB7 and optimizing polymer solar cells based on it.

    PubMed

    Lu, Luyao; Yu, Luping

    2014-07-09

    Solution processed single junction polymer solar cells (PSCs) have been developed from less than 1% power conversion efficiency (PCE) to beyond 9% PCE in the last decade. The significant efficiency improvement comes from progress in both rational design of donor polymers and innovation of device architectures. Among all the novel high efficient donor polymers, PTB7 stands out as the most widely used one for solar cell studies. Herein the recent development of PTB7 solar cells is reviewed. Detailed discussion of basic property, structure property relationship, morphology study, interfacial engineering, and inorganic nanomaterials incorporation is provided. Possible future directions for further increasing the performance of PTB7 solar cells are discussed.

  18. Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence

    PubMed Central

    Martínez, Eriel; Campos-Gómez, Javier

    2016-01-01

    The oxygenation of unsaturated fatty acids by dioxygenases occurs in all kingdoms of life and produces physiologically important lipids called oxylipins. The biological roles of oxylipins have been extensively studied in animals, plants, algae and fungi, but remain largely unidentified in prokaryotes. The bacterium Pseudomonas aeruginosa displays a diol synthase activity that transforms several monounsaturated fatty acids into mono- and di-hydroxylated derivatives. Here we show that oxylipins derived from this activity inhibit flagellum-driven motility and upregulate type IV pilus-dependent twitching motility of P. aeruginosa. Consequently, these oxylipins promote bacterial organization in microcolonies, increasing the ability of P. aeruginosa to form biofilms in vitro and in vivo (in Drosophila flies). We also demonstrate that oxylipins produced by P. aeruginosa promote virulence in Drosophila flies and lettuce. Our study thus uncovers a role for prokaryotic oxylipins in the physiology and pathogenicity of bacteria. PMID:27929111

  19. MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development

    PubMed Central

    Boutz, Paul L.; Chawla, Geetanjali; Stoilov, Peter; Black, Douglas L.

    2007-01-01

    Alternative pre-mRNA splicing determines many changes in gene expression during development. Two regulators known to control splicing patterns during neuron and muscle differentiation are the polypyrimidine tract-binding protein (PTB) and its neuronal homolog nPTB. These proteins repress certain exons in early myoblasts, but upon differentiation of mature myotubes PTB/nPTB expression is reduced, leading to increased inclusion of their target exons. We show here that the repression of nPTB expression during myoblast differentiation results from its targeting by the muscle-restricted microRNA miR-133. During differentiation of C2C12 myoblasts, nPTB protein but not mRNA expression is strongly reduced, concurrent with the up-regulation of miR-133 and the induction of splicing for several PTB-repressed exons. Introduction of synthetic miR-133 into undifferentiated C2C12 cells leads to a decrease in endogenous nPTB expression. Both the miR-133 and the coexpressed miR-1/206 microRNAs are extremely conserved across animal species, and PTB proteins are predicted targets for these miRNAs in Drosophila, mice, and humans. There are two potential miR-133-responsive elements (MRE) within the nPTB 3′ untranslated region (UTR), and a luciferase reporter carrying this 3′ UTR is repressed by miR-133 in an MRE-dependent manner. Transfection of locked nucleic acid (LNA) oligonucleotides designed to block the function of miR-133 and miR-1/206 increases expression of nPTB and decreases the inclusion of PTB dependent exons. These results indicate that miR-133 directly down-regulates a key splicing factor during muscle development and establishes a role for microRNAs in the control of a developmentally dynamic splicing program. PMID:17210790

  20. Induced dicentric chromosome formation promotes genomic rearrangements and tumorigenesis.

    PubMed

    Gascoigne, Karen E; Cheeseman, Iain M

    2013-07-01

    Chromosomal rearrangements can radically alter gene products and their function, driving tumor formation or progression. However, the molecular origins and evolution of such rearrangements are varied and poorly understood, with cancer cells often containing multiple, complex rearrangements. One mechanism that can lead to genomic rearrangements is the formation of a "dicentric" chromosome containing two functional centromeres. Indeed, such dicentric chromosomes have been observed in cancer cells. Here, we tested the ability of a single dicentric chromosome to contribute to genomic instability and neoplastic conversion in vertebrate cells. We developed a system to transiently and reversibly induce dicentric chromosome formation on a single chromosome with high temporal control. We find that induced dicentric chromosomes are frequently damaged and mis-segregated during mitosis, and that this leads to extensive chromosomal rearrangements including translocations with other chromosomes. Populations of pre-neoplastic cells in which a single dicentric chromosome is induced acquire extensive genomic instability and display hallmarks of cellular transformation including anchorage-independent growth in soft agar. Our results suggest that a single dicentric chromosome could contribute to tumor initiation.

  1. PTB’s Time and Frequency Activities in 2008 and 2009

    DTIC Science & Technology

    2009-11-01

    A. Bauch, 2005, “The PTB primary clocks CS1 and CS2,” Metrologia , 42, S43-S54. [2] R. Wynands and S. Weyers, 2005, “Atomic fountain clocks... Metrologia , 42, S64-S79. [3] V. Gerginov, N. Nemitz, S. Weyers, R. Schröder, D. Griebsch, and R. Wynands, 2010, “Uncertainty evaluation of the caesium...fountain clock PTB-CSF2,” Metrologia , 47, 65-79. [4] W. Lewandowski, G. Panfilo, A Harmegnies, and F. Arias, 2010, “BIPM time section update,” in

  2. Methylglyoxal induces tau hyperphosphorylation via promoting AGEs formation.

    PubMed

    Li, Xiao-Hong; Xie, Jia-Zhao; Jiang, Xia; Lv, Bing-Ling; Cheng, Xiang-Shu; Du, Lai-Ling; Zhang, Jia-Yu; Wang, Jian-Zhi; Zhou, Xin-Wen

    2012-12-01

    The hyperphosphorylated tau is a major protein component of neurofibrillary tangle, which is one of hallmarks of Alzheimer's disease (AD). While the level of methylglyoxal (MG) is significantly increased in the AD brains, the role of MG in tau phosphorylation is still not reported. Here, we found that MG could induce tau hyperphosphorylation at multiple AD-related sites in neuroblastoma 2a cells under maintaining normal cell viability. MG treatment increased the level of advanced glycation end products (AGEs) and the receptor of AGEs (RAGE). Glycogen synthesis kinase-3β (GSK-3β) and p38 MAPK were activated, whereas the level and activity of JNK, Erk1/2, cdk5, and PP2A were not altered after MG treatment. Simultaneous inhibition of GSK-3β or p38 attenuated the MG-induced tau hyperphosphorylation. Aminoguanidine, a blocker of AGEs formation, could effectively reverse the MG-induced tau hyperphosphorylation. These data suggest that MG induces AD-like tau hyperphosphorylation through AGEs formation involving RAGE up-regulation and GSK-3β activation and p38 activation is also partially involved in MG-induced tau hyperphosphorylation. Thus, targeting MG may be a promising therapeutic strategy to prevent AD-like tau hyperphosphorylation.

  3. Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation

    PubMed Central

    Bernut, Audrey; Herrmann, Jean-Louis; Kissa, Karima; Dubremetz, Jean-François; Gaillard, Jean-Louis; Lutfalla, Georges; Kremer, Laurent

    2014-01-01

    Mycobacterium abscessus is a rapidly growing Mycobacterium causing a wide spectrum of clinical syndromes. It now is recognized as a pulmonary pathogen to which cystic fibrosis patients have a particular susceptibility. The M. abscessus rough (R) variant, devoid of cell-surface glycopeptidolipids (GPLs), causes more severe clinical disease than the smooth (S) variant, but the underlying mechanisms of R-variant virulence remain obscure. Exploiting the optical transparency of zebrafish embryos, we observed that the increased virulence of the M. abscessus R variant compared with the S variant correlated with the loss of GPL production. The virulence of the R variant involved the massive production of serpentine cords, absent during S-variant infection, and the cords initiated abscess formation leading to rapid larval death. Cording occurred within the vasculature and was highly pronounced in the central nervous system (CNS). It appears that M. abscessus is transported to the CNS within macrophages. The release of M. abscessus from apoptotic macrophages initiated the formation of cords that grew too large to be phagocytized by macrophages or neutrophils. This study is a description of the crucial role of cording in the in vivo physiopathology of M. abscessus infection and emphasizes cording as a mechanism of immune evasion. PMID:24567393

  4. PTH-IGF SIGNALING PROMOTES BONE FORMATION THROUGH GLYCOLYSIS

    PubMed Central

    Esen, Emel; Lee, Seung-Yon; Wice, Burton M; Long, Fanxin

    2016-01-01

    Teriparatide, a recombinant peptide corresponding to amino acids 1-34 of human parathyroid hormone (PTH), has been an effective bone anabolic drug for over a decade. However, the mechanism whereby PTH stimulates bone formation remains poorly understood. Here we report that in cultures of osteoblast-lineage cells, PTH stimulates glucose consumption and lactate production in the presence of oxygen, a hallmark of aerobic glycolysis, also known as Warburg effect. Experiments with radioactively labeled glucose demonstrate that PTH suppresses glucose entry into the tricarboxylic acid cycle (TCA cycle). Mechanistically, the increase in aerobic glycolysis is secondary to insulin-like growth factor (Igf) signaling induced by PTH, whereas the metabolic effect of Igf is dependent on activation of mammalian target of rapamycin complex 2 (mTORC2). Importantly, pharmacological perturbation of glycolysis suppresses the bone anabolic effect of intermittent PTH in the mouse. Thus, stimulation of aerobic glycolysis via Igf signaling contributes to bone anabolism in response to PTH. PMID:25990470

  5. Exercise promotes positive impression formation towards both men and women.

    PubMed

    Kanarek, Robin B; Mathes, Wendy Foulds; D'Anci, Kristen E

    2012-06-01

    Exercise is endorsed for its physiological and psychological benefits, and has been proposed to have positive effects on impression formation. To test this proposal, 62 female and 44 male college students read one of three brief descriptions of either a fictitious male or female "target" student. The descriptions varied only in exercise level: no exercise; moderate exercise and intensive exercise. Participants then rated the fictitious student on 38 personality traits. Ratings of characteristics that are associated with exercise (e.g. athletic; energetic) increased, while ratings associated with the lack of exercise (e.g. lazy; weak) decreased as a function of the reported level of exercise. Exercise level also positively influenced ratings of characteristics not related to exercise. These data show that even minimal information about exercise is an important component of first impressions in both men and women.

  6. Promoting cooperation by reputation-driven group formation

    NASA Astrophysics Data System (ADS)

    Yang, Han-Xin; Wang, Zhen

    2017-02-01

    In previous studies of the spatial public goods game, each player is able to establish a group. However, in real life, some players cannot successfully organize groups for various reasons. In this paper, we propose a mechanism of reputation-driven group formation, in which groups can only be organized by players whose reputation reaches or exceeds a threshold. We define a player’s reputation as the frequency of cooperation in the last T time steps. We find that the highest cooperation level can be obtained when groups are only established by pure cooperators who always cooperate in the last T time steps. Effects of the memory length T on cooperation are also studied.

  7. Tropoelastin coated PLLA-PLGA scaffolds promote vascular network formation.

    PubMed

    Landau, Shira; Szklanny, Ariel A; Yeo, Giselle C; Shandalov, Yulia; Kosobrodova, Elena; Weiss, Anthony S; Levenberg, Shulamit

    2017-04-01

    The robust repair of large wounds and tissue defects relies on blood flow. This vascularization is the major challenge faced by tissue engineering on the path to forming thick, implantable tissue constructs. Without this vasculature, oxygen and nutrients cannot reach the cells located far from host blood vessels. To make viable constructs, tissue engineering takes advantage of the mechanical properties of synthetic materials, while combining them with ECM proteins to create a natural environment for the tissue-specific cells. Tropoelastin, the precursor of the elastin, is the ECM protein responsible for elasticity in diverse tissues, including robust blood vessels. Here, we seeded endothelial cells with supporting cells on PLLA/PLGA scaffolds treated with tropoelastin, and examined the morphology, expansion and maturity of the newly formed vessels. Our results demonstrate that the treated scaffolds elicit a more expanded, complex and developed vascularization in comparison to the untreated group. Implantation of tropoelastin-treated scaffolds into mouse abdominal muscle resulted in enhanced perfusion of the penetrating vasculature and improved integration. This study points to the great potential of these combined materials in promoting the vascularization of implanted engineered constructs, which can be further exploited in the fabrication of clinically relevant engineered tissues.

  8. NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis

    PubMed Central

    Röhm, Marc; Grimm, Melissa J.; D'Auria, Anthony C.; Almyroudis, Nikolaos G.

    2014-01-01

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47phox−/−) mice which had resolved in wild-type mice by day 5 but progressed in p47phox−/− mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47phox−/− mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance. PMID:24549323

  9. Native low density lipoprotein promotes lipid raft formation in macrophages

    PubMed Central

    SONG, JIAN; PING, LING-YAN; DUONG, DUC M.; GAO, XIAO-YAN; HE, CHUN-YAN; WEI, LEI; WU, JUN-ZHU

    2016-01-01

    Oxidized low-density lipoprotein (LDL) has an important role in atherogenesis; however, the mechanisms underlying cell-mediated LDL oxidation remain to be elucidated. The present study investigated whether native-LDL induced lipid raft formation, in order to gain further insight into LDL oxidation. Confocal microscopic analysis revealed that lipid rafts were aggregated or clustered in the membrane, which were colocalized with myeloperoxidase (MPO) upon native LDL stimulation; however, in the presence of methyl-β-cyclodextrin (MβCD), LDL-stimulated aggregation, translocation, and colocalization of lipid rafts components was abolished.. In addition, lipid raft disruptors MβCD and filipin decreased malondialdehyde expression levels. Density gradient centrifugation coupled to label-free quantitative proteomic analysis identified 1,449 individual proteins, of which 203 were significantly upregulated following native-LDL stimulation. Functional classification of the proteins identified in the lipid rafts revealed that the expression levels of translocation proteins were upregulated. In conclusion, the results of the present study indicated that native-LDL induced lipid raft clustering in macrophages, and the expression levels of several proteins were altered in the stimulated macrophages, which provided novel insights into the mechanism underlying LDL oxidation. PMID:26781977

  10. Chlorogenic acid increased acrylamide formation through promotion of HMF formation and 3-aminopropionamide deamination.

    PubMed

    Cai, Yun; Zhang, Zhenhua; Jiang, Shanshan; Yu, Miao; Huang, Caihuan; Qiu, Ruixia; Zou, Yueyu; Zhang, Qirui; Ou, Shiyi; Zhou, Hua; Wang, Yong; Bai, Weibing; Li, Yiqun

    2014-03-15

    This research was aimed to investigate why chlorogenic acid, presents at high concentrations in some food raw material, influences acrylamide formation. In the asparagine/glucose Maillard reaction system (pH=6.8), addition of chlorogenic acid significantly increased acrylamide formation and inhibited its elimination. In contrast, the quinone derivative of chlorogenic acid decreased acrylamide formation. Three mechanisms may be involved for increasing acrylamide formation by chlorogenic acid. Firstly, it increased the formation of HMF, which acts as a more efficient precursor than glucose to form acrylamide. Secondly, it decreased activation energy for conversion of 3-aminopropionamide (3-APA) to acrylamide (from 173.2 to 136.6kJ/mol), and enhances deamination from 3-APA. And thirdly, it prevented attack of the produced acrylamide from free radicals by keeping high redox potential during the Maillard reaction.

  11. Insight into the mechanism of Sb promoted Cu(In,Ga)Se{sub 2} formation

    SciTech Connect

    Xiang, Yong Zhang, Xiaokun; Zhang, Shu

    2013-08-15

    Sb-doping has been demonstrated to be a new approach to promote Cu(In,Ga)Se{sub 2} (CIGS) thin film formation. To study the mechanism of Sb-promoted CIGS formation, we investigated the influence of Sb on the evolution of the critical intermediate Cu–Se phases, and found that Cu{sub 3}SbSe{sub 3} species was formed. Phase change of the as-prepared Cu–Se compounds at elevated temperature was determined using the differential scanning calorimetry analysis. For samples containing Sb, the melting decomposition occurred at 507.1 °C, along with aggregation of nanocrystals into a bulk, while the morphology of the sample without Sb barely changed after heating. This result suggests that the mobile Cu{sub 3}SbSe{sub 3} is likely the key intermediate responsible for Sb-promoted CIGS formation. Furthermore, we extended the scope of Sb-doping approach to solvothermal synthesis and CIGS nanocrystals were synthesized with significantly promoted kinetics in the presence of Sb. Based on these results, we propose the mechanism of Sb promoted CIGS formation. - Graphical abstract: Cu{sub 3}SbSe{sub 3} mobile phase is likely the key species to promote the formation of Cu(In,Ga)Se{sub 2}, and significantly promoting effect by Sb is also found in the synthesis of Cu(In,Ga)Se{sub 2} nanocrystals. Highlights: • In the presence of Sb, Cu{sub 3}SbSe{sub 3} is formed while synthesizing the essential intermediate Cu–Se compounds for Cu(In,Ga)Se{sub 2} materials. • Cu{sub 3}SbSe{sub 3} shows high mobility at elevated temperature. • Cu{sub 3}SbSe{sub 3} mobile phase is likely the key species to improve Cu(In,Ga)Se{sub 2} thin film formation. • A synthesis methodology is developed to produce Cu(In,Ga)Se{sub 2} nanocrystals with significantly promoted reaction kinetics.

  12. Structural Analysis of G-Quadruplex Formation at the Human MEST Promoter

    PubMed Central

    Kennedy, Martin A.

    2017-01-01

    The promoter region of the imprinted gene MEST contains several motifs capable of forming G-quadruplex (G4) structures, which appear to contribute to consistent allelic dropout during polymerase chain reaction (PCR) analysis of this region. Here, we extend our previous analysis of MEST G4 structures by applying fluorescent footprinting techniques to assess non B-DNA structure and topology in dsDNA from the full MEST promoter region, under conditions that mimic PCR. We demonstrate that the buffer used for PCR provides an extremely favourable milieu for G4 formation, and that cytosine methylation helps maintain G4 structures during PCR. Additionally, we demonstrate G4 formation at motifs not previously identified through bioinformatic analysis of the MEST promoter, and provide nucleotide level resolution for topological reconstruction of these structures. These observations increase our understanding of the mechanisms through which methylation and G4 contribute towards allelic drop-out during PCR. PMID:28052120

  13. Modeling the Free Carrier Recombination Kinetics in PTB7:PCBM Organic Photovoltaics

    SciTech Connect

    Oosterhout, Stefan D.; Ferguson, Andrew J.; Larson, Bryon W.; Olson, Dana C.; Kopidakis, Nikos

    2016-11-03

    Currently the exact recombination mechanism of free carriers in organic photovoltaic (OPV) devices is poorly understood. Often a reduced Langevin model is used to describe the decay behavior of electrons and holes. Here we propose a novel, simple kinetic model that accurately describes the decay behavior of free carriers in the PTB7:PCBM organic photovoltaic blend. This model needs to only take into account free and trapped holes in the polymer, and free electrons in the fullerene, to accurately describe the recombination behavior of free carriers as measured by time-resolved microwave conductivity (TRMC). The model is consistent for different PTB7:PCBM blend ratios and spans a light intensity range of over 3 orders of magnitude. The model demonstrates that dark carriers exist in the polymer and interact with photoinduced charge carriers, and that the trapping and detrapping rates of the holes are of high importance to the overall carrier lifetime.

  14. Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB

    PubMed Central

    Coelho, Miguel B; Attig, Jan; Bellora, Nicolás; König, Julian; Hallegger, Martina; Kayikci, Melis; Eyras, Eduardo; Ule, Jernej; Smith, Christopher WJ

    2015-01-01

    Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3. PMID:25599992

  15. Providing radiometric traceability for the calibration home base of DLR by PTB

    NASA Astrophysics Data System (ADS)

    Taubert, D. R.; Hollandt, J.; Sperfeld, P.; Pape, S.; Höpe, A.; Hauer, K.-O.; Gege, P.; Schwarzmaier, T.; Lenhard, K.; Baumgartner, A.

    2013-05-01

    A dedicated calibration technique was applied for the calibration of the spectral radiance transfer standard (RASTA) of the German Aerospace Center (DLR) at the Physikalisch-Technische Bundesanstalt (PTB), consisting of two independent but complementing calibration procedures to provide redundancy and smallest possible calibration uncertainties. Procedure I included two calibration steps: In a first step the optical radiation source of RASTA, an FEL lamp, was calibrated in terms of its spectral irradiance Eλ(λ) in the wavelength range from 350 nm to 2400 nm using the PTB Spectral Irradiance Calibration Equipment (SPICE), while in a second step the spectral radiance factor β0°:45°(λ) of the RASTA reflection standard was calibrated in a 0°:45°-viewing geometry in the wavelength range from 350 nm to 1700 nm at the robot-based gonioreflectometer facility of PTB. The achieved relative standard uncertainties (k = 1) range from 0.6 % to 3.2 % and 0.1 % to 0.6 % respectively. Procedure II was completely independent from procedure I and allowed to cover the entire spectral range of RASTA from 350 nm to 2500 nm. In the second procedure, the 0°:45°-viewing geometry spectral radiance Lλ,0°:45°(λ) of RASTA was directly calibrated at the Spectral Radiance Comparator Facility (SRCF) of PTB. The relative uncertainties for this calibration procedure range from 0.8 % in the visible up to 7.5 % at 2500 nm (k = 1). In the overlapping spectral range of both calibration procedures the calculated spectral radiance Lλ,0°:45°,calc(λ) from procedure I is in good agreement with the direct measurement of procedure II, i.e. well within the combined expanded uncertainties (k = 2) of both procedures.

  16. Providing radiometric traceability for the calibration home base of DLR by PTB

    SciTech Connect

    Taubert, D. R.; Hollandt, J.; Sperfeld, P.; Pape, S.; Hoepe, A.; Hauer, K.-O.; Gege, P.; Schwarzmaier, T.; Lenhard, K.; Baumgartner, A.

    2013-05-10

    A dedicated calibration technique was applied for the calibration of the spectral radiance transfer standard (RASTA) of the German Aerospace Center (DLR) at the Physikalisch-Technische Bundesanstalt (PTB), consisting of two independent but complementing calibration procedures to provide redundancy and smallest possible calibration uncertainties. Procedure I included two calibration steps: In a first step the optical radiation source of RASTA, an FEL lamp, was calibrated in terms of its spectral irradiance E{sub {lambda}}({lambda}) in the wavelength range from 350 nm to 2400 nm using the PTB Spectral Irradiance Calibration Equipment (SPICE), while in a second step the spectral radiance factor {beta}{sub 0 Degree-Sign :45 Degree-Sign }({lambda}) of the RASTA reflection standard was calibrated in a 0 Degree-Sign :45 Degree-Sign -viewing geometry in the wavelength range from 350 nm to 1700 nm at the robot-based gonioreflectometer facility of PTB. The achieved relative standard uncertainties (k= 1) range from 0.6 % to 3.2 % and 0.1 % to 0.6 % respectively. Procedure II was completely independent from procedure I and allowed to cover the entire spectral range of RASTA from 350 nm to 2500 nm. In the second procedure, the 0 Degree-Sign :45 Degree-Sign -viewing geometry spectral radiance L{sub {lambda},0 Degree-Sign :45 Degree-Sign }({lambda}) of RASTA was directly calibrated at the Spectral Radiance Comparator Facility (SRCF) of PTB. The relative uncertainties for this calibration procedure range from 0.8 % in the visible up to 7.5 % at 2500 nm (k= 1). In the overlapping spectral range of both calibration procedures the calculated spectral radiance L{sub {lambda},0 Degree-Sign :45 Degree-Sign ,calc}({lambda}) from procedure I is in good agreement with the direct measurement of procedure II, i.e. well within the combined expanded uncertainties (k= 2) of both procedures.

  17. Circadian glucocorticoid oscillations promote learning-dependent synapse formation and maintenance.

    PubMed

    Liston, Conor; Cichon, Joseph M; Jeanneteau, Freddy; Jia, Zhengping; Chao, Moses V; Gan, Wen-Biao

    2013-06-01

    Excessive glucocorticoid exposure during chronic stress causes synapse loss and learning impairment. Under normal physiological conditions, glucocorticoid activity oscillates in synchrony with the circadian rhythm. Whether and how endogenous glucocorticoid oscillations modulate synaptic plasticity and learning is unknown. Here we show that circadian glucocorticoid peaks promote postsynaptic dendritic spine formation in the mouse cortex after motor skill learning, whereas troughs are required for stabilizing newly formed spines that are important for long-term memory retention. Conversely, chronic and excessive exposure to glucocorticoids eliminates learning-associated new spines and disrupts previously acquired memories. Furthermore, we show that glucocorticoids promote rapid spine formation through a non-transcriptional mechanism by means of the LIM kinase-cofilin pathway and increase spine elimination through transcriptional mechanisms involving mineralocorticoid receptor activation. Together, these findings indicate that tightly regulated circadian glucocorticoid oscillations are important for learning-dependent synaptic formation and maintenance. They also delineate a new signaling mechanism underlying these effects.

  18. Sleep promotes branch-specific formation of dendritic spines after learning.

    PubMed

    Yang, Guang; Lai, Cora Sau Wan; Cichon, Joseph; Ma, Lei; Li, Wei; Gan, Wen-Biao

    2014-06-06

    How sleep helps learning and memory remains unknown. We report in mouse motor cortex that sleep after motor learning promotes the formation of postsynaptic dendritic spines on a subset of branches of individual layer V pyramidal neurons. New spines are formed on different sets of dendritic branches in response to different learning tasks and are protected from being eliminated when multiple tasks are learned. Neurons activated during learning of a motor task are reactivated during subsequent non-rapid eye movement sleep, and disrupting this neuronal reactivation prevents branch-specific spine formation. These findings indicate that sleep has a key role in promoting learning-dependent synapse formation and maintenance on selected dendritic branches, which contribute to memory storage.

  19. PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

    PubMed

    Prieto-Echagüe, Victoria; Chan, Perry M; Craddock, Barbara P; Manser, Edward; Miller, W Todd

    2011-04-26

    Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

  20. Thyroid dysfunction, either hyper or hypothyroidism, promotes gallstone formation by different mechanisms*

    PubMed Central

    Wang, Yong; Yu, Xing; Zhao, Qun-zi; Zheng, Shu; Qing, Wen-jie; Miao, Chun-di; Sanjay, Jaiswal

    2016-01-01

    We have investigated comprehensively the effects of thyroid function on gallstone formation in a mouse model. Gonadectomized gallstone-susceptible male C57BL/6 mice were randomly distributed into three groups each of which received an intervention to induce hyperthyroidism, hypothyroidism, or euthyroidism. After 5 weeks of feeding a lithogenic diet of 15% (w/w) butter fat, 1% (w/w) cholesterol, and 0.5% (w/w) cholic acid, mice were killed for further experiments. The incidence of cholesterol monohydrate crystal formation was 100% in mice with hyperthyroidism, 83% in hypothyroidism, and 33% in euthyroidism, the differences being statistically significant. Among the hepatic lithogenic genes, Trβ was found to be up-regulated and Rxr down-regulated in the mice with hypothyroidism. In contrast, Lxrα, Rxr, and Cyp7α1 were up-regulated and Fxr down-regulated in the mice with hyperthyroidism. In conclusion, thyroid dysfunction, either hyperthyroidism or hypothyroidism, promotes the formation of cholesterol gallstones in C57BL/6 mice. Gene expression differences suggest that thyroid hormone disturbance leads to gallstone formation in different ways. Hyperthyroidism induces cholesterol gallstone formation by regulating expression of the hepatic nuclear receptor genes such as Lxrα and Rxr, which are significant in cholesterol metabolism pathways. However, hypothyroidism induces cholesterol gallstone formation by promoting cholesterol biosynthesis. PMID:27381728

  1. Protein tyrosine phosphatase SHP2 promotes invadopodia formation through suppression of Rho signaling

    PubMed Central

    Tsai, Wan-Chen; Chen, Chien-Lin; Chen, Hong-Chen

    2015-01-01

    Invadopodia are actin-enriched membrane protrusions that are important for extracellular matrix degradation and invasive cell motility. Src homolog domain-containing phosphatase 2 (SHP2), a non-receptor protein tyrosine phosphatase, has been shown to play an important role in promoting cancer metastasis, but the underlying mechanism is unclear. In this study, we found that depletion of SHP2 by short-hairpin RNA suppressed invadopodia formation in several cancer cell lines, particularly in the SAS head and neck squamous cell line. In contrast, overexpression of SHP2 promoted invadopodia formation in the CAL27 head and neck squamous cell line, which expresses low levels of endogenous SHP2. The depletion of SHP2 in SAS cells significantly decreased their invasive motility. The suppression of invadopodia formation by SHP2 depletion was restored by the Clostridium botulinum C3 exoenzyme (a Rho GTPase inhibitor) or Y27632 (a specific inhibitor for Rho-associated kinase). Together, our results suggest that SHP2 may promote invadopodia formation through inhibition of Rho signaling in cancer cells. PMID:26204488

  2. The calcium-dependent ribonuclease XendoU promotes ER network formation through local RNA degradation

    PubMed Central

    Schwarz, Dianne S.

    2014-01-01

    How cells shape and remodel organelles in response to cellular signals is a poorly understood process. Using Xenopus laevis egg extract, we found that increases in cytosolic calcium lead to the activation of an endogenous ribonuclease, XendoU. A fraction of XendoU localizes to the endoplasmic reticulum (ER) and is required for nuclear envelope assembly and ER network formation in a catalysis-dependent manner. Using a purified vesicle fusion assay, we show that XendoU functions on the surface of ER membranes to promote RNA cleavage and ribonucleoprotein (RNP) removal. Additionally, RNA removal from the surface of vesicles by RNase treatment leads to increased ER network formation. Using human tissue culture cells, we found that hEndoU localizes to the ER, where it promotes the formation of ER tubules in a catalysis-dependent manner. Together, these results demonstrate that calcium-activated removal of RNA from membranes by XendoU promotes and refines ER remodeling and the formation of tubular ER. PMID:25287301

  3. Transcriptional inhibition of the bacteriophage T7 early promoter region by oligonucleotide triple helix formation.

    PubMed

    Ross, C; Samuel, M; Broitman, S L

    1992-12-30

    We have identified a purine-rich triplex binding sequence overlapping a -35 transcriptional early promoter region of the bacteriophage T7. Triplex-forming oligonucleotide designed to bind this target was annealed to T7 templates and introduced into in vitro transcription systems under conditions favoring specific initiation from this promoter. These templates demonstrated significant transcriptional inhibition relative to naked genomic templates and templates mixed with non-triplex-forming oligonucleotide. It is suggested that triplex formation along this target interferes with transcriptional initiation, and this mechanism may hold potential to disrupt bacteriophage T7 early transcription in vivo.

  4. SCCmec-associated psm-mec mRNA promotes Staphylococcus epidermidis biofilm formation.

    PubMed

    Yang, Yongchang; Zhang, Xuemei; Huang, Wenfang; Yin, Yibing

    2016-10-01

    Biofilm formation is considered the major pathogenic mechanism of Staphylococcus epidermidis-associated nosocomial infections. Reports have shown that SCCmec-associated psm-mec regulated methicillin-resistant Staphylococcus aureus virulence and biofilm formation. However, the role of psm-mec in S. epidermidis remains unclear. To this purpose, we analysed 165 clinical isolates of S. epidermidis to study the distribution, mutation and expression of psm-mec and the relationship between this gene and biofilm formation. Next, we constructed three psm-mec deletion mutants, one psm-mec transgene expression strain (p221) and two psm-mec point mutant strains (pM, pAG) to explore its effects on S. epidermidis biofilm formation. Then, the amount of biofilm formation, extracellular DNA (eDNA) and Triton X-100-induced autolysis of the constructed strains was measured. Results of psm-mec deletion and transgene expression showed that the gene regulated S. epidermidis biofilm formation. Compared with the control strains, the ability to form biofilm, Triton X-100-induced autolysis and the amount of eDNA increased in the p221 strain and the two psm-mec mutants pM and pAG expressed psm-mec mRNA without its protein, whereas no differences were observed among the three constructed strains, illustrating that psm-mec mRNA promoted S. epidermidis biofilm formation through up-regulation of bacterial autolysis and the release of eDNA. Our results reveal that acquisition of psm-mec promotes S. epidermidis biofilm formation.

  5. Rapid formation of N-Glycopeptides via Cu(II)-promoted glycosylative ligation.

    PubMed

    Joseph, Ryan; Dyer, Frank Brock; Garner, Philip

    2013-02-15

    Herein is described the chemoselective Cu(II)-HOBt promoted chemical ligation of glycosylamines and peptide thioacids to give N-glycosylated peptides. The method is distinguished from other chemical approaches to peptide N-glycosylation in that (1) it can be employed in the presence of unprotected N-terminal and Lys side chain amines; (2) it is remarkably fast, going to completion in under 30 min; and (3) it produces glycopeptides without attendant aspartimide formation.

  6. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  7. Nitrite promotes protein carbonylation and Strecker aldehyde formation in experimental fermented sausages: are both events connected?

    PubMed

    Villaverde, A; Ventanas, J; Estévez, M

    2014-12-01

    The role played by curing agents (nitrite, ascorbate) on protein oxidation and Strecker aldehyde formation is studied. To fulfill this objective, increasing concentrations of nitrite (0, 75 and 150ppm) and ascorbate (0, 250 and 500ppm) were added to sausages subjected to a 54day drying process. The concurrence of intense proteolysis, protein carbonylation and formation of Strecker aldehydes during processing of sausages suggests that α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) may be implicated in the formation of Strecker aldehydes. The fact that nitrite (150ppm, ingoing amount) significantly promoted the formation of protein carbonyls at early stages of processing and the subsequent formation of Strecker aldehydes provides strength to this hypothesis. Ascorbate (125 and 250ppm) controlled the overall extent of protein carbonylation in sausages without declining the formation of Strecker aldehydes. These results may contribute to understanding the chemistry fundamentals of the positive influence of nitrite on the flavor and overall acceptability of cured muscle foods.

  8. Gait, cost and time implications for changing from PTB to ICEX sockets.

    PubMed

    Datta, D; Harris, I; Heller, B; Howitt, J; Martin, R

    2004-08-01

    The ICEX system (Ossur, Iceland), allows a socket to be manufactured directly onto the stump and is thought to provide improved comfort due to better pressure distribution whilst being easier to fit and manufacture. The aims of this project were to a) compare gait performance by measuring several gait characteristics, b) compare production and fitting times, c) investigate financial implications and d) attempt to gauge the amputees' subjective opinions of socket comfort. A randomised, controlled trial was conducted on 27 trans-tibial amputees with an existing patellar tendon bearing (PTB) socket on the Endolite system (Chas A. Blatchford, UK). Twenty one (21) subjects completed the study. Of these, 10 in the control group received new PTB sockets while 11 in the experimental group received ICEX. Gait analysis wearing existing sockets was performed and kinetic data obtained from a force plate. This was repeated with the new sockets after a 6 week period of adjustment. Mann-Whitney tests were used in statistical evaluations with a significance level of 5%. Subjects were asked to score their prosthesis for comfort using the Socket Comfort Score (Hanspal et al., 2003) and the frequency of visits for socket adjustments over a three-month period post-delivery of the sockets was recorded. This study demonstrates no significant difference in any of the gait parameters measured. Though the time required to manufacture a PTB prosthesis was found to be considerably longer than the ICEX, the overall cost for producing the ICEX was significantly greater. Subjects showed only minor comfort preference for the ICEX design and there was no significant difference in the mean number of visits for socket adjustments. In view of the considerable additional cost of providing ICEX and the lack of evidence of improvement in any parameter tested, the routine provision of ICEX prostheses to unselected trans-tibial amputees cannot be recommended.

  9. HMGA2 regulates CD44 expression to promote gastric cancer cell motility and sphere formation

    PubMed Central

    Sun, Junying; Sun, Baocun; Zhu, Dongwang; Zhao, Xiulan; Zhang, Yanhui; Dong, Xueyi; Che, Na; Li, Jing; Liu, Fang; Zhao, Nan; Zhang, Danfang; Liu, Tieju; Lin, Xian

    2017-01-01

    High mobility group AT-hook 2 (HMGA2) is a transcriptional modulator that mediates motility and self-renewal in cancer stem cells. Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC contains a population of stem-like cells that promote tumor invasion and resistance to therapy. In the current study, we investigated the expression of HMGA2 and the cancer stem cell marker CD44 in 200 GC samples and found that HMGA2 and CD44 were significantly associated with distant metastasis, histological differentiation and poor prognosis in GC patients. Positive clinical correlations of HMGA2 with CD44 were also observed in tissue sections. In vitro, overexpression of HMGA2 promoted GC sphere formation and migration in MKN74/MKN28 cells, whereas downregulation of HMGA2 decreased GC sphere formation and migration in MKN45/MGC803 cells. In addition, western blot and immunofluorescent analyses showed that HMGA2 increased the expression of the stem cell markers CD44, ALDH1, Sox2, and Oct4 and the EMT-related factors Snail and β-catenin. In a xenograft mouse model, overexpression of HMGA2 promoted tumor growth. Further immunohistochemical (IHC) analysis showed that HMGA2 increased the expression of CD44 and β-catenin, resulting in the promotion of tumor growth. Taken together, our findings indicate that HMGA2 promotes GC cancer stem cell induction and cell motility by regulating the expression of CD44. Therefore, targeting HMGA2 in GC may be therapeutically beneficial. PMID:28337375

  10. SH2B1 and IRSp53 proteins promote the formation of dendrites and dendritic branches.

    PubMed

    Chen, Chien-Jen; Shih, Chien-Hung; Chang, Yu-Jung; Hong, Shao-Jing; Li, Tian-Neng; Wang, Lily Hui-Ching; Chen, Linyi

    2015-03-06

    SH2B1 is an adaptor protein known to enhance neurite outgrowth. In this study, we provide evidence suggesting that the SH2B1 level is increased during in vitro culture of hippocampal neurons, and the β isoform (SH2B1β) is the predominant isoform. The fact that formation of filopodia is prerequisite for neurite initiation suggests that SH2B1 may regulate filopodium formation and thus neurite initiation. To investigate whether SH2B1 may regulate filopodium formation, the effect of SH2B1 and a membrane and actin regulator, IRSp53 (insulin receptor tyrosine kinase substrate p53), is investigated. Overexpressing both SH2B1β and IRSp53 significantly enhances filopodium formation, neurite outgrowth, and branching. Both in vivo and in vitro data show that SH2B1 interacts with IRSp53 in hippocampal neurons. This interaction depends on the N-terminal proline-rich domains of SH2B1. In addition, SH2B1 and IRSp53 co-localize at the plasma membrane, and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These findings suggest that SH2B1-IRSp53 complexes promote the formation of filopodia, neurite initiation, and neuronal branching.

  11. SH2B1 and IRSp53 Proteins Promote the Formation of Dendrites and Dendritic Branches*

    PubMed Central

    Chen, Chien-Jen; Shih, Chien-Hung; Chang, Yu-Jung; Hong, Shao-Jing; Li, Tian-Neng; Wang, Lily Hui-Ching; Chen, Linyi

    2015-01-01

    SH2B1 is an adaptor protein known to enhance neurite outgrowth. In this study, we provide evidence suggesting that the SH2B1 level is increased during in vitro culture of hippocampal neurons, and the β isoform (SH2B1β) is the predominant isoform. The fact that formation of filopodia is prerequisite for neurite initiation suggests that SH2B1 may regulate filopodium formation and thus neurite initiation. To investigate whether SH2B1 may regulate filopodium formation, the effect of SH2B1 and a membrane and actin regulator, IRSp53 (insulin receptor tyrosine kinase substrate p53), is investigated. Overexpressing both SH2B1β and IRSp53 significantly enhances filopodium formation, neurite outgrowth, and branching. Both in vivo and in vitro data show that SH2B1 interacts with IRSp53 in hippocampal neurons. This interaction depends on the N-terminal proline-rich domains of SH2B1. In addition, SH2B1 and IRSp53 co-localize at the plasma membrane, and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These findings suggest that SH2B1-IRSp53 complexes promote the formation of filopodia, neurite initiation, and neuronal branching. PMID:25586189

  12. Nickel Promotes Biofilm Formation by Escherichia coli K-12 Strains That Produce Curli▿

    PubMed Central

    Perrin, Claire; Briandet, Romain; Jubelin, Gregory; Lejeune, Philippe; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès; Dorel, Corinne

    2009-01-01

    The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads Escherichia coli bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in E. coli is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated. PMID:19168650

  13. Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation.

    PubMed

    Ma, Benyu; Cao, Weipeng; Li, Wenxia; Gao, Chan; Qi, Zhen; Zhao, Yan; Du, Jun; Xue, Hua; Peng, Junya; Wen, Jun; Chen, Hua; Ning, Yuanheng; Huang, Lei; Zhang, Hong; Gao, Xiang; Yu, Li; Chen, Ye-Guang

    2014-08-01

    Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.

  14. Nickel promotes biofilm formation by Escherichia coli K-12 strains that produce curli.

    PubMed

    Perrin, Claire; Briandet, Romain; Jubelin, Gregory; Lejeune, Philippe; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès; Dorel, Corinne

    2009-03-01

    The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads Escherichia coli bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in E. coli is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated.

  15. Mesenchymal Deletion of Histone Demethylase NO66 in Mice Promotes Bone Formation

    PubMed Central

    Chen, Qin; Sinha, Krishna; Deng, Jian Min; Yasuda, Hideyo; Krahe, Ralf; Behringer, Richard R.; de Crombrugghe, Benoit

    2016-01-01

    Our previous studies indicated that the Jumonji C (JmjC)-domain-containing NO66 is a histone demethylase with specificity for methylated histone H3K4 and H3K36. NO66 binds to the transcription factor Osterix (Osx) and inhibits its transcriptional activity in promoter assays. However, the physiological role of NO66 in formation of mammalian bones is unknown. Here, using a genetically engineered mouse model, we show that during early skeletal development, Prx1-Cre dependent mesenchymal deletion of NO66 promotes osteogenesis and formation of both endochondral as well as intramembranous skeletal elements, leading to a larger skeleton and a high bone mass phenotype in adult mice. The excess bone formation in mice where NO66 was deleted in cells of mesenchymal origin is associated with an increase in the number of preosteoblasts and osteoblasts. Further analysis revealed that in the embryonic limbs and adult calvaria of mice with deletion of NO66 in cells of mesenchymal origin, expression of several genes including bone morphogenetic protein 2, insulin-like growth factor 1 and osteoclast inhibitor osteoprotegerin was increased, concurrent with an increase in expression of bone formation markers such as Osx, type I collagen and bone sialoprotein. Taken together, our results provide the first in vivo evidence that NO66 histone demethylase plays an important role in mammalian osteogenesis during early development as well as in adult bone homeostasis. We postulate that NO66 regulates bone formation, at least in part, via regulating the number of bone-forming cells and expression of multiple genes that are critical for these processes. PMID:25736226

  16. Kartogenin with PRP promotes the formation of fibrocartilage zone in the tendon-bone interface.

    PubMed

    Zhou, Yiqin; Zhang, Jianying; Yang, Jinsong; Narava, Manoj; Zhao, Guangyi; Yuan, Ting; Wu, Haishan; Zheng, Nigel; Hogan, MaCalus V; Wang, James H-C

    2017-01-27

    Treatment of tendon-bone junction injuries is a challenge because tendon-bone interface often heals poorly and the fibrocartilage zone, which reduces stress concentration, at the interface is not formed. In this study, we used a compound called kartogenin (KGN) with platelet-rich plasma (PRP) to induce the formation of fibrocartilage zone in a rat tendon graft-bone tunnel model. The experimental rats received KGN-PRP or PRP injections in the tendon graft-bone tunnel interface. The control group received saline. After 4, 8 and 12 weeks, Safranin O staining of the tendon graft-bone tunnels revealed abundant proteoglycans in the KGN-PRP group indicating the formation of cartilage-like transition zone. Immunohistochemical and immuno-fluorescence staining revealed collagen types I (Col-I) and II (Col-II) in the newly formed fibrocartilage zone. Both fibrocartilage zone formation and maturation were healing time dependent. In contrast, the PRP and saline control groups had no cartilage-like tissues and minimal Col-I and Col-II staining. Some gaps were also present in the saline control group. Finally, pull-out strength in the KGN-PRP-treated group at 8 weeks was 1.4-fold higher than the PRP-treated group and 1.6-fold higher than the saline control group. These findings indicate that KGN, with PRP as a carrier, promotes the formation of fibrocartilage zone between the tendon graft and bone interface. Thus, KGN-PRP may be used as a convenient cell-free therapy in clinics to promote fibrocartilage zone formation in rotator calf repair and anterior cruciate ligament reconstruction, thereby enhancing the mechanical strength of the tendon-bone interface and hence the clinical outcome of these procedures. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling.

    PubMed

    Li, C; Shi, C; Kim, J; Chen, Y; Ni, S; Jiang, L; Zheng, C; Li, D; Hou, J; Taichman, R S; Sun, H

    2015-03-01

    Recent studies have demonstrated that erythropoietin (EPO) has extensive nonhematopoietic biological functions. However, little is known about how EPO regulates bone formation, although several studies suggested that EPO can affect bone homeostasis. In this study, we investigated the effects of EPO on the communication between osteoclasts and osteoblasts through the ephrinB2/EphB4 signaling pathway. We found that EPO slightly promotes osteoblastic differentiation with the increased expression of EphB4 in ST2 cells. However, EPO increased the expression of Nfatc1 and ephrinB2 but decreased the expression of Mmp9 in RAW264.7 cells, resulting in an increase of ephrinB2-expressing osteoclasts and a decrease in resorption activity. The stimulation of ephrinB2/EphB4 signaling via ephrinB2-Fc significantly promoted EPO-mediated osteoblastic differentiation in ST2 cells. EphB4 knockdown through EphB4 shRNA inhibited EPO-mediated osteoblastic phenotypes. Furthermore, in vivo assays clearly demonstrated that EPO efficiently induces new bone formation in the alveolar bone regeneration model. Taken together, these results suggest that ephrinB2/EphB4 signaling may play an important role in EPO-mediated bone formation.

  18. Semaphorin3A regulates neuronal polarization by suppressing axon formation and promoting dendrite growth.

    PubMed

    Shelly, Maya; Cancedda, Laura; Lim, Byung Kook; Popescu, Andrei T; Cheng, Pei-lin; Gao, Hongfeng; Poo, Mu-ming

    2011-08-11

    Semaphorin 3A (Sema3A) is a secreted factor known to guide axon/dendrite growth and neuronal migration. We found that it also acts as a polarizing factor for axon/dendrite development in cultured hippocampal neurons. Exposure of the undifferentiated neurite to localized Sema3A suppressed its differentiation into axon and promoted dendrite formation, resulting in axon formation away from the Sema3A source, and bath application of Sema3A to polarized neurons promoted dendrite growth but suppressed axon growth. Fluorescence resonance energy transfer (FRET) imaging showed that Sema3A elevated the cGMP but reduced cAMP and protein kinase A (PKA) activity, and its axon suppression is attributed to the downregulation of PKA-dependent phosphorylation of axon determinants LKB1 and GSK-3β. Downregulating Sema3A signaling in rat embryonic cortical progenitors via in utero electroporation of siRNAs against the Sema3A receptor neuropilin-1 also resulted in polarization defects in vivo. Thus, Sema3A regulates the earliest step of neuronal morphogenesis by polarizing axon/dendrite formation.

  19. Erythropoietin Promotes Bone Formation through EphrinB2/EphB4 Signaling

    PubMed Central

    Li, C.; Shi, C.; Kim, J.; Chen, Y.; Ni, S.; Jiang, L.; Zheng, C.; Li, D.; Hou, J.; Sun, H.

    2015-01-01

    Recent studies have demonstrated that erythropoietin (EPO) has extensive nonhematopoietic biological functions. However, little is known about how EPO regulates bone formation, although several studies suggested that EPO can affect bone homeostasis. In this study, we investigated the effects of EPO on the communication between osteoclasts and osteoblasts through the ephrinB2/EphB4 signaling pathway. We found that EPO slightly promotes osteoblastic differentiation with the increased expression of EphB4 in ST2 cells. However, EPO increased the expression of Nfatc1 and ephrinB2 but decreased the expression of Mmp9 in RAW264.7 cells, resulting in an increase of ephrinB2-expressing osteoclasts and a decrease in resorption activity. The stimulation of ephrinB2/EphB4 signaling via ephrinB2-Fc significantly promoted EPO-mediated osteoblastic differentiation in ST2 cells. EphB4 knockdown through EphB4 shRNA inhibited EPO-mediated osteoblastic phenotypes. Furthermore, in vivo assays clearly demonstrated that EPO efficiently induces new bone formation in the alveolar bone regeneration model. Taken together, these results suggest that ephrinB2/EphB4 signaling may play an important role in EPO-mediated bone formation. PMID:25586589

  20. Liquid eutectic GaIn as an alternative electrode for PTB7:PCBM organic solar cells

    NASA Astrophysics Data System (ADS)

    Thanh Hau Pham, Viet; Kieu Trinh, Thanh; Tam Nguyen Truong, Nguyen; Park, Chinho

    2017-04-01

    Conventional vacuum deposition process of aluminum (Al) is costly, time-consuming and difficult to apply to the large-scale production of organic photovoltaic devices (OPV). This paper reports a vacuum-free fabrication process of poly[[4,8-bis(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b‧]dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thienophenediyl]:[6,6]-phenyl C71 butyric acid methyl ester (PTB7:PCBM) bulk heterojunction organic solar cell with liquid eutectic gallium–indium (EGaIn) electrode as an alternative to the common Al electrode. The insertion of a thin poly(ethylene oxide) (PEO) layer after depositing organic photoactive layer could help prevent the diffusion of liquid EGaIn into the active layer and allow the deposition of the EGaIn electrode. The PEO interfacial layer was formed by spin-coating from a mixed solvent of alcohol and water. Among different alcohol+water (methanol, ethanol, ethylene glycol, n-propanol, isopropanol, and isobutanol) mixed solvent tested, the n-propanol+water mixed solvent showed the greatest enhancement to the performance of OPVs. The improved device performance was attributed to the reactivity of mixed solvent n-propanol+water toward the surface of PTB7:PCBM active layer, which could help optimize surface morphology.

  1. New PTB Setup for the Absolute Calibration of the Spectral Responsivity of Radiation Thermometers

    NASA Astrophysics Data System (ADS)

    Anhalt, K.; Zelenjuk, A.; Taubert, D. R.; Keawprasert, T.; Hartmann, J.

    2009-02-01

    The paper describes the new experimental setup assembled at the PTB for the absolute spectral responsivity measurement of radiation thermometers. The concept of this setup is to measure the relative spectral responsivity of the radiation thermometer using the conventional monochromator-based spectral comparator facility also used for the calibration of filter radiometers. The absolute spectral responsivity is subsequently measured at one wavelength, supplied by the radiation of a diode laser, using the new setup. The radiation of the diode laser is guided with an optical fiber into an integrating sphere source that is equipped with an aperture of absolutely known area. The spectral radiance of this integrating sphere source is determined via the spectral irradiance measured by a trap detector with an absolutely calibrated spectral responsivity traceable to the primary detector standard of the PTB, the cryogenic radiometer. First results of the spectral responsivity calibration of the radiation thermometer LP3 are presented, and a provisional uncertainty budget of the absolute spectral responsivity is given.

  2. Mercury-induced crystallization and SAD phasing of the human Fe65-PTB1 domain

    SciTech Connect

    Radzimanowski, Jens; Ravaud, Stéphanie; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Crystals of the phosphotyrosine-binding domain 1 (PTB1) of the neuronal adaptor protein Fe65 grown in the presence of a mercury derivative show a dramatic improvement in resolution, permitting SAD phasing. Fe65 is a three-domain neuronal adaptor protein involved in brain development and amyloid precursor protein (APP) signalling. The phosphotyrosine-binding domain 1 (PTB1) of human Fe65 has been cloned, overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals belong to the space group R3 and diffract to 2.6 Å resolution. This crystal form suffered from high thermal B factors and pseudo-symmetry, resulting in a bisection of the c axis. Co-crystallization with a mercury compound under similar conditions induced an orthorhombic crystal form in the space group P2{sub 1}2{sub 1}2{sub 1} diffracting to 2.2 Å resolution. SAD phases have been computed to the diffraction limit at the wavelength of maximum absorption (L{sub III} edge)

  3. Properties of synthetic ferrihydrite as an amino acid adsorbent and a promoter of peptide bond formation.

    PubMed

    Matrajt, G; Blanot, D

    2004-03-01

    Ferrihydrite, an iron oxide hydroxide, is found in all kinds of environments, from hydrothermal hot springs to extraterrestrial materials. It has been shown that this material is nanoporous, and because of its high surface area, it has outstanding adsorption properties and in some cases catalysis properties. In this work we studied the adsorption properties of ferrihydrite with respect to amino acids. Samples of pure ferrihydrite were synthesised and exposed to solutions of amino acids including both proteinaceous and non-proteinaceous species. These experiments revealed important characteristics of this mineral as both an adsorbent of amino acids and a promoter of peptide bond formation.

  4. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation.

    PubMed

    Burke, Michael C; Li, Feng-Qian; Cyge, Benjamin; Arashiro, Takeshi; Brechbuhl, Heather M; Chen, Xingwang; Siller, Saul S; Weiss, Matthew A; O'Connell, Christopher B; Love, Damon; Westlake, Christopher J; Reynolds, Susan D; Kuriyama, Ryoko; Takemaru, Ken-Ichi

    2014-10-13

    Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

  5. Collagen-derived matricryptins promote inhibitory nerve terminal formation in the developing neocortex

    PubMed Central

    Su, Jianmin; Chen, Jiang; Lippold, Kumiko; Monavarfeshani, Aboozar; Carrillo, Gabriela Lizana; Jenkins, Rachel

    2016-01-01

    Inhibitory synapses comprise only ∼20% of the total synapses in the mammalian brain but play essential roles in controlling neuronal activity. In fact, perturbing inhibitory synapses is associated with complex brain disorders, such as schizophrenia and epilepsy. Although many types of inhibitory synapses exist, these disorders have been strongly linked to defects in inhibitory synapses formed by Parvalbumin-expressing interneurons. Here, we discovered a novel role for an unconventional collagen—collagen XIX—in the formation of Parvalbumin+ inhibitory synapses. Loss of this collagen results not only in decreased inhibitory synapse number, but also in the acquisition of schizophrenia-related behaviors. Mechanistically, these studies reveal that a proteolytically released fragment of this collagen, termed a matricryptin, promotes the assembly of inhibitory nerve terminals through integrin receptors. Collectively, these studies not only identify roles for collagen-derived matricryptins in cortical circuit formation, but they also reveal a novel paracrine mechanism that regulates the assembly of these synapses. PMID:26975851

  6. Formation of a seed germination promoter from carbohydrates and amino acids.

    PubMed

    Light, Marnie E; Burger, Barend V; van Staden, Johannes

    2005-07-27

    The ability of plant-derived smoke to act as a germination cue in many species has led to widespread interest in this aspect of seed biology. Recently, 3-methyl-2H-furo[2,3-c]pyran-2-one was identified as the main germination cue from smoke. Here, we report on the formation of this compound from reactions of sugars with amino acids. Heating proteins or amino acids with sugars at 180 degrees C for 30 min produces water soluble extracts that promote germination. High-performance liquid chromatography indicated that the active compound(s) derived from these reactions coeluted with the active fraction from a smoke solution. Gas chromatography-mass spectroscopy showed that the active constituent is identical to the germination cue from plant-derived smoke. The results presented in this paper provide evidence for the formation of the major germination cue found in smoke from ubiquitously occurring organic compounds.

  7. Interferon-β promotes macrophage foam cell formation by altering both cholesterol influx and efflux mechanisms.

    PubMed

    Boshuizen, Marieke C S; Hoeksema, Marten A; Neele, Annette E; van der Velden, Saskia; Hamers, Anouk A J; Van den Bossche, Jan; Lutgens, Esther; de Winther, Menno P J

    2016-01-01

    Foam cell formation is a crucial event in atherogenesis. While interferon-β (IFNβ) is known to promote atherosclerosis in mice, studies on the role of IFNβ on foam cell formation are minimal and conflicting. We therefore extended these studies using both in vitro and in vivo approaches and examined IFNβ's function in macrophage foam cell formation. To do so, murine bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages were loaded with acLDL overnight, followed by 6h IFNβ co-treatment. This increased lipid content as measured by Oil red O staining. We next analyzed the lipid uptake pathways of IFNβ-stimulated BMDMs and observed increased endocytosis of DiI-acLDL as compared to controls. These effects were mediated via SR-A, as its gene expression was increased and inhibition of SR-A with Poly(I) blocked the IFNβ-induced increase in Oil red O staining and DiI-acLDL endocytosis. The IFNβ-induced increase in lipid content was also associated with decreased ApoA1-mediated cholesterol efflux, in response to decreased ABCA1 protein and gene expression. To validate our findings in vivo, LDLR(-/-) mice were put on chow or a high cholesterol diet for 10weeks. 24 and 8h before sacrifice mice were injected with IFNβ or PBS, after which thioglycollate-elicited peritoneal macrophages were collected and analyzed. In accordance with the in vitro data, IFNβ increased lipid accumulation. In conclusion, our experimental data support the pro-atherogenic role of IFNβ, as we show that IFNβ promotes macrophage foam cell formation by increasing SR-A-mediated cholesterol influx and decreasing ABCA1-mediated efflux mechanisms.

  8. Cisplatin upregulates mitochondrial nitric oxide synthase and peroxynitrite formation to promote renal injury.

    PubMed

    Jung, Michaela; Hotter, Georgina; Viñas, Jose Luis; Sola, Anna

    2009-01-15

    The mitochondria are a critical target for cisplatin-associated nephrotoxicity. Though nitric oxide formation has been implicated in the toxicity of cisplatin, this formation has not so far been related to a possible activation of mitochondrial nitric oxide synthase (mNOS). We show here that the upregulation of oxide mNOS and peroxynitrite formation in cisplatin treatment are key events that influence the development of the harmful parameters described in cisplatin-associated kidney failure. We confirm this by isolating the mitochondrial fraction of the kidney and across different access routes such as the use of a specific inhibitor of neuronal NOS, L-NPA, a peroxynitrite scavenger, FeTMPyP, and a peroxynitrite donor, SIN-1. The in vitro studies corroborated the information obtained in the in vivo experiments. The administration of cisplatin reveals a clear upregulation in the transcription of neuronal NOS and an increase in the levels of nitrites in the mitochondrial fractions of the kidneys. The upregulated transcription directly affects the cytoskeleton structure and the apoptosis. The inhibition of neuronal NOS reduces the levels of nitrites, cell death, and cytoskeleton derangement. Peroxynitrite is involved in the mechanism promoting the NOS transcription. In addition, in controls SIN-1 imitates the effects of cisplatin. In summary, we demonstrate that upregulation of mNOS in cisplatin treatment is a key component in both the initiation and the spread of cisplatin-associated damage in the kidney. Furthermore, peroxynitrite formation is directly involved in this process.

  9. Cisplatin upregulates mitochondrial nitric oxide synthase and peroxynitrite formation to promote renal injury

    SciTech Connect

    Jung, Michaela; Sola, Anna

    2009-01-15

    The mitochondria are a critical target for cisplatin-associated nephrotoxicity. Though nitric oxide formation has been implicated in the toxicity of cisplatin, this formation has not so far been related to a possible activation of mitochondrial nitric oxide synthase (mNOS). We show here that the upregulation of oxide mNOS and peroxynitrite formation in cisplatin treatment are key events that influence the development of the harmful parameters described in cisplatin-associated kidney failure. We confirm this by isolating the mitochondrial fraction of the kidney and across different access routes such as the use of a specific inhibitor of neuronal NOS, L-NPA, a peroxynitrite scavenger, FeTMPyP, and a peroxynitrite donor, SIN-1. The in vitro studies corroborated the information obtained in the in vivo experiments. The administration of cisplatin reveals a clear upregulation in the transcription of neuronal NOS and an increase in the levels of nitrites in the mitochondrial fractions of the kidneys. The upregulated transcription directly affects the cytoskeleton structure and the apoptosis. The inhibition of neuronal NOS reduces the levels of nitrites, cell death, and cytoskeleton derangement. Peroxynitrite is involved in the mechanism promoting the NOS transcription. In addition, in controls SIN-1 imitates the effects of cisplatin. In summary, we demonstrate that upregulation of mNOS in cisplatin treatment is a key component in both the initiation and the spread of cisplatin-associated damage in the kidney. Furthermore, peroxynitrite formation is directly involved in this process.

  10. Biosurfactant as a Promoter of Methane Hydrate Formation: Thermodynamic and Kinetic Studies

    PubMed Central

    Arora, Amit; Cameotra, Swaranjit Singh; Kumar, Rajnish; Balomajumder, Chandrajit; Singh, Anil Kumar; Santhakumari, B.; Kumar, Pushpendra; Laik, Sukumar

    2016-01-01

    Natural gas hydrates (NGHs) are solid non-stoichiometric compounds often regarded as a next generation energy source. Successful commercialization of NGH is curtailed by lack of efficient and safe technology for generation, dissociation, storage and transportation. The present work studied the influence of environment compatible biosurfactant on gas hydrate formation. Biosurfactant was produced by Pseudomonas aeruginosa strain A11 and was characterized as rhamnolipids. Purified rhamnolipids reduced the surface tension of water from 72 mN/m to 36 mN/m with Critical Micelle Concentration (CMC) of 70 mg/l. Use of 1000 ppm rhamnolipids solution in C type silica gel bed system increased methane hydrate formation rate by 42.97% and reduced the induction time of hydrate formation by 22.63% as compared to water saturated C type silica gel. Presence of rhamnolipids also shifted methane hydrate formation temperature to higher values relative to the system without biosurfactant. Results from thermodynamic and kinetic studies suggest that rhamnolipids can be applied as environment friendly methane hydrate promoter. PMID:26869357

  11. Biosurfactant as a Promoter of Methane Hydrate Formation: Thermodynamic and Kinetic Studies.

    PubMed

    Arora, Amit; Cameotra, Swaranjit Singh; Kumar, Rajnish; Balomajumder, Chandrajit; Singh, Anil Kumar; Santhakumari, B; Kumar, Pushpendra; Laik, Sukumar

    2016-02-12

    Natural gas hydrates (NGHs) are solid non-stoichiometric compounds often regarded as a next generation energy source. Successful commercialization of NGH is curtailed by lack of efficient and safe technology for generation, dissociation, storage and transportation. The present work studied the influence of environment compatible biosurfactant on gas hydrate formation. Biosurfactant was produced by Pseudomonas aeruginosa strain A11 and was characterized as rhamnolipids. Purified rhamnolipids reduced the surface tension of water from 72 mN/m to 36 mN/m with Critical Micelle Concentration (CMC) of 70 mg/l. Use of 1000 ppm rhamnolipids solution in C type silica gel bed system increased methane hydrate formation rate by 42.97% and reduced the induction time of hydrate formation by 22.63% as compared to water saturated C type silica gel. Presence of rhamnolipids also shifted methane hydrate formation temperature to higher values relative to the system without biosurfactant. Results from thermodynamic and kinetic studies suggest that rhamnolipids can be applied as environment friendly methane hydrate promoter.

  12. Biosurfactant as a Promoter of Methane Hydrate Formation: Thermodynamic and Kinetic Studies

    NASA Astrophysics Data System (ADS)

    Arora, Amit; Cameotra, Swaranjit Singh; Kumar, Rajnish; Balomajumder, Chandrajit; Singh, Anil Kumar; Santhakumari, B.; Kumar, Pushpendra; Laik, Sukumar

    2016-02-01

    Natural gas hydrates (NGHs) are solid non-stoichiometric compounds often regarded as a next generation energy source. Successful commercialization of NGH is curtailed by lack of efficient and safe technology for generation, dissociation, storage and transportation. The present work studied the influence of environment compatible biosurfactant on gas hydrate formation. Biosurfactant was produced by Pseudomonas aeruginosa strain A11 and was characterized as rhamnolipids. Purified rhamnolipids reduced the surface tension of water from 72 mN/m to 36 mN/m with Critical Micelle Concentration (CMC) of 70 mg/l. Use of 1000 ppm rhamnolipids solution in C type silica gel bed system increased methane hydrate formation rate by 42.97% and reduced the induction time of hydrate formation by 22.63% as compared to water saturated C type silica gel. Presence of rhamnolipids also shifted methane hydrate formation temperature to higher values relative to the system without biosurfactant. Results from thermodynamic and kinetic studies suggest that rhamnolipids can be applied as environment friendly methane hydrate promoter.

  13. Autophagy is induced by anti-neutrophil cytoplasmic Abs and promotes neutrophil extracellular traps formation.

    PubMed

    Sha, Li-Li; Wang, Huan; Wang, Chen; Peng, Hong-Ying; Chen, Min; Zhao, Ming-Hui

    2016-11-01

    Dysregulated neutrophil extracellular traps (NETs) formation contributes to the pathogenesis of anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV). Increasing evidence indicates that autophagy is involved in the process of NETs formation. In this study, we aimed to investigate whether ANCA could induce autophagy in the process of NETs formation. Autophagy was detected using live cell imaging, microtubule-associated protein light chain 3B (LC3B) accumulation and Western blotting. The results showed that autophagy vacuolization was detected in neutrophils treated with ANCA-positive IgG by live cell imaging. This effect was enhanced by rapamycin, the autophagy inducer, and weakened by 3-methyladenine (3-MA), the autophagy inhibitor. In line with these results, the autophagy marker, LC3B, showed a punctate distribution pattern in the neutrophils stimulated with ANCA-positive IgG. In the presence of rapamycin, LC3B accumulation was further increased; however, this effect was attenuated by 3-MA. Moreover, incubated with ANCA-positive IgG, the NETosis rate significantly increased compared with the unstimulated group. And, the rate significantly increased or decreased in the neutrophils pretreated with rapamycin or 3-MA, respectively, as compared with the cells incubated with ANCA-positive IgG. Overall, this study demonstrates that autophagy is induced by ANCA and promotes ANCA-induced NETs formation.

  14. Incorporation of RANKL promotes osteoclast formation and osteoclast activity on β-TCP ceramics.

    PubMed

    Choy, John; Albers, Christoph E; Siebenrock, Klaus A; Dolder, Silvia; Hofstetter, Wilhelm; Klenke, Frank M

    2014-12-01

    β-Tricalcium phosphate (β-TCP) ceramics are approved for the repair of osseous defects. In large defects, however, the substitution of the material by authentic bone is inadequate to provide sufficient long-term mechanical stability. We aimed to develop composites of β-TCP ceramics and receptor activator of nuclear factor κ-B ligand (RANKL) to enhance the formation of osteoclasts and promote cell mediated calcium phosphate resorption. RANKL was adsorbed superficially onto β-TCP ceramics or incorporated into a crystalline layer of calcium phosphate by the use of a co-precipitation technique. Murine osteoclast precursors were seeded onto the ceramics. After 15 days, the formation of osteoclasts was quantified cytologically and colorimetrically with tartrate-resistant acidic phosphatase (TRAP) staining and TRAP activity measurements, respectively. Additionally, the expression of transcripts encoding the osteoclast gene products cathepsin K, calcitonin receptor, and of the sodium/hydrogen exchanger NHA2 were quantified by real-time PCR. The activity of newly formed osteoclasts was evaluated by means of a calcium phosphate resorption assay. Superficially adsorbed RANKL did not induce the formation of osteoclasts on β-TCP ceramics. When co-precipitated onto β-TCP ceramics RANKL supported the formation of mature osteoclasts. The development of osteoclast lineage cells was further confirmed by the increased expression of cathepsin K, calcitonin receptor, and NHA2. Incorporated RANKL stimulated the cells to resorb crystalline calcium phosphate. Our in vitro study shows that RANKL incorporated into β-TCP ceramics induces the formation of active, resorbing osteoclasts on the material surface. Once formed, osteoclasts mediate the release of RANKL thereby perpetuating their differentiation and activation. In vivo, the stimulation of osteoclast-mediated resorption may contribute to a coordinated sequence of material resorption and bone formation. Further in vivo studies

  15. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  16. Mechanical strain promotes osteoblast ECM formation and improves its osteoinductive potential

    PubMed Central

    2012-01-01

    Background The extracellular matrix (ECM) provides a supportive microenvironment for cells, which is suitable as a tissue engineering scaffold. Mechanical stimulus plays a significant role in the fate of osteoblast, suggesting that it regulates ECM formation. Therefore, we investigated the influence of mechanical stimulus on ECM formation and bioactivity. Methods Mouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with mechanical tensile strain. After removing the cells, the ECMs coated on dishes were prepared. The ECM protein and calcium were assayed and MC3T3-E1 cells were re-seeded on the ECM-coated dishes to assess osteoinductive potential of the ECM. Results The cyclic tensile strain increased collagen, bone morphogenetic protein 2 (BMP-2), BMP-4, and calcium levels in the ECM. Compared with the ECM produced by unstrained osteoblasts, those of mechanically stimulated osteoblasts promoted alkaline phosphatase activity, elevated BMP-2 and osteopontin levels and mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and increased secreted calcium of the re-seeded MC3T3-E1 cells. Conclusion Mechanical strain promoted ECM production of osteoblasts in vitro, increased BMP-2/4 levels, and improved osteoinductive potential of the ECM. This study provided a novel method to enhance bioactivity of bone ECM in vitro via mechanical strain to osteoblasts. PMID:23098360

  17. Antheraea pernyi silk sericin mediating biomimetic nucleation and growth of hydroxylapatite crystals promoting bone matrix formation.

    PubMed

    Jiayao, Zhuang; Guanshan, Zhou; Jinchi, Zhang; Yuyin, Chen; Yongqiang, Zhu

    2017-03-01

    Bone biomineralization is well-regulated processes mediated by extracellular matrix proteins. The materials that can direct nucleation of hydroxylapatite (HAp) crystals and assembly of well-structured material-minerals complex are the key to mimicking the natural mineralization. This study used sericin from Antheraea pernyi (A.pernyi), non-mulberry silkworm cocoon as template to mediate nucleation of HAp crystals. Here we find out that AS (Antheraea pernyi sericin) can nucleate the formation HAp crystals in simulated body fluid verified by XRD and FTIR observations. The HAp crystals are organized into nano-rods oriented with c-axis preferentially parallel to the long axis of AS due to hydrogen bonds and electrostatic interaction and finally aggregated into HAp globule. The cell culture of human bone marrow-derived mesenchymal stem cells (BMSCs) showed that the HAp crystals mediated by AS not only stimulate cell adhesion and proliferation but also promote 0f osteogenic differentiation, suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. Thus our work will provide significant implication on biomineralization of A. pernyi silk sericin as a potential scaffold for tissue engineering.

  18. Interruptions between the triple helix peptides can promote the formation of amyloid-like fibrils

    NASA Astrophysics Data System (ADS)

    Parmar, Avanish; Hwang, Eileen; Brodsky, Barbara

    2010-03-01

    It has been reported that collagen can initiate or accelerate the formation of amyloid fibrils. Non-fibrillar collagen types have sites where the repeating (Gly-Xaa-Yaa)n sequences are interrupted by non- Gly-Xaa-Yaa sequences, and we are investigating the hypothesis that some of these interruptions can promote amyloid formation. Our experimental data show that model peptides containing an 8 or 9 residue interruption sequence between (Gly-Pro-Hyp)n domains have a strong propensity for self association to form fibrous structures. A peptide containing only the 9-residue interruption sequence forms amyloid like fibrils with anti-parallel β sheet. Computational analysis predicts that 33 out of 374 naturally occurring human non-fibrillar collagen sequences within or between triple-helical sequences have significant cross-β aggregation potential, including the 8 and 9 residue sequences studied in peptides. Further studies are in progress to investigate whether a triple-helix peptide promotes amyloidogenesis and whether amyloid interferes with collagen fibrillogenesis.

  19. An invasive podosome-like structure promotes fusion pore formation during myoblast fusion

    PubMed Central

    Sens, Kristin L.; Zhang, Shiliang; Jin, Peng; Duan, Rui; Zhang, Guofeng; Luo, Fengbao; Parachini, Lauren

    2010-01-01

    Recent studies in Drosophila have implicated actin cytoskeletal remodeling in myoblast fusion, but the cellular mechanisms underlying this process remain poorly understood. Here we show that actin polymerization occurs in an asymmetric and cell type–specific manner between a muscle founder cell and a fusion-competent myoblast (FCM). In the FCM, a dense F-actin–enriched focus forms at the site of fusion, whereas a thin sheath of F-actin is induced along the apposing founder cell membrane. The FCM-specific actin focus invades the apposing founder cell with multiple finger-like protrusions, leading to the formation of a single-channel macro fusion pore between the two muscle cells. Two actin nucleation–promoting factors of the Arp2/3 complex, WASP and Scar, are required for the formation of the F-actin foci, whereas WASP but not Scar promotes efficient foci invasion. Our studies uncover a novel invasive podosome-like structure (PLS) in a developing tissue and reveal a previously unrecognized function of PLSs in facilitating cell membrane juxtaposition and fusion. PMID:21098115

  20. Subinhibitory concentrations of triclosan promote Streptococcus mutans biofilm formation and adherence to oral epithelial cells.

    PubMed

    Bedran, Telma Blanca Lombardo; Grignon, Louis; Spolidorio, Denise Palomari; Grenier, Daniel

    2014-01-01

    Triclosan is a general membrane-active agent with a broad-spectrum antimicrobial activity that is commonly used in oral care products. In this study, we investigated the effect of sub-minimum inhibitory concentrations (MICs) of triclosan on the capacity of the cariogenic bacterium Streptococcus mutans to form biofilm and adhere to oral epithelial cells. As quantified by crystal violet staining, biofilm formation by two reference strains of S. mutans was dose-dependently promoted, in the range of 2.2- to 6.2-fold, by 1/2 and 1/4 MIC of triclosan. Observations by scanning electron microscopy revealed the presence of a dense biofilm attached to the polystyrene surface. Growth of S. mutans in the presence of triclosan at sub-MICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. The expression of several genes involved in adherence and biofilm formation in S. mutans was investigated by quantitative RT-PCR. It was found that sub-MICs of triclosan significantly increased the expression of comD, gtfC, and luxS, and to a lesser extent of gtfB and atlA genes. These findings stress the importance of maintaining effective bactericidal concentrations of therapeutic triclosan since sub-MICs may promote colonization of the oral cavity by S. mutans.

  1. Examining the Factors That Promote Long-Term Change in Elementary Teachers' Instructional Practices: Sustaining Formative Assessment Reform

    ERIC Educational Resources Information Center

    Sherbinko, Thomas, Jr.

    2011-01-01

    The purpose of this holistic case study with a mixed methods approach was two-fold: first, to investigate the extent to which elementary teachers in the BTSD sustained the use of formative assessment reform to regulate instruction, and second, to explore the factors that promoted fidelity to formative assessment reform. Although this study…

  2. Amyloid Aβ 42, a promoter of magnetite nanoparticle formation in Alzheimer’s disease

    NASA Astrophysics Data System (ADS)

    Bogachan Tahirbegi, Islam; Pardo, Wilmer Alfonso; Alvira, Margarita; Mir, Mònica; Samitier, Josep

    2016-11-01

    The accumulation of iron oxides—mainly magnetite—with amyloid peptide is a key process in the development of Alzheimer’s disease (AD). However, the mechanism for biogeneration of magnetite inside the brain of someone with AD is still unclear. The iron-storing protein ferritin has been identified as the main magnetite-storing molecule. However, accumulations of magnetite in AD are not correlated with an increase in ferritin, leaving this question unresolved. Here we demonstrate the key role of amyloid peptide Aβ 42, one of the main hallmarks of AD, in the generation of magnetite nanoparticles in the absence of ferritin. The capacity of amyloid peptide to bind and concentrate iron hydroxides, the basis for the formation of magnetite, benefits the spontaneous synthesis of these nanoparticles, even under unfavorable conditions for their formation. Using scanning and transmission electron microscopy, electron energy loss spectroscopy and magnetic force microscopy we characterized the capacity of amyloid peptide Aβ 42 to promote magnetite formation.

  3. WNT7B promotes bone formation in part through mTORC1.

    PubMed

    Chen, Jianquan; Tu, Xiaolin; Esen, Emel; Joeng, Kyu Sang; Lin, Congxin; Arbeit, Jeffrey M; Rüegg, Markus A; Hall, Michael N; Ma, Liang; Long, Fanxin

    2014-01-01

    WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation.

  4. Ethylene promotes induction of aerenchyma formation and ethanolic fermentation in waterlogged roots of Dendranthema spp.

    PubMed

    Yin, Dongmei; Chen, Sumei; Chen, Fadi; Jiang, Jiafu

    2013-07-01

    The role of ethylene in induction of aerenchyma formation and ethanolic fermentation in waterlogged roots of Dendranthema zawadskii and D. nankingense, two species that differ with respect to waterlogging tolerance, was examined. In the more tolerant D. zawadskii, but not in D. nankingense, ethylene accelerated programmed cell death and promoted formation of lysigenous aerenchyma, both of which were inhibited by treatment with the ethylene inhibitor 1-methylcyclopropene. Waterlogged D. zawadskii roots generated a higher quantity of endogenous ethylene than did those of D. nankingense. In waterlogged D. zawadskii roots, transcription of the genes encoding alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.1) increased rapidly but transiently, whereas expression of these genes in D. nankingense increased gradually and over a longer period. In D. nankingense, waterlogging elevated both alcohol dehydrogenase and pyruvate decarboxylase activity, and the production of ethanol and acetaldehyde was increased in the presence of exogenous ethylene and inhibited by 1-methylcyclopropene. In D. zawadskii, in contrast, after a prolonged episode of waterlogging stress, exogenous supply of ethylene suppressed the production of ethanol and acetaldehyde, whereas exogenous 1-methylcyclopropene enhanced their production. In the more tolerant Dendranthema species, ethylene appeared to signal an acceleration of both waterlogging-induced programmed cell death and aerenchyma formation and to alleviate ethanolic fermentation, whereas in the more sensitive species ethylene activated fermentation and increased the release of ethanol and acetaldehyde, which are by-products probably responsible for the collapse of the waterlogging-damaged root.

  5. Mercury-induced crystallization and SAD phasing of the human Fe65-PTB1 domain.

    PubMed

    Radzimanowski, Jens; Ravaud, Stéphanie; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Fe65 is a three-domain neuronal adaptor protein involved in brain development and amyloid precursor protein (APP) signalling. The phosphotyrosine-binding domain 1 (PTB1) of human Fe65 has been cloned, overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals belong to the space group R3 and diffract to 2.6 A resolution. This crystal form suffered from high thermal B factors and pseudo-symmetry, resulting in a bisection of the c axis. Co-crystallization with a mercury compound under similar conditions induced an orthorhombic crystal form in the space group P2(1)2(1)2(1) diffracting to 2.2 A resolution. SAD phases have been computed to the diffraction limit at the wavelength of maximum absorption (L(III) edge).

  6. Interferometric determination of the topographies of absolute sphere radii using the sphere interferometer of PTB

    NASA Astrophysics Data System (ADS)

    Bartl, Guido; Krystek, Michael; Nicolaus, Arnold; Giardini, Walter

    2010-11-01

    This paper presents a method to reconstruct the absolute shape of a sphere—i.e. a topography of radii—using the sphere interferometer of PTB in combination with a stitching approach. The method allows for the reconstruction of absolute radii instead of the relative shape deviations which result from conventional sphericity measurements. The sphere interferometer was developed for the volume determination of spherical material measures—in particular the spheres of the Avogadro project—by precise diameter measurements with an uncertainty of 1 nm or less. In the scope of the present work a procedure has been implemented that extends the applicability of the interferometer to fields where not the volume or diameter but the direction-dependent radii are of interest. The results of the reconstruction were compared quantitatively to the independent results of sphericity measurements from CSIRO.

  7. Characterizing the performance of the PTB line scale interferometer by measuring photoelectric incremental encoders

    NASA Astrophysics Data System (ADS)

    Koening, Rainer; Fluegge, Jens; Bosse, Harald

    2005-08-01

    Until today one dimensional length comparators or line scale interferometers are used to realize and disseminate the unit of length. The performance of the vacuum length comparator of the PTB, the Nanometer Comparator, was characterized by measuring photoelectric incremental encoders. In some respects the measurements were used to optimize the performance of the instrument, e.g. with respect to its noise characteristics. The non-linearity of its vacuum interferometer could be determined to show an amplitude of 0.2 nm. The reproducibility of the measurement of an incremental encoder system with 280 mm measuring range was 0.3 nm. Currently, the relative expanded measurement uncertainty for the calibration of incremental encoder systems is in the range of 2x10-8. These results show that incremental encoders are well suited to characterize one dimensional length measuring machines.

  8. Promoter-Specific Effects of DREADD Modulation on Hippocampal Synaptic Plasticity and Memory Formation

    PubMed Central

    López, Alberto J.; Kramár, Enikö; Matheos, Dina P.; White, André O.; Kwapis, Janine; Vogel-Ciernia, Annie; Sakata, Keith; Espinoza, Monica

    2016-01-01

    Designer receptors exclusively activated by designer drug (DREADDs) are a novel tool with the potential to bidirectionally drive cellular, circuit, and ultimately, behavioral changes. We used DREADDs to evaluate memory formation in a hippocampus-dependent task in mice and effects on synaptic physiology in the dorsal hippocampus. We expressed neuron-specific (hSyn promoter) DREADDs that were either excitatory (HM3D) or inhibitory (HM4D) in the dorsal hippocampus. As predicted, hSyn–HM3D was able to transform a subthreshold learning event into long-term memory (LTM), and hSyn–HM4D completely impaired LTM formation. Surprisingly, the opposite was observed during experiments examining the effects on hippocampal long-term potentiation (LTP). hSyn–HM3D impaired LTP and hSyn–HM4D facilitated LTP. Follow-up experiments indicated that the hSyn–HM3D-mediated depression of fEPSP appears to be driven by presynaptic activation of inhibitory currents, whereas the hSyn–HM4D-mediated increase of fEPSP is induced by a reduction in GABAA receptor function. To determine whether these observations were promoter specific, we next examined the effects of using the CaMKIIα promoter that limits expression to forebrain excitatory neurons. CaMKIIα–HM3D in the dorsal hippocampus led to the transformation of a subthreshold learning event into LTM, whereas CaMKIIα–HM4D blocked LTM formation. Consistent with these findings, baseline synaptic transmission and LTP was increased in CaMKIIα–HM3D hippocampal slices, whereas slices from CaMKIIα–HM4D mice produced expected decreases in baseline synaptic transmission and LTP. Together, these experiments further demonstrate DREADDs as being a robust and reliable means of modulating neuronal function to manipulate long-term changes in behavior, while providing evidence for specific dissociations between LTM and LTP. SIGNIFICANCE STATEMENT This study evaluates the efficacy of designer receptors exclusively activated by designer

  9. Final results of bilateral comparison between NIST and PTB for flows of high pressure natural gas

    NASA Astrophysics Data System (ADS)

    Mickan, B.; Toebben, H.; Johnson, A.; Kegel, T.

    2013-01-01

    In 2009 NIST developed a US national flow standard to provide traceability for flow meters used for custody transfer of pipeline quality natural gas. NIST disseminates the SI unit of flow by calibrating a customer flow meter against a parallel array of turbine meter working standards, which in turn are traceable to a pressure-volume-temperature-time (PVTt) primary standard. The calibration flow range extends from 0.125 actual m3/s to 9 actual m3/s with an expanded uncertainty as low as 0.22% at high flows, and increasing to almost 0.40% at the lowest flows. Details regarding the traceability chain and uncertainty analysis are documented in prior publications. The current manuscript verifies NIST's calibration uncertainty via a bilateral comparison with the German National Metrology Institute PTB. The results of the bilateral are linked to the 2006 key comparison results between three EURAMET national metrology institutes (i.e., PTB, VSL and LNE). Linkage is accomplished in spite of using a different transfer standard in the bilateral versus the key comparison. A mathematical proof is included that demonstrates that the relative difference between a laboratory's measured flow and the key comparison reference value is independent of the transfer package for most flow measurement applications. The bilateral results demonstrate that NIST's natural gas flow measurements are within their specified uncertainties and are equivalent to those of the EURAMET National Metrology Institutes. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

  10. Characterization of optical material parameters for EUV Lithography applications at PTB

    NASA Astrophysics Data System (ADS)

    Laubis, Christian; Haase, Anton; Soltwisch, Victor; Scholze, Frank

    2015-09-01

    EUV Lithography now reaches the fab floor. The technology ramp up and integration with existing processes will require evolutionary steps in many aspects of the technology. For instance will it be necessary to reduce 3D mask effects like shadowing e.g. by introducing a thinner absorber structure. Continuous progress will be based on using new materials, adapted multilayers, and new reticle designs. Many of these developments are based on simulations and computer models for the design of the required structures and thus require data on the optical properties of the materials involved. In particular when addressing the reticle where the optical function is the target value. Using its more than 25 years of expertise in EUV metrology1, PTB operates instrumentation for reflectometry and scatterometry2 in the EUV and adjacent wavelength ranges and can provide the data for the determination of optical material parameters for individual thin layers. The need for sound optical parameter characterization for the development of alternative EUV materials was thoroughly motivated during the 2015 SPIE Advanced Lithography conference3. The data required is not readily available from databases, as thin film properties - depending on their deposition method and interfaces - may deviate significantly from standard bulk data4. Therefore, better optical constants and a continuous availability of the associated measurement tools are vital for further progress in EUV reticle and optical system design. The ability to vary relevant parameters like wavelength, angle of incidence (AOI), the plane of incidence and polarization is a prerequisite to gather sufficient data to model optical constants. We give details on PTB's measurement capabilities and accessible parameter space for optical material parameter characterization and show some representative data and results.

  11. Plasticity-related Gene 5 Promotes Spine Formation in Murine Hippocampal Neurons*

    PubMed Central

    Coiro, Pierluca; Stoenica, Luminita; Strauss, Ulf; Bräuer, Anja Ursula

    2014-01-01

    The transmembrane protein plasticity-related genes 3 and 5 (PRG3 and PRG5) increase filopodial formation in various cell lines, independently of Cdc42. However, information on the effects of PRG5 during neuronal development is sparse. Here, we present several lines of evidence for the involvement of PRG5 in the genesis and stabilization of dendritic spines. First, PRG5 was strongly expressed during mouse brain development from embryonic day 14 (E14), peaked around the time of birth, and remained stable at least until early adult stages (i.e. P30). Second, on a subcellular level, PRG5 expression shifted from an equal distribution along all neurites toward accumulation only along dendrites during hippocampal development in vitro. Third, overexpression of PRG5 in immature hippocampal neurons induced formation of spine-like structures ahead of time. Proper amino acid sequences in the extracellular domains (D1 to D3) of PRG5 were a prerequisite for trafficking and induction of spine-like structures, as shown by mutation analysis. Fourth, at stages when spines are present, knockdown of PRG5 reduced the number but not the length of protrusions. This was accompanied by a decrease in the number of excitatory synapses and, consequently, by a reduction of miniature excitatory postsynaptic current frequencies, although miniature excitatory postsynaptic current amplitudes remained similar. In turn, overexpressing PRG5 in mature neurons not only increased Homer-positive spine numbers but also augmented spine head diameters. Mechanistically, PRG5 interacts with phosphorylated phosphatidylinositols, phospholipids involved in dendritic spine formation by different lipid-protein assays. Taken together, our data propose that PRG5 promotes spine formation. PMID:25074937

  12. Testosterone promotes tube formation of endothelial cells isolated from veins via activation of Smad1 protein.

    PubMed

    Liu, Pei; Li, Xiaosa; Song, Fuhu; Li, Ping; Wei, Jinzhi; Yan, Qing; Xu, Xingyan; Yang, Jun; Li, Chuanxiang; Fu, Xiaodong

    2017-05-05

    Testosterone (T) deficiency is positively correlated with the increased incidence of cardiovascular disease. However, the effects of T on vascular endothelial cells remain obscure. Tube formation capacity is critical for vascular regeneration/repair and Smad1 plays an important role in these events. In this study, we investigated the effects of T on Smad1 activation and tube formation of cultured human umbilical endothelial cells (HUVECs). Our results showed that T rapidly increased endothelial Smad1 phosphorylation. This effect was mimicked by cell-impermeable T-BSA conjugates and was not altered by transcriptional inhibitor actinomycin D or translational inhibitor cycloheximide. T-induced Smad1 phosphorylation was blocked by ERK1/2 and c-Src inhibitors or their specific siRNAs, while it was reinforced by ERK1/2 or c-Src overexpression. Indeed, T rapidly activated ERK1/2 and c-Src signalings and c-Src was confirmed as the upstream of ERK1/2. Moreover, caveolae disruptor methyl-β-cyclodextrin (β-MCD) blocked Smad1 activation induced by T. The association of caveolin-1 with androgen receptor (AR) or c-Src was detected by immunoprecipitation and it was significantly increased by rapid T stimulation. Furthermore, fractional analysis showed that AR and c-Src were expressed in caveolae-enriched membrane fractions. T promoted tube formation of HUVECs, which was blocked by c-Src and ERK1/2 inhibitors or by the knockdown of Smad1. In conclusion, T increased tube formation of endothelial cells isolated from veins by stimulating Smad1 phosphorylation in a nongenomic manner, which was mediated by signals from AR/c-Src located in caveolae to ERK1/2 cascade. These findings may shed new light on the relevance of T to its vascular functions.

  13. Precise time and frequency intercomparison between NPL, India and PTB, Federal Republic of Germany via satellite symphonie-1

    NASA Technical Reports Server (NTRS)

    Mathur, B. S.; Banerjee, P.; Sood, P. C.; Saxena, M.; Kumar, N.; Suri, A. K.

    1981-01-01

    A time and frequency intercomparison experiment conducted using Earth stations in New Delhi, India and Raisting, FRG is described. The NPL clock was placed at New Delhi Earth Station and the Raisting Clock was calibrated with PTB/Primary standard via LORAN-C and travelling clocks. The random uncertainity of time comparisons, represented by two sample Allan Variance sigma (30 seconds), was less than 10 nanoseconds. The relative frequency difference between the NPL and Raisting Clocks, SNPL, RAIS, as measured over the 44 days period was found to be -15.7 x 10 to the -13th power. The relative frequency difference between PTB Primary Standard and Raisting Clock, SPTB, RAIS, during this period, was measured to be -22.8 x 10 to the -13th power. The relative frequency difference between NPL clock and PTB Primary Standard, SNPL, PTB, thus, is +7.1 x 10 to the -13th power. The clock rate (UTC, India) of +7.1 + or - 0.5 x 10 to the -13th power, agrees well with that obtained via VLF phase measurements over one year period and with USNO travelling clock time comparisons made in September, 1980.

  14. A pro-chelator triggered by hydrogen peroxide inhibits iron-promoted hydroxyl radical formation.

    PubMed

    Charkoudian, Louise K; Pham, David M; Franz, Katherine J

    2006-09-27

    The synthesis and structural characterization of a new pro-chelating agent, isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide (BSIH), are presented. BSIH only weakly interacts with iron unless hydrogen peroxide (H2O2) is present to remove the boronic ester protecting group to reveal a phenol that is a key metal-binding group of tridentate salicylaldehyde isonicotinoyl hydrazone (SIH). BSIH prevents deoxyribose degradation caused by hydroxyl radicals that are generated from H2O2 and redox-active iron by sequestering Fe3+ and preventing iron-promoted hydroxyl radical formation. The rate-determining step for iron sequestration is conversion of BSIH to SIH, followed by rapid Fe3+ complexation. The pro-chelate approach of BSIH represents a promising strategy for chelating a specific pool of detrimental metal ions without disturbing healthy metal ion distribution.

  15. Communication between nitric oxide synthase and positively-charged surface and bone formation promotion.

    PubMed

    Zhang, Wei; Liu, Jun; Shi, Haigang; Yang, Kun; Wang, Pingli; Wang, Gexia; Liu, Na; Wang, Huaiyu; Ji, Junhui; Chu, Paul K

    2016-12-01

    Despite the effects on physiology of bone marrow mesenchymal stem cells (BMSCs) and bone tissue, biological signal communication between bone implants and them is seldom employed as a guidance to create an osteo-inductive interface. Herein, the positively-charged surface is constructed on bone implant from the perspective of mediation of nitric oxide synthase (NOS) expression to signal BMSCs osteo-differentiation. In vitro and in vivo results indicate that the proper surface potential on the positively-charged surface affects NOS to express a high level of inducible nitric oxide synthase (iNOS) in three NOS isoforms of the contacted BMSCs, upregulates their osteogenetic expression, and ultimately foster new bone growth. However, an excessively high surface potential produces substantial immunomodulatory effects thereby offsetting the aforementioned advantages. This study demonstrates that fine-tuning of the positively-charged surface and proper utilization of the communication between NOS and bone implants promote bone formation.

  16. Monoallelic Loss of the Imprinted Gene Grb10 Promotes Tumor Formation in Irradiated Nf1+/- Mice

    PubMed Central

    Mroue, Rana; Huang, Brian; Braunstein, Steve; Firestone, Ari J.; Nakamura, Jean L.

    2015-01-01

    Imprinted genes are expressed from only one parental allele and heterozygous loss involving the expressed allele is sufficient to produce complete loss of protein expression. Genetic alterations are common in tumorigenesis but the role of imprinted genes in this process is not well understood. In earlier work we mutagenized mice heterozygous for the Neurofibromatosis I tumor suppressor gene (NF1) to model radiotherapy-associated second malignant neoplasms that arise in irradiated NF1 patients. Expression analysis of tumor cell lines established from our mouse models identified Grb10 expression as widely absent. Grb10 is an imprinted gene and polymorphism analysis of cell lines and primary tumors demonstrates that the expressed allele is commonly lost in diverse Nf1 mutant tumors arising in our mouse models. We performed functional studies to test whether Grb10 restoration or loss alter fundamental features of the tumor growth. Restoring Grb10 in Nf1 mutant tumors decreases proliferation, decreases soft agar colony formation and downregulates Ras signaling. Conversely, Grb10 silencing in untransformed mouse embryo fibroblasts significantly increased cell proliferation and increased Ras-GTP levels. Expression of a constitutively activated MEK rescued tumor cells from Grb10-mediated reduction in colony formation. These studies reveal that Grb10 loss can occur during in vivo tumorigenesis, with a functional consequence in untransformed primary cells. In tumors, Grb10 loss independently promotes Ras pathway hyperactivation, which promotes hyperproliferation, an early feature of tumor development. In the context of a robust Nf1 mutant mouse model of cancer this work identifies a novel role for an imprinted gene in tumorigenesis. PMID:26000738

  17. Topical hemostatic powder promotes reepithelialization and reduces scar formation after extensive esophageal mucosal resection.

    PubMed

    Beye, B; Barret, M; Alatawi, A; Beuvon, F; Nicco, C; Pratico, C A; Chereau, C; Chaussade, S; Batteux, F; Prat, F

    2016-08-01

    The development of techniques for endoscopic resection has provided new strategies for radical conservative treatment of superficial esophageal neoplasms, even those that are circumferential, such as Barrett's neoplasia. However, it is necessary to prevent the formation of scar tissue that can be responsible for esophageal strictures following circumferential resection. Preliminary data have suggested the possible efficacy of a hemostatic powder in the promotion of wound healing. The study aims to assess the effectiveness of Hemospray (Cook Medical) in a swine model of post-endoscopic esophageal stricture. Our prospective controlled study included 21 pigs. A 6-cm circumferential submucosal dissection of the esophagus (CESD) was performed in each pig. Group 1 (n = 11) only underwent CESD and Group 2 (n = 10) had repeated Hemospray applications after CESD. Clinical, endoscopic, and radiological monitoring were performed, blood levels of four inflammatory or pro-fibrotic cytokines were assessed, and histological analysis was performed. Median esophageal diameter was greater in the group treated with Hemospray (2 mm [1-3] vs. 3 mm [2-4], P = 0.01), and the rate of symptomatic esophageal stricture was 100% and 60% in Groups 1 and 2, respectively (P = 0.09). The thicknesses of esophageal fibrosis and inflammatory cell infiltrate were significantly lower in Group 2 than in Group 1 (P = 0.002 and 0.0003, respectively). The length of the neoepithelium was greater in Group 2 than in Group 1 (P = 0.0004). Transforming growth factor-β levels were significantly lower in Group 2 than in Group 1 (P = 0.01). The application of Hemospray after esophageal CESD reduces scar tissue formation and promotes reepithelialization, and therefore is a promising therapeutic approach in the prevention of post-endoscopic esophageal stricture.

  18. Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

    PubMed Central

    He, Yingpei; Ren, Yuan; Wu, Bingbing; Decourt, Boris; Lee, Aih Cheun; Taylor, Aaron; Suter, Daniel M.

    2015-01-01

    Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia. PMID:26224308

  19. Detergent-Mediated Formation of β-Hematin: Heme Crystallization Promoted by Detergents Implicates Nanostructure Formation for Use as a Biological Mimic

    PubMed Central

    2016-01-01

    Hemozoin is a unique biomineral that results from the sequestration of toxic free heme liberated as a consequence of hemoglobin degradation in the malaria parasite. Synthetic neutral lipid droplets (SNLDs) and phospholipids were previously shown to support the rapid formation of β-hematin, abiological hemozoin, under physiologically relevant pH and temperature, though the mechanism by which heme crystallization occurs remains unclear. Detergents are particularly interesting as a template because they are amphiphilic molecules that spontaneously organize into nanostructures and have been previously shown to mediate β-hematin formation. Here, 11 detergents were investigated to elucidate the physicochemical properties that best recapitulate crystal formation in the parasite. A strong correlation between the detergent’s molecular structure and the corresponding kinetics of β-hematin formation was observed, where higher molecular weight polar chains promoted faster reactions. The larger hydrophilic chains correlated to the detergent’s ability to rapidly sequester heme into the lipophilic core, allowing for crystal nucleation to occur. The data presented here suggest that detergent nanostructures promote β-hematin formation in a similar manner to SNLDs and phospholipids. Through understanding mediator properties that promote optimal crystal formation, we are able to establish an in vitro assay to probe this drug target pathway. PMID:27175104

  20. Urokinase Receptor Promotes Skin Tumor Formation by Preventing Epithelial Cell Activation of Notch1.

    PubMed

    Mazzieri, Roberta; Pietrogrande, Giovanni; Gerasi, Laura; Gandelli, Alessandro; Colombo, Piergiuseppe; Moi, Davide; Brombin, Chiara; Ambrosi, Alessandro; Danese, Silvio; Mignatti, Paolo; Blasi, Francesco; D'Alessio, Silvia

    2015-11-15

    The urokinase-type plasminogen activator receptor (uPAR) has a well-established role in cancer progression, but it has been little studied at earlier stages of cancer initiation. Here, we show that uPAR deficiency in the mouse dramatically reduces susceptibility to the classical two-stage protocol of inflammatory skin carcinogenesis. uPAR genetic deficiency decreased papilloma formation and accelerated keratinocyte differentiation, effects mediated by Notch1 hyperactivation. Notably, Notch1 inhibition in uPAR-deficient mice rescued their susceptibility to skin carcinogenesis. Clinically, we found that human differentiated keratoacanthomas expressed low levels of uPAR and high levels of activated Notch1, with opposite effects in proliferating tumors, confirming the relevance of the observations in mice. Furthermore, we found that TACE-dependent activation of Notch1 in basal kerantinocytes was modulated by uPAR. Mechanistically, uPAR sequestered TACE within lipid rafts to prevent Notch1 activation, thereby promoting cell proliferation and tumor formation. Given that uPAR signaling is nonessential for normal epidermal homeostasis, our results argue that uPAR may present a promising disease-specific target for preventing skin cancer development.

  1. Myeloid STAT3 promotes formation of colitis-associated colorectal cancer in mice

    PubMed Central

    Pathria, Paulina; Gotthardt, Dagmar; Prchal-Murphy, Michaela; Putz, Eva-Maria; Holcmann, Martin; Schlederer, Michaela; Grabner, Beatrice; Crncec, Ilija; Svinka, Jasmin; Musteanu, Monica; Hoffmann, Thomas; Filipits, Martin; Berger, Walter; Poli, Valeria; Kenner, Lukas; Bilban, Martin; Casanova, Emilio; Müller, Mathias; Strobl, Birgit; Bayer, Editha; Mohr, Thomas; Sexl, Veronika; Eferl, Robert

    2015-01-01

    Myeloid cells lacking STAT3 promote antitumor responses of NK and T cells but it is unknown if this crosstalk affects development of autochthonous tumors. We deleted STAT3 in murine myeloid cells (STAT3Δm) and examined the effect on the development of autochthonous colorectal cancers (CRCs). Formation of Azoxymethane/Dextransulfate (AOM/DSS)-induced CRCs was strongly suppressed in STAT3Δm mice. Gene expression profiling showed strong activation of T cells in the stroma of STAT3Δm CRCs. Moreover, STAT3Δm host mice were better able to control the growth of transplanted MC38 colorectal tumor cells which are known to be killed in a T cell-dependent manner. These data suggest that myeloid cells lacking STAT3 control formation of CRCs mainly via cross activation of T cells. Interestingly, the few CRCs that formed in STAT3Δm mice displayed enhanced stromalization but appeared normal in size indicating that they have acquired ways to escape enhanced tumor surveillance. We found that CRCs in STAT3Δm mice consistently activate STAT3 signaling which is implicated in immune evasion and might be a target to prevent tumor relapse. PMID:26137415

  2. The transcription factor OBP4 controls root growth and promotes callus formation.

    PubMed

    Ramirez-Parra, Elena; Perianez-Rodriguez, Juan; Navarro-Neila, Sara; Gude, Inmaculada; Moreno-Risueno, Miguel A; Del Pozo, Juan C

    2017-03-01

    Plant growth and development require a continuous balance between cell division and differentiation. In root meristems, differentiated cells acquire specialized functions, losing their mitotic potential. Some plant cells, such as pericycle cells, have a remarkable plasticity to regenerate new organs. The molecular mechanisms underlying cell reprogramming are not completely known. In this work, a functional screening of transcription factors identified Arabidopsis OBP4 (OBF Binding Protein 4) as a novel regulator of root growth and cell elongation and differentiation. Overexpression of OBP4 regulates the levels of a large number of transcripts in roots, many involved in hormonal signaling and callus formation. OBP4 controls cell elongation and differentiation in root cells. OBP4 does not induce cell division in the root meristem, but promotes pericycle cell proliferation, forming callus-like structures at the root tip, as shown by the expression of stem cell markers. Callus formation is enhanced by ectopic expression of OBP4 in the wild-type or alf4-1, but is significantly reduced in roots that have lower levels of OBP4. Our data provide molecular insights into how differentiated root cells acquire the potential to generate callus, a pluripotent mass of cells that can regenerate fully functional plant organs.

  3. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.

    PubMed

    Parkhurst, Christopher N; Yang, Guang; Ninan, Ipe; Savas, Jeffrey N; Yates, John R; Lafaille, Juan J; Hempstead, Barbara L; Littman, Dan R; Gan, Wen-Biao

    2013-12-19

    Microglia are the resident macrophages of the CNS, and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and function, however, remain unclear. To address this question, we generated CX3CR1(CreER) mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function in microglia. Using CX3CR1(CreER) to drive diphtheria toxin receptor expression in microglia, we found that microglia could be specifically depleted from the brain upon diphtheria toxin administration. Mice depleted of microglia showed deficits in multiple learning tasks and a significant reduction in motor-learning-dependent synapse formation. Furthermore, Cre-dependent removal of brain-derived neurotrophic factor (BDNF) from microglia largely recapitulated the effects of microglia depletion. Microglial BDNF increases neuronal tropomyosin-related kinase receptor B phosphorylation, a key mediator of synaptic plasticity. Together, our findings reveal that microglia serve important physiological functions in learning and memory by promoting learning-related synapse formation through BDNF signaling.

  4. Sfp-type PPTase inactivation promotes bacterial biofilm formation and ability to enhance wheat drought tolerance

    PubMed Central

    Timmusk, Salme; Kim, Seong-Bin; Nevo, Eviatar; Abd El Daim, Islam; Ek, Bo; Bergquist, Jonas; Behers, Lawrence

    2015-01-01

    Paenibacillus polymyxa is a common soil bacterium with broad range of practical applications. An important group of secondary metabolites in P. polymyxa are non-ribosomal peptide and polyketide derived metabolites (NRPs/PKs). Modular non-ribosomal peptide synthetases catalyze main steps in the biosynthesis of the complex secondary metabolites. Here we report on the inactivation of an A26 Sfp-type 4'-phosphopantetheinyl transferase (Sfp-type PPTase). The inactivation of the gene resulted in loss of NRPs/PKs production. In contrast to the former Bacillus spp. model the mutant strain compared to wild type showed greatly enhanced biofilm formation ability. A26Δsfp biofilm promotion is directly mediated by NRPs/PKs, as exogenous addition of the wild type metabolite extracts restores its biofilm formation level. Wheat inoculation with bacteria that had lost their Sfp-type PPTase gene resulted in two times higher plant survival and about three times increased biomass under severe drought stress compared to wild type. Challenges with P. polymyxa genetic manipulation are discussed. PMID:26052312

  5. BipA Is Associated with Preventing Autoagglutination and Promoting Biofilm Formation in Bordetella holmesii

    PubMed Central

    Hiramatsu, Yukihiro; Saito, Momoko; Otsuka, Nao; Suzuki, Eri; Watanabe, Mineo; Shibayama, Keigo; Kamachi, Kazunari

    2016-01-01

    Bordetella holmesii causes both invasive and respiratory diseases in humans. Although the number of cases of pertussis-like respiratory illnesses due to B. holmesii infection has increased in the last decade worldwide, little is known about the virulence factors of the organism. Here, we analyzed a B. holmesii isolate that forms large aggregates and precipitates in suspension, and subsequently demonstrated that the autoagglutinating isolate is deficient in Bordetella intermediate protein A (BipA) and that this deletion is caused by a frame-shift mutation in the bipA gene. A BipA-deficient mutant generated by homologous recombination also exhibited the autoagglutination phenotype. Moreover, the BipA mutant adhered poorly to an abiotic surface and failed to form biofilms, as did two other B. holmesii autoagglutinating strains, ATCC 51541 and ATCC 700053, which exhibit transcriptional down-regulation of bipA gene expression, indicating that autoagglutination indirectly inhibits biofilm formation. In a mouse intranasal infection model, the BipA mutant showed significantly lower levels of initial lung colonization than did the parental strain (P < 0.01), suggesting that BipA might be a critical virulence factor in B. holmesii respiratory infection. Together, our findings suggest that BipA production plays an essential role in preventing autoagglutination and indirectly promoting biofilm formation by B. holmesii. PMID:27448237

  6. Hand2 inhibits kidney specification while promoting vein formation within the posterior mesoderm

    PubMed Central

    Perens, Elliot A; Garavito-Aguilar, Zayra V; Guio-Vega, Gina P; Peña, Karen T; Schindler, Yocheved L; Yelon, Deborah

    2016-01-01

    Proper organogenesis depends upon defining the precise dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that set the boundaries of the IM are poorly understood. Here, we show that the bHLH transcription factor Hand2 limits the size of the embryonic kidney by restricting IM dimensions. The IM is expanded in zebrafish hand2 mutants and is diminished when hand2 is overexpressed. Within the posterior mesoderm, hand2 is expressed laterally adjacent to the IM. Venous progenitors arise between these two territories, and hand2 promotes venous development while inhibiting IM formation at this interface. Furthermore, hand2 and the co-expressed zinc-finger transcription factor osr1 have functionally antagonistic influences on kidney development. Together, our data suggest that hand2 functions in opposition to osr1 to balance the formation of kidney and vein progenitors by regulating cell fate decisions at the lateral boundary of the IM. DOI: http://dx.doi.org/10.7554/eLife.19941.001 PMID:27805568

  7. VEGF expression in mesenchymal stem cells promotes bone formation of tissue-engineered bones.

    PubMed

    Liu, Boling; Li, Xihai; Liang, Guiqing; Liu, Xianxiang

    2011-01-01

    The purpose of this study was to investigate the in vivo vascularization and bone formation activity of tissue-engineered bone constructed using bone marrow mesenchymal stem cells (MSCs) transfected with vascular endothelial growth factor (VEGF). The expression of VEGF165 in rat bone marrow MSCs was confirmed using RT-PCR and immunohistochemistry. The MSCs were cultured together with nano-hydroxyapatite/collagen (NHAC) to form tissue-engineered bone. Untransfected MSCs were used as controls. The mice were sacrificed, and the bone xenografts were analyzed using immunohistochemistry and quantified for the degree of vascularization and new bone formation. Based on our results, expression of the VEGF165 gene was detected using RT-PCR and immunohistochemistry following transfection and 4 weeks of selection. The co-cultured NHAC- and VEGF-transfected MSCs had significantly higher alkaline phosphatase (AP) activity compared to the controls (P<0.05). In the mice that received the tissue-engineered bone xenografts, clumps of cartilage cells, irregular bone-like tissue and microvessels were observed. The growth of these structures progressed with time. In the control mice, however, only small amounts of bone-like and fibrotic tissue were observed. The differences between the control and experimental groups were statistically significant (P<0.05). In conclusion, VEGF165‑transfected bone marrow MSCs promotes vascularization of tissue-engineered bone and ectopic osteogenesis.

  8. The MinCDJ System in Bacillus subtilis Prevents Minicell Formation by Promoting Divisome Disassembly

    PubMed Central

    van Baarle, Suey; Bramkamp, Marc

    2010-01-01

    Background Cell division in Bacillus subtilis takes place precisely at midcell, through the action of Noc, which prevents division from occurring over the nucleoids, and the Min system, which prevents cell division from taking place at the poles. Originally it was thought that the Min system acts directly on FtsZ, preventing the formation of a Z-ring and, therefore, the formation of a complete cytokinetic ring at the poles. Recently, a new component of the B. subtilis Min system was identified, MinJ, which acts as a bridge between DivIVA and MinCD. Methodology/Principal Findings We used fluorescence microscopy and molecular genetics to examine the molecular role of MinJ. We found that in the absence of a functional Min system, FtsA, FtsL and PBP-2B remain associated with completed division sites. Evidence is provided that MinCDJ are responsible for the failure of these proteins to localize properly, indicating that MinCDJ can act on membrane integral components of the divisome. Conclusions/Significance Taken together, we postulate that the main function of the Min system is to prevent minicell formation adjacent to recently completed division sites by promoting the disassembly of the cytokinetic ring, thereby ensuring that cell division occurs only once per cell cycle. Thus, the role of the Min system in rod-shaped bacteria seems not to be restricted to an inhibitory function on FtsZ polymerization, but can act on different levels of the divisome. PMID:20352045

  9. Ezrin/Radixin/Moesin proteins and flotillins cooperate to promote uropod formation in T cells.

    PubMed

    Martinelli, Sibylla; Chen, Emily J H; Clarke, Fiona; Lyck, Ruth; Affentranger, Sarah; Burkhardt, Janis K; Niggli, Verena

    2013-01-01

    T cell uropods are enriched in specific proteins including adhesion receptors such as P-selectin glycoprotein ligand-1 (PSGL-1), lipid raft-associated proteins such as flotillins and ezrin/radixin/moesin (ERM) proteins which associate with cholesterol-rich raft domains and anchor adhesion receptors to the actin cytoskeleton. Using dominant mutants and siRNA technology we have tested the interactions among these proteins and their role in shaping the T cell uropod. Expression of wild type (WT) ezrin-EGFP failed to affect the morphology of human T cells or chemokine-induced uropod recruitment of PSGL-1 and flotillin-1 and -2. In contrast, expression of constitutively active T567D ezrin-EGFP induced a motile, polarized phenotype in some of the transfected T cells, even in the absence of chemokine. These cells featured F-actin-rich ruffles in the front and uropod enrichment of PSGL-1 and flotillins. T567D ezrin-EGFP was itself strongly enriched in the rear of the polarized T cells. Uropod formation induced by T567D ezrin-EGFP was actin-dependent as it was attenuated by inhibition of Rho-kinase or myosin II, and abolished by disruption of actin filaments. While expression of constitutively active ezrin enhanced cell polarity, expression of a dominant-negative deletion mutant of ezrin, 1-310 ezrin-EGFP, markedly reduced uropod formation induced by the chemokine SDF-1, T cell front-tail polarity, and capping of PSGL-1 and flotillins. Transfection of T cells with WT or T567D ezrin did not affect chemokine-mediated chemotaxis whereas 1-310 ezrin significantly impaired spontaneous 2D migration and chemotaxis. siRNA-mediated downregulation of flotillins in murine T cells attenuated moesin capping and uropod formation, indicating that ERM proteins and flotillins cooperate in uropod formation. In summary, our results indicate that activated ERM proteins function together with flotillins to promote efficient chemotaxis of T cells by structuring the uropod of migrating T cells.

  10. Hydroxyl radical induced by lipid in Maillard reaction model system promotes diet-derived N(ε)-carboxymethyllysine formation.

    PubMed

    Han, Lipeng; Li, Lin; Li, Bing; Zhao, Di; Li, Yuting; Xu, Zhenbo; Liu, Guoqin

    2013-10-01

    N(ε)-carboxymethyllysine (CML) is commonly found in food, and is considered as a potential hazard to human health. However, the effect of lipids on CML formation in Maillard reaction is still not clarified. In this study, the content of diet-derived CML and its key intermediates, epsilon-fructoselysine (FL) and glyoxal (GO), is determined with high performance liquid chromatography mass spectrum (HPLC-MS) in model system containing lipid compounds. According to the results, hydroxyl radical (OH) induced by Fenton reagent can promote the three pathways of CML formation. Moreover, in the Maillard reaction system, linoleic acid (Lin), oleic acid (Ole) and glycerol trioleate (Tri) can induce more OH·, which promotes CML formation. Their level of promoting CML formation is in the order of Ole>Lin>Tri. On the contrary, glycerol (Gly) can scavenge OH·, which inhibit the CML formation. Finally, it is proved that FL content and GO content decreases with heating time in model system, while CML content increases with heating time. Thus, it is concluded that in the Maillard reaction system lipids can induce more OH·, which promotes the conversion from FL and GO to CML. Our research may contribute to the development of inhibitory methods for diet-derived CML by scavenging OH·.

  11. EPO Promotes Bone Repair through Enhanced Cartilaginous Callus Formation and Angiogenesis

    PubMed Central

    Wan, Lin; Zhang, Fengjie; He, Qiling; Tsang, Wing Pui; Lu, Li; Li, Qingnan; Wu, Zhihong; Qiu, Guixing; Zhou, Guangqian; Wan, Chao

    2014-01-01

    Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration. PMID:25003898

  12. EPO promotes bone repair through enhanced cartilaginous callus formation and angiogenesis.

    PubMed

    Wan, Lin; Zhang, Fengjie; He, Qiling; Tsang, Wing Pui; Lu, Li; Li, Qingnan; Wu, Zhihong; Qiu, Guixing; Zhou, Guangqian; Wan, Chao

    2014-01-01

    Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.

  13. WHI-131 Promotes Osteoblast Differentiation and Prevents Osteoclast Formation and Resorption in Mice.

    PubMed

    Cheon, Yoon-Hee; Kim, Ju-Young; Baek, Jong Min; Ahn, Sung-Jun; Jun, Hong Young; Erkhembaatar, Munkhsoyol; Kim, Min Seuk; Lee, Myeung Su; Oh, Jaemin

    2016-02-01

    levels of genes related to osteoblast differentiation, and induced the phosphorylation of Akt, p38, and Smad1/5/8. Furthermore, 5-week-old ICR mice treated with WHI-131 exhibited antiresorbing effects in a lipopolysaccharide-induced calvaria bone loss model in vivo and increased bone-forming activity in a calvarial bone formation model. Therefore, the results of this study show that WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Thus, WHI-131 could be a useful pharmacological agent to treat osteoporosis by promoting bone growth and inhibiting resorption.

  14. Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells

    NASA Astrophysics Data System (ADS)

    Berny-Lang, M. A.; Aslan, J. E.; Tormoen, G. W.; Patel, I. A.; Bock, P. E.; Gruber, A.; McCarty, O. J. T.

    2011-02-01

    The routine observation of tumor emboli in the peripheral blood of patients with carcinomas raises questions about the clinical relevance of these circulating tumor cells. Thrombosis is a common clinical manifestation of cancer, and circulating tumor cells may play a pathogenetic role in this process. The presence of coagulation-associated molecules on cancer cells has been described, but the mechanisms by which circulating tumor cells augment or alter coagulation remains unclear. In this study we utilized suspensions of a metastatic adenocarcinoma cell line, MDA-MB-231, and a non-metastatic breast epithelial cell line, MCF-10A, as models of circulating tumor cells to determine the thromobogenic activity of these blood-foreign cells. In human plasma, both metastatic MDA-MB-231 cells and non-metastatic MCF-10A cells significantly enhanced clotting kinetics. The effect of MDA-MB-231 and MCF-10A cells on clotting times was cell number-dependent and inhibited by a neutralizing antibody to tissue factor (TF) as well as inhibitors of activated factor X and thrombin. Using fluorescence microscopy, we found that both MDA-MB-231 and MCF-10A cells supported the binding of fluorescently labeled thrombin. Furthermore, in a model of thrombus formation under pressure-driven flow, MDA-MB-231 and MCF-10A cells significantly decreased the time to occlusion. Our findings indicate that the presence of breast epithelial cells in blood can stimulate coagulation in a TF-dependent manner, suggesting that tumor cells that enter the circulation may promote the formation of occlusive thrombi under shear flow conditions.

  15. Arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells

    SciTech Connect

    Liao Weiting; Lin Pinpin; Cheng, T.-S.; Yu, H.-S.; Chang, Louis W.

    2007-12-01

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-{alpha}-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 {mu}M) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-{alpha} and 5 {mu}M sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

  16. GLUTAMATERGIC SYNAPSE FORMATION IS PROMOTED BY α7-CONTAINING NICOTINIC ACETYLCHOLINE RECEPTORS

    PubMed Central

    Lozada, Adrian F.; Wang, Xulong; Gounko, Natalia V.; Massey, Kerri A.; Duan, Jingjing; Liu, Zhaoping; Berg, Darwin K.

    2012-01-01

    Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in wild-type organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction. PMID:22649244

  17. Absence of keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice

    PubMed Central

    Toivola, Diana M.; Habtezion, Aida; Misiorek, Julia O.; Zhang, Linxing; Nyström, Joel H.; Sharpe, Orr; Robinson, William H.; Kwan, Raymond; Omary, M. Bishr

    2015-01-01

    Human mutations in keratin 8 (K8) and keratin 18 (K18), the intermediate filament proteins of hepatocytes, predispose to several liver diseases. K8-null mice develop chronic liver injury and fragile hepatocytes, dysfunctional mitochondria, and Th2-type colitis. We tested the hypothesis that autoantibody formation accompanies the liver damage that associates with K8/K18 absence. Sera from wild-type control, K8-null, and K18-null mice were analyzed by immunoblotting and immunofluorescence staining of cell and mouse tissue homogenates. Autoantibodies to several antigens were identified in 81% of K8-null male mice 8 mo or older. Similar autoantibodies were detected in aging K18-null male mice that had a related liver phenotype but normal colon compared with K8-null mice, suggesting that the autoantibodies are linked to liver rather than colonic disease. However, these autoantibodies were not observed in nontransgenic mice subjected to 4 chronic injury models. The autoantigens are ubiquitous and partition with mitochondria. Mass spectrometry and purified protein analysis identified, mitochondrial HMG-CoA synthase, aldehyde dehydrogenase, and catalase as the primary autoantigens, and glutamate dehydrogenase and epoxide hydrolase-2 as additional autoantigens. Therefore, absence of the hepatocyte keratins results in production of anti-mitochondrial autoantibodies (AMA) that recognize proteins involved in energy metabolism and oxidative stress, raising the possibility that AMA may be found in patients with keratin mutations that associate with liver and other diseases.—Toivola, D. M., Habtezion, A., Misiorek, J. O., Zhang, L., Nyström, J. H., Sharpe, O., Robinson, W. H., Kwan, R., Omary, M. B. Absence of keratin 8 or 18 promotes antimitochondrial autoantibody formation in aging male mice. PMID:26399787

  18. Spp1, a member of the Set1 Complex, promotes meiotic DSB formation in promoters by tethering histone H3K4 methylation sites to chromosome axes.

    PubMed

    Sommermeyer, Vérane; Béneut, Claire; Chaplais, Emmanuel; Serrentino, Maria Elisabetta; Borde, Valérie

    2013-01-10

    Meiotic chromosomes are organized into arrays of loops that are anchored to the chromosome axis structure. Programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination, catalyzed by Spo11 and accessory DSB proteins, form in loop sequences in promoters, whereas the DSB proteins are located on chromosome axes. Mechanisms bridging these two chromosomal regions for DSB formation have remained elusive. Here we show that Spp1, a conserved member of the histone H3K4 methyltransferase Set1 complex, is required for normal levels of DSB formation and is associated with chromosome axes during meiosis, where it physically interacts with the Mer2 DSB protein. The PHD finger module of Spp1, which reads H3K4 methylation close to promoters, promotes DSB formation by tethering these regions to chromosome axes and activating cleavage by the DSB proteins. This paper provides the molecular mechanism linking DSB sequences to chromosome axes and explains why H3K4 methylation is important for meiotic recombination.

  19. Nanoscale Morphology of PTB7 Based Organic Photovoltaics as a Function of Fullerene Size

    PubMed Central

    Roehling, John D.; Baran, Derya; Sit, Joseph; Kassar, Thaer; Ameri, Tayebeh; Unruh, Tobias; Brabec, Christoph J.; Moulé, Adam J.

    2016-01-01

    High efficiency polymer:fullerene photovoltaic device layers self-assemble with hierarchical features from ångströms to 100’s of nanometers. The feature size, shape, composition, orientation, and order all contribute to device efficiency and are simultaneously difficult to study due to poor contrast between carbon based materials. This study seeks to increase device efficiency and simplify morphology measurements by replacing the typical fullerene acceptor with endohedral fullerene Lu3N@PC80BEH. The metal atoms give excellent scattering contrast for electron beam and x-ray experiments. Additionally, Lu3N@PC80BEH has a lower electron affinity than standard fullerenes, which can raise the open circuit voltage of photovoltaic devices. Electron microscopy techniques are used to produce a detailed account of morphology evolution in mixtures of Lu3N@PC80BEH with the record breaking donor polymer, PTB7 and coated using solvent mixtures. We demonstrate that common solvent additives like 1,8-diiodooctane or chloronapthalene do not improve the morphology of endohedral fullerene devices as expected. The poor device performance is attributed to the lack of mutual miscibility between this particular polymer:fullerene combination and to co-crystallization of Lu3N@PC80BEH with 1,8-diiodooctane. This negative result explains why solvent additives mixtures are not necessarily a morphology cure-all. PMID:27498880

  20. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants.

    PubMed

    Hao, Yu-Jun; Wei, Wei; Song, Qing-Xin; Chen, Hao-Wei; Zhang, Yu-Qin; Wang, Fang; Zou, Hong-Feng; Lei, Gang; Tian, Ai-Guo; Zhang, Wan-Ke; Ma, Biao; Zhang, Jin-Song; Chen, Shou-Yi

    2011-10-01

    NAC transcription factors play important roles in plant growth, development and stress responses. Previously, we identified multiple NAC genes in soybean (Glycine max). Here, we identify the roles of two genes, GmNAC11 and GmNAC20, in stress responses and other processes. The two genes were differentially induced by multiple abiotic stresses and plant hormones, and their transcripts were abundant in roots and cotyledons. Both genes encoded proteins that localized to the nucleus and bound to the core DNA sequence CGT[G/A]. In the protoplast assay system, GmNAC11 acts as a transcriptional activator, whereas GmNAC20 functions as a mild repressor; however, the C-terminal end of GmANC20 has transcriptional activation activity. Over-expression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 over-expression only improves salt tolerance. Over-expression of GmNAC20 also promotes lateral root formation. GmNAC20 may regulate stress tolerance through activation of the DREB/CBF-COR pathway, and may control lateral root development by altering auxin signaling-related genes. GmNAC11 probably regulates DREB1A and other stress-related genes. The roles of the two GmNAC genes in stress tolerance were further analyzed in soybean transgenic hairy roots. These results provide a basis for genetic manipulation to improve the agronomic traits of important crops.

  1. MEKK2 mediates an alternative β-catenin pathway that promotes bone formation

    PubMed Central

    Greenblatt, Matthew Blake; Shin, Dong Yeon; Oh, Hwanhee; Lee, Ki-Young; Zhai, Bo; Gygi, Steven P.; Lotinun, Sutada; Baron, Roland; Liu, Dou; Su, Bing; Glimcher, Laurie H.; Shim, Jae-Hyuck

    2016-01-01

    Proper tuning of β-catenin activity in osteoblasts is required for bone homeostasis, because both increased and decreased β-catenin activity have pathologic consequences. In the classical pathway for β-catenin activation, stimulation with WNT ligands suppresses constitutive phosphorylation of β-catenin by glycogen synthase kinase 3β, preventing β-catenin ubiquitination and proteasomal degradation. Here, we have found that mitogen-activated protein kinase kinase kinase 2 (MAP3K2 or MEKK2) mediates an alternative pathway for β-catenin activation in osteoblasts that is distinct from the canonical WNT pathway. FGF2 activates MEKK2 to phosphorylate β-catenin at serine 675, promoting recruitment of the deubiquitinating enzyme, ubiquitin-specific peptidase 15 (USP15). USP15 in turn prevents the basal turnover of β-catenin by inhibiting its ubiquitin-dependent proteasomal degradation, thereby enhancing WNT signaling. Analysis of MEKK2-deficient mice and genetic interaction studies between Mekk2- and β-catenin–null alleles confirm that this pathway is an important physiologic regulator of bone mass in vivo. Thus, an FGF2/MEKK2 pathway mediates an alternative nonclassical pathway for β-catenin activation, and this pathway is a key regulator of bone formation by osteoblasts. PMID:26884171

  2. NGF promotes long-term memory formation by activating poly(ADP-ribose)polymerase-1.

    PubMed

    Wang, Shao-Hui; Liao, Xiao-Mei; Liu, Dan; Hu, Juan; Yin, Yang-Yang; Wang, Jian-Zhi; Zhu, Ling-Qiang

    2012-11-01

    Nerve growth factor (NGF) is a critical secreted protein that plays an important role in development, survival, and function of the mammalian nervous system. Previously reports suggest that endogenous NGF is essential for the hippocampal plasticity/memory and NGF deprivation induces the impairment of hippocampus-related memory and synaptic plasticity. However, whether exogenous supplement of NGF could promote the hippocampus-dependent synaptic plasticity/memory and the possible underlying mechanisms are not clear. In this study we found that NGF administration facilitates the hippocampus-dependent long-term memory and synaptic plasticity by increasing the activity of PARP-1, a polymerase mediating the PolyADP-ribosylation and important for the memory formation. Co-application of 3-Aminobenzamide (3-AB), a specific inhibitor of PARP-1, distinctly blocked the boosting effect of NGF on memory and synaptic plasticity, and the activation of downstream PKA-CREB signal pathway. Our data provide the first evidence that NGF supplement facilitates synaptic plasticity and the memory ability through PARP-1-mediated protein polyADP-ribosylation and activation of PKA-CREB pathway.

  3. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity.

    PubMed

    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li

    2016-04-22

    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  4. Comparison of the NIST and PTB Air-Kerma Standards for Low-Energy X-Rays.

    PubMed

    O'Brien, Michelle; Bueermann, Ludwig

    2009-01-01

    A comparison has been made of the air-kerma standards for low-energy x rays at the National Institute of Standards and Technology (NIST) and the Physikalisch-Technische Bundesanstalt (PTB). The comparison involved a series of measurements at the PTB and the NIST using the air-kerma standards and two NIST reference-class transfer ionization chamber standards. Results are presented for the reference radiation beam qualities in the range from 25 kV to 50 kV for low energy x rays, including the techniques used for mammography dose traceability. The tungsten generated reference radiation qualities, between 25 kV and 50 kV used for this comparison, are new to NIST; therefore this comparison will serve as the preliminary comparison for NIST and a verification of the primary standard correction factors. The mammography comparison will repeat two previously unpublished comparisons between PTB and NIST. The results show the standards to be in reasonable agreement within the standard uncertainty of the comparison of about 0.4 %.

  5. Frequency Comparison of [Formula: see text] Ion Optical Clocks at PTB and NPL via GPS PPP.

    PubMed

    Leute, J; Huntemann, N; Lipphardt, B; Tamm, Christian; Nisbet-Jones, P B R; King, S A; Godun, R M; Jones, J M; Margolis, H S; Whibberley, P B; Wallin, A; Merimaa, M; Gill, P; Peik, E

    2016-07-01

    We used precise point positioning, a well-established GPS carrier-phase frequency transfer method to perform a direct remote comparison of two optical frequency standards based on single laser-cooled [Formula: see text] ions operated at the National Physical Laboratory (NPL), U.K. and the Physikalisch-Technische Bundesanstalt (PTB), Germany. At both institutes, an active hydrogen maser serves as a flywheel oscillator which is connected to a GPS receiver as an external frequency reference and compared simultaneously to a realization of the unperturbed frequency of the (2)S1/2(F=0)-(2)D3/2(F=2) electric quadrupole transition in [Formula: see text] via an optical femtosecond frequency comb. To profit from long coherent GPS-link measurements, we extrapolate the fractional frequency difference over the various data gaps in the optical clock to maser comparisons which introduces maser noise to the frequency comparison but improves the uncertainty from the GPS-link instability. We determined the total statistical uncertainty consisting of the GPS-link uncertainty and the extrapolation uncertainties for several extrapolation schemes. Using the extrapolation scheme with the smallest combined uncertainty, we find a fractional frequency difference [Formula: see text] of -1.3×10(-15) with a combined uncertainty of 1.2×10(-15) for a total measurement time of 67 h. This result is consistent with an agreement of the frequencies realized by both optical clocks and with recent absolute frequency measurements against caesium fountain clocks within the corresponding uncertainties.

  6. Biomarker evidence for shallow water marine euxinia through the PTB in the Panthalassic Ocean (Peace River Basin Embayment, Canada)

    NASA Astrophysics Data System (ADS)

    Hays, L. E.; Beatty, T.; Henderson, C. M.; Summons, R. E.; Love, G. D.

    2007-12-01

    Protracted euxinic conditions in the late Paleozoic and early Mesozoic oceans may have been an important paleoenvironmental factor in the Permian-Triassic Boundary (PTB) extinction. Release of hydrogen sulfide during upwelling or transgressive events from such an ocean (Kump et al. 2005; Riccardi et al. 2006) may have been a driver of the extinction in both marine and terrestrial environments. Worldwide marine PTB sections show evidence for a stratified water column and the presence of sulfidic deep water, at least episodically (Isozaki 1997; Grice et al. 2005). Taxa that are particularly characteristic of such an environment are the green sulfur bacteria, or Chlorobiaceae. These anoxygenic phototrophic bacteria utilize sulfide as an electron donor for photosynthesis and live in modern stratified water columns where euxinia extends into the photic zone. Indeed, biomarkers derived from these organisms have been identified at a number of the PTB sections. The Peace River embayment in western Canada has been identified as a section that spans the PTB based on conodont biostratigraphy (Henderson 1997). Samples from five drill cores in this section provide new insight into the state of the Panthalassic Ocean during this time of unprecedented turnover in Earth's biota. Using standard biomarker protocols, we identified aromatic hydrocarbons that are diagentic products of the carotenoids isorenieratene and chlorobactene, which are diagnostic for the brown and green strains, respectively, of the Chlorobiaceae. The occurrence of chlorobactane is especially notable since the green-pigmented varieties of the Chlorobiaceae require higher light intensities than the brown-pigmented forms and, in modern environments where they have been found, occur between 13 and 30 m of the surface. This is the first time that chlorobactane has been reported from a PTB section and it suggests a particularly shallow chemocline periodically at this location. The δ13C values for the aryl

  7. Dramatic Enhancement of Power Conversion Efficiency in Polymer Solar Cells by Conjugating Very Low Ratio of Triplet Iridium Complexes to PTB7.

    PubMed

    Qian, Min; Zhang, Ran; Hao, Jingyu; Zhang, Wenjun; Zhang, Qin; Wang, Jianpu; Tao, Youtian; Chen, Shufen; Fang, Junfeng; Huang, Wei

    2015-06-17

    Various low ratios of triplet iridium complexes (0, 0.5, 1, 1.5, 2.5, and 5 mol%) are conjugated to the backbone of the famous champion donor polymer PTB7. At the same conditions, the power conversion efficiency for polymer containing 1% of Ir increases by 45%, 39%, and 31% in three batches of devices compared with control Ir-free PTB7.

  8. Intercomparison exercise of the PTB, BfS, MPA and PSI calibration facilities for radon gas concentration.

    PubMed

    Butterweck, G; Schuler, C; Paul, A; Honig, A; Dersch, R; Schmidt, V; Hamel, P; Buchröder, H; Rox, A; Herzog, W

    2002-01-01

    The traceability chain of one national reference laboratory (PTB) and three accredited radon calibration laboratories (BfS MPA and PSI) to internationally acknowledged radon gas standards is specified. As an additional tool for quality assurance, interchange of an electronic radon measuring instrument was used as a means for a relative comparison of the radon gas reference atmospheres. The instrument was exposed to radon gas activity concentrations between 500 Bq.m(-3) and 15 kBq.m(-3). Measured sensitivities of the participants agree well inside the range of specified calibration uncertainties.

  9. In vitro oxidation of fibrinogen promotes functional alterations and formation of advanced oxidation protein products, an inflammation mediator.

    PubMed

    Torbitz, Vanessa Dorneles; Bochi, Guilherme Vargas; de Carvalho, José Antônio Mainardi; de Almeida Vaucher, Rodrigo; da Silva, José Edson Paz; Moresco, Rafael Noal

    2015-01-01

    Fibrinogen (FB) is a soluble blood plasma protein and is a key molecule involved in coagulation. Oxidative modification of proteins, such as the formation of advanced oxidation protein products (AOPP), a heterogeneous family of protein compounds structurally modified and derived from oxidative stress, may be associated with the pathophysiology of a number of chronic inflammatory diseases. Therefore, the aim of this study was to determine whether the formation of this mediator of inflammation occurs from FB and whether its generation is associated with structural changes. Results of the present study suggest that the oxidation of FB may provoke the formation of AOPP, which in turn, may promote functional alterations in FB, thus causing changes in its structural domains and increasing its procoagulant activity.

  10. Azelnidipine, Not Amlodipine, Induces Secretion of Vascular Endothelial Growth Factor From Smooth Muscle Cells and Promotes Endothelial Tube Formation

    PubMed Central

    Kawamura, Akira; Miura, Shin-ichiro; Matsuo, Yoshino; Tanigawa, Hiroyuki; Saku, Keijiro

    2014-01-01

    Background We previously reported that the calcium channel blocker (CCB) nifedipine-induced secretion of vascular endothelial growth factor (VEGF) from human coronary smooth muscle cells (HCSMCs) promoted human coronary endothelial cell (HCEC) tube formation. Therefore, we analyzed whether other CCBs, azelnidipine and amlodipine, also induced the secretion of VEGF and promoted HCEC tube formation, and the underlying molecular mechanisms. Methods To evaluate the tube formation, HCECs were grown on Matrigel for 18 hours in the supernatants from HCSMCs that had been treated with different kinds of reagents. Concentrations of VEGF in cultured HCSMCs were determined by specific enzyme immunoassays. Nuclear extracts from HCSMCs were prepared, and nuclear factor-kappa B (NF-κB) activation was measured by EZ-DetectTM Transcription Factor Kits for NF-κB p50 or p65. Results Although azelnidipine dose-dependently stimulated the significant secretion of VEGF from HCSMCs and this stimulation was abolished by a protein kinase C inhibitor, amlodipine-induced secretion of VEGF was significantly lower than that induced by azelnidipine. The medium derived from azelnidipine (at up to 2 μM)-treated HCSMCs led to HCEC tube formation, whereas that obtained with amlodipine did not. Azelnidipine-induced tube formation was blocked by an inhibitor of kinase insert domain-containing receptor/fetal liver kinase-1 tyrosine kinase. Azelnidipine at up to 2 μM induced NF-κB activation. Conclusions Azelnidipine, but not amlodipine, stimulated the secretion of VEGF from HCSMCs and induced HCEC tube formation. This secretion is mediated at least in part via the activation of NF-κB. Azelnidipine may have a novel beneficial effect in improving coronary microvascular blood flow in addition to its main effect of lowering blood pressure.

  11. DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.

    PubMed

    Marilley, M; Radebaugh, C A; Geiss, G K; Laybourn, P J; Paule, M R

    2002-08-01

    Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.

  12. Surfactant process for promoting gas hydrate formation and application of the same

    DOEpatents

    Rogers, Rudy E.; Zhong, Yu

    2002-01-01

    This invention relates to a method of storing gas using gas hydrates comprising forming gas hydrates in the presence of a water-surfactant solution that comprises water and surfactant. The addition of minor amounts of surfactant increases the gas hydrate formation rate, increases packing density of the solid hydrate mass and simplifies the formation-storage-decomposition process of gas hydrates. The minor amounts of surfactant also enhance the potential of gas hydrates for industrial storage applications.

  13. The "Balance Intervention" for Promoting Caloric Compensatory Behaviours in Response to Overeating: A Formative Evaluation

    ERIC Educational Resources Information Center

    Wammes, Birgitte; Breedveld, Boudewijn; Kremers, Stef; Brug, Johannes

    2006-01-01

    To help people prevent weight gain, the Netherlands Nutrition Centre initiated the "balance intervention", which promotes moderation of food intake and/or increased physical activity in response to occasions of overeating. The aim of this study was to determine whether intervention materials were appreciated, encouraged information…

  14. A Vygotskian Approach to Promote and Formatively Assess Academic Concept Learning

    ERIC Educational Resources Information Center

    Edens, Kellah; Shields, Carol

    2015-01-01

    Promoting students' conceptual understanding and academic language development is a primary goal of instructors in all subject areas. These goals, however, are sometimes hindered by the way students' learning is assessed. In many college-level courses, knowledge-level tests that assess concrete thinking associated with superficial approaches to…

  15. A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis

    PubMed Central

    Abraham, Sabu; Scarcia, Margherita; Bagshaw, Richard D.; McMahon, Kathryn; Grant, Gary; Harvey, Tracey; Yeo, Maggie; Esteves, Filomena O.G.; Thygesen, Helene H.; Jones, Pamela F.; Speirs, Valerie; Hanby, Andrew M.; Selby, Peter J.; Lorger, Mihaela; Dear, T. Neil; Pawson, Tony; Marshall, Christopher J.; Mavria, Georgia

    2015-01-01

    During angiogenesis, Rho-GTPases influence endothelial cell migration and cell–cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell–cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis. PMID:26129894

  16. Ubiquilin/Dsk2 promotes inclusion body formation and vacuole (lysosome)-mediated disposal of mutated huntingtin.

    PubMed

    Chuang, Kun-Han; Liang, Fengshan; Higgins, Ryan; Wang, Yanchang

    2016-07-01

    Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.

  17. A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis.

    PubMed

    Abraham, Sabu; Scarcia, Margherita; Bagshaw, Richard D; McMahon, Kathryn; Grant, Gary; Harvey, Tracey; Yeo, Maggie; Esteves, Filomena O G; Thygesen, Helene H; Jones, Pamela F; Speirs, Valerie; Hanby, Andrew M; Selby, Peter J; Lorger, Mihaela; Dear, T Neil; Pawson, Tony; Marshall, Christopher J; Mavria, Georgia

    2015-07-01

    During angiogenesis, Rho-GTPases influence endothelial cell migration and cell-cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell-cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis.

  18. The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation.

    PubMed

    van Rijssel, Jos; Kroon, Jeffrey; Hoogenboezem, Mark; van Alphen, Floris P J; de Jong, Renske J; Kostadinova, Elena; Geerts, Dirk; Hordijk, Peter L; van Buul, Jaap D

    2012-08-01

    Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.

  19. The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA.

    PubMed Central

    Giraldo, R; Rhodes, D

    1994-01-01

    The protein RAP1 is essential for the maintenance of the telomeres of Saccharomyces cerevisiae and binds in vitro to multiple sites found within the TG1-3 telomeric repeats. We show here that, in addition to its known binding activity for double-stranded DNA, RAP1 binds sequence-specifically to the GT-strands. This indicates that RAP1 is the protein that binds to the telomeric terminal GT-tails. Furthermore, we have found that RAP1 binds to and promotes the formation of G-tetrads, i.e. DNA quadruplexes, in GT-strand oligonucleotides at nanomolar concentrations. The formation of DNA quadruplexes appears to involve the intermolecular association of GT-strands. The minimal DNA-binding domain of RAP1 (DBD) binds only to double-stranded DNA, so that the novel DNA-binding activity we have found involves regions of the protein located outside of the DBD. The finding that a telomeric protein promotes the formation of G-tetrads argues for the use of DNA quadruplexes in telomere association. Images PMID:8194531

  20. Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation

    PubMed Central

    Onaka, Atsushi T.; Toyofuku, Naoko; Inoue, Takahiro; Okita, Akiko K.; Sagawa, Minami; Su, Jie; Shitanda, Takeshi; Matsuyama, Rei; Zafar, Faria; Takahashi, Tatsuro S.; Masukata, Hisao; Nakagawa, Takuro

    2016-01-01

    Centromeres consist of DNA repeats in many eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres. PMID:27697832

  1. Molecular interactions position Mso1p, a novel PTB domain homologue, in the interface of the exocyst complex and the exocytic SNARE machinery in yeast.

    PubMed

    Knop, Michael; Miller, K Juha; Mazza, Massimiliano; Feng, DeJiang; Weber, Marion; Keränen, Sirkka; Jäntti, Jussi

    2005-10-01

    In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p-Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins. These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery.

  2. Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin

    PubMed Central

    Wu, Chao; Cheng, Yuan-Yuan; Yin, Hao; Song, Xiang-Ning; Li, Wen-Wei; Zhou, Xian-Xuan; Zhao, Li-Ping; Tian, Li-Jiao; Han, Jun-Cheng; Yu, Han-Qing

    2013-01-01

    Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32. PMID:23736081

  3. Sam37 is crucial for formation of the mitochondrial TOM–SAM supercomplex, thereby promoting β-barrel biogenesis

    PubMed Central

    Wenz, Lena-Sophie; Ellenrieder, Lars; Qiu, Jian; Bohnert, Maria; Zufall, Nicole; van der Laan, Martin; Becker, Thomas

    2015-01-01

    Biogenesis of mitochondrial β-barrel proteins requires two preprotein translocases, the general translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). TOM and SAM form a supercomplex that promotes transfer of β-barrel precursors. The SAM core complex contains the channel protein Sam50, which cooperates with Sam35 in precursor recognition, and the peripheral membrane protein Sam37. The molecular function of Sam37 has been unknown. We report that Sam37 is crucial for formation of the TOM–SAM supercomplex. Sam37 interacts with the receptor domain of Tom22 on the cytosolic side of the mitochondrial outer membrane and links TOM and SAM complexes. Sam37 thus promotes efficient transfer of β-barrel precursors to the SAM complex. We conclude that Sam37 functions as a coupling factor of the translocase supercomplex of the mitochondrial outer membrane. PMID:26416958

  4. The C-Terminal of Nucleolin Promotes the Formation of the c-MYC G-Quadruplex and Inhibits c-MYC Promoter Activity†

    PubMed Central

    González, Verónica; Hurley, Laurence H.

    2010-01-01

    Nucleolin, the most abundant nucleolar phosphoprotein of eukaryotic cells, is known primarily for its role in ribosome biogenesis and cell proliferation. It is, however, a multifunctional protein that, depending on the cellular context, can drive either cell proliferation or apoptosis. Our laboratory recently demonstrated that nucleolin can function as a repressor of c-MYC transcription by binding to and stabilizing the formation of a G-quadruplex structure in a region of the c-MYC promoter responsible for controlling 85–90% of c-MYC’s transcriptional activity. In this study, we investigate the structural elements of nucleolin that are required for c-MYC repression. The effect of nucleolin deletion mutants on the formation and stability of the c-MYC G-quadruplex, as well as c-MYC transcriptional activity, was assessed by circular dichroic spectropolarimetry (CD),1 thermal stability, and in vitro transcription. Here we report that nucleolin’s RNA binding domains (RBDs) 3 and 4, as well as the arginine-glycine-glycine (RGG) domain, are required to repress c-MYC transcription. PMID:20932061

  5. Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules

    PubMed Central

    Brunello, Cecilia A.; Yan, Xu; Huttunen, Henri J.

    2016-01-01

    Stress granules are membrane-less RNA- and RNA-binding protein-containing complexes that are transiently assembled in stressful conditions to promote cell survival. Several stress granule-associated RNA-binding proteins have been associated with neurodegenerative diseases. In addition, a close link was recently identified between the stress granule core-nucleating protein TIA-1 and Tau. Tau is a central pathological protein in Alzheimer’s disease and other tauopathies, and misfolded, aggregated Tau is capable of propagating pathology via cell-to-cell transmission. Here we show that following internalization hyperphosphorylated extracellular Tau associates with stress granules in a TIA-1 dependent manner. Cytosolic Tau normally only weakly interacts with TIA-1 but mutations mimicking abnormal phosphorylation promote this interaction. We show that internalized Tau significantly delays normal clearance of stress granules in the recipient cells sensitizing them to secondary stress. These results suggest that secreted Tau species may have properties, likely related to its hyperphosphorylation and oligomerization, which promote pathological association of internalized Tau with stress granules altering their dynamics and reducing cell viability. We suggest that stress granules and TIA-1 play a central role in the cell-to-cell transmission of Tau pathology. PMID:27460788

  6. Induction of mutation and differentiation in mammalian cells by chemicals which initiate or promote tumor formation

    SciTech Connect

    Huberman, E.

    1980-01-01

    A cell-mediated mutagenesis assay was developed to predict the potential carcinogenic hazard of some environmental chemicals. In this assay, cells with appropriate markers for mutagenesis, such as the Chinese hamster V79 cells, are co-cultivated with cells capable of metabolizing chemical carcinogens. Use of this assay made it possible to demonstrate a relationship between the degree of carcinogenicity and mutagenicity of a series of polycyclic hydrocarbons, nitrosamines and aflatoxins, and to establish means to study organ specificity of some chemical carcinogens. However, most short term in vitro assays are designed to detect mutagenic activity and therefore do not detect tumor promoting agents which are devoid of this activity. By analyzing various markers of terminal differentiation in cultured human melanoma and myeloid leukemia cells, we have established a relationship between the activity of a series of tumor promoting phorbol diesters in the mouse skin and their ability to induce terminal differentiation. We suggest that measuring alterations in the differentiation characteristics of some cultured cells may represent an approach by which environmental tumor promoting agents can be studied and detected.

  7. Control of mammalian cell mutagenesis and differentiation by chemicals which initiate or promote tumor formation

    SciTech Connect

    Jones, C. A.; Huberman, E.

    1980-01-01

    A cell-mediated mutagenesis assay was developed to predict the potential carcinogenic hazard of some environmental chemicals. In this assay, Chinese hamster V79 cells, which are susceptible to mutagenesis, are co-cultivated with cells capable of metabolizing chemical carcinogens. Use of this assay made it possible to demonstrate a relationship between the degree of carcinogenicity and mutagenicity of a series of polycyclic hydrocarbons and nitrosamines and to study the organ specificity exhibited by some chemical carcinogens. However, most short-term in vitro assays are designed to detect mutagenic activity and therefore do not detect tumor promoting agents which are devoid of this activity. By analyzing various markers of terminal differentiation in cultured human melanoma and myeloid leukemia cells, we have established a relationship between the activity of a series of tumor promoting phorbol diesters in the mouse skin and their ability to induce terminal differentiation. We suggest that measuring alterations in the differentiation characteristics of some cultured cells may represent an approach by which environmental tumor promoting agents can be studied and detected.

  8. An Undergraduate Study of Two Transcription Factors that Promote Lateral Root Formation

    ERIC Educational Resources Information Center

    Bargmann, Bastiaan O. R.; Birnbaum, Kenneth D.; Brenner, Eric D.

    2014-01-01

    We present a lab that enables students to test the role of genes involved in the regulation of lateral roots growth in the model plant "Arabidopsis thaliana." Here, students design an experiment that follows the effects of the hormone auxin on the stimulation of genes involved in the formation of lateral root initials. These genes, known…

  9. Using Common Formative Assessments to Promote Student Achievement: A Case Study of Practice, Leadership, and Culture

    ERIC Educational Resources Information Center

    Wall, Patricia T. C.

    2012-01-01

    It is the moral responsibility of educators to work diligently to provide every student with rich, challenging coursework in efforts to prepare them for post high school careers and education. The use of common formative assessments provides teachers with the valuable, timely information they need to make instructional decisions that will better…

  10. Embedded Formative Assessment and Classroom Process Quality: How Do They Interact in Promoting Science Understanding?

    ERIC Educational Resources Information Center

    Decristan, Jasmin; Klieme, Eckhard; Kunter, Mareike; Hochweber, Jan; Büttner, Gerhard; Fauth, Benjamin; Hondrich, A. Lena; Rieser, Svenja; Hertel, Silke; Hardy, Ilonca

    2015-01-01

    In this study we examine the interplay between curriculum-embedded formative assessment--a well-known teaching practice--and general features of classroom process quality (i.e., cognitive activation, supportive climate, classroom management) and their combined effect on elementary school students' understanding of the scientific concepts of…

  11. Acetoacetate promotes the formation of fluorescent advanced glycation end products (AGEs).

    PubMed

    Bohlooli, Mousa; Ghaffari-Moghaddam, Mansour; Khajeh, Mostafa; Aghashiri, Zohre; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

    2016-12-01

    Acetoacetate (AA) is an important ketone body, which produces reactive oxygen species (ROS). Advanced glycation end products (AGEs) are defined as final products of glycation process whose production is influenced by the levels of ROS. The accumulation of AGEs in the body contributes to pathogenesis of many diseases including complications of diabetes, and Alzheimer's and Parkinson's disease. Here, we evaluated the impact of AA on production of AGEs upon incubation of human serum albumin (HSA) with glucose. The effect of AA on the AGEs formation of HSA was studied under physiological conditions after incubation with glucose for 35 days. The physical techniques including circular dichroism (CD) and fluorescence spectroscopy were used to assess the impact of AA on formation and structural changes of glycated HSA (GHSA). Our results indicated that the secondary and tertiary structural changes of GHSA were increased in the presence of AA. The fluorescence intensity measurements of AGEs also showed an increase in AGEs formation. Acetoacetate has an activator effect in formation of AGEs through ROS production. The presence of AA may result in enhanced glycation in the presence of glucose and severity of complications associated with accumulation of AGEs.

  12. A Needs Assessment, Development, and Formative Evaluation of a Health Promotion Smartphone Application for College Students

    ERIC Educational Resources Information Center

    Miller, Tiffany; Chandler, Laura; Mouttapa, Michele

    2015-01-01

    Background: Approximately half of college students who completed the National College Health Assessment 2013 indicated a greater need for health-related information. University-based smartphone applications may help students better access this information. Purpose: This study describes the needs assessment, development, and formative evaluation of…

  13. Promoting Prospective Elementary Teachers' Learning to Use Formative Assessment for Life Science Instruction

    NASA Astrophysics Data System (ADS)

    Sabel, Jaime L.; Forbes, Cory T.; Zangori, Laura

    2015-06-01

    To support elementary students' learning of core, standards-based life science concepts highlighted in the Next Generation Science Standards, prospective elementary teachers should develop an understanding of life science concepts and learn to apply their content knowledge in instructional practice to craft elementary science learning environments grounded in students' thinking. To do so, teachers must learn to use high-leverage instructional practices, such as formative assessment, to engage students in scientific practices and connect instruction to students' ideas. However, teachers may not understand formative assessment or possess sufficient science content knowledge to effectively engage in related instructional practices. To address these needs, we developed and conducted research within an innovative course for preservice elementary teachers built upon two pillars—life science concepts and formative assessment. An embedded mixed methods study was used to evaluate the effect of the intervention on preservice teachers' (n = 49) content knowledge and ability to engage in formative assessment practices for science. Findings showed that increased life content knowledge over the semester helped preservice teachers engage more productively in anticipating and evaluating students' ideas, but not in identifying effective instructional strategies to respond to those ideas.

  14. Identification of Genes That Promote or Inhibit Olfactory Memory Formation in Drosophila

    PubMed Central

    Walkinshaw, Erica; Gai, Yunchao; Farkas, Caitlin; Richter, Daniel; Nicholas, Eric; Keleman, Krystyna; Davis, Ronald L.

    2015-01-01

    Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources. PMID:25644700

  15. The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development

    PubMed Central

    Kamiuchi, Yuri; Yamamoto, Kayo; Furutani, Masahiko; Tasaka, Masao; Aida, Mitsuhiro

    2014-01-01

    Carpel margin meristems (CMMs), a pair of meristematic tissues present along the margins of two fused carpel primordia of Arabidopsis thaliana, are essential for the formation of ovules and the septum, two major internal structures of the gynoecium. Although a number of regulatory factors involved in shoot meristem activity are known to be required for the formation of these gynoecial structures, their direct roles in CMM development have yet to be addressed. Here we show that the CUP-SHAPED COTYLEDON genes CUC1 and CUC2, which are essential for shoot meristem initiation, are also required for formation and stable positioning of the CMMs. Early in CMM formation, CUC1 and CUC2 are also required for expression of the SHOOT MERISTEMLESS gene, a central regulator for stem cell maintenance in the shoot meristem. Moreover, plants carrying miR164-resistant forms of CUC1 and CUC2 resulted in extra CMM activity with altered positioning. Our results thus demonstrate that the two regulatory proteins controlling shoot meristem activity also play critical roles in elaboration of the female reproductive organ through the control of meristematic activity. PMID:24817871

  16. Down-regulation of MUC1 in cancer cells inhibits cell migration by promoting E-cadherin/catenin complex formation

    SciTech Connect

    Yuan Zhenglong; Wong, Sandy; Borrelli, Alexander; Chung, Maureen A.

    2007-10-26

    MUC1, a tumor associated glycoprotein, is over-expressed in most cancers and can promote proliferation and metastasis. The objective of this research was to study the role of MUC1 in cancer metastasis and its potential mechanism. Pancreatic (PANC1) and breast (MCF-7) cancer cells with stable 'knockdown' of MUC1 expression were created using RNA interference. {beta}-Catenin and E-cadherin protein expression were upregulated in PANC1 and MCF-7 cells with decreased MUC1 expression. Downregulation of MUC1 expression also induced {beta}-catenin relocation from the nucleus to the cytoplasm, increased E-cadherin/{beta}-catenin complex formation and E-cadherin membrane localization in PANC1 cells. PANC1 cells with 'knockdown' MUC1 expression had decreased in vitro cell invasion. This study suggested that MUC1 may affect cancer cell migration by increasing E-cadherin/{beta}-catenin complex formation and restoring E-cadherin membrane localization.

  17. Down-regulation of MUC1 in cancer cells inhibits cell migration by promoting E-cadherin/catenin complex formation.

    PubMed

    Yuan, Zhenglong; Wong, Sandy; Borrelli, Alexander; Chung, Maureen A

    2007-10-26

    MUC1, a tumor associated glycoprotein, is over-expressed in most cancers and can promote proliferation and metastasis. The objective of this research was to study the role of MUC1 in cancer metastasis and its potential mechanism. Pancreatic (PANC1) and breast (MCF-7) cancer cells with stable 'knockdown' of MUC1 expression were created using RNA interference. beta-Catenin and E-cadherin protein expression were upregulated in PANC1 and MCF-7 cells with decreased MUC1 expression. Downregulation of MUC1 expression also induced beta-catenin relocation from the nucleus to the cytoplasm, increased E-cadherin/beta-catenin complex formation and E-cadherin membrane localization in PANC1 cells. PANC1 cells with 'knockdown' MUC1 expression had decreased in vitro cell invasion. This study suggested that MUC1 may affect cancer cell migration by increasing E-cadherin/beta-catenin complex formation and restoring E-cadherin membrane localization.

  18. Preformed template fluctuations promote fibril formation: Insights from lattice and all-atom models

    SciTech Connect

    Kouza, Maksim Kolinski, Andrzej; Co, Nguyen Truong; Nguyen, Phuong H.; Li, Mai Suan

    2015-04-14

    Fibril formation resulting from protein misfolding and aggregation is a hallmark of several neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Despite the fact that the fibril formation process is very slow and thus poses a significant challenge for theoretical and experimental studies, a number of alternative pictures of molecular mechanisms of amyloid fibril formation have been recently proposed. What seems to be common for the majority of the proposed models is that fibril elongation involves the formation of pre-nucleus seeds prior to the creation of a critical nucleus. Once the size of the pre-nucleus seed reaches the critical nucleus size, its thermal fluctuations are expected to be small and the resulting nucleus provides a template for sequential (one-by-one) accommodation of added monomers. The effect of template fluctuations on fibril formation rates has not been explored either experimentally or theoretically so far. In this paper, we make the first attempt at solving this problem by two sets of simulations. To mimic small template fluctuations, in one set, monomers of the preformed template are kept fixed, while in the other set they are allowed to fluctuate. The kinetics of addition of a new peptide onto the template is explored using all-atom simulations with explicit water and the GROMOS96 43a1 force field and simple lattice models. Our result demonstrates that preformed template fluctuations can modulate protein aggregation rates and pathways. The association of a nascent monomer with the template obeys the kinetics partitioning mechanism where the intermediate state occurs in a fraction of routes to the protofibril. It was shown that template immobility greatly increases the time of incorporating a new peptide into the preformed template compared to the fluctuating template case. This observation has also been confirmed by simulation using lattice models and may be invoked to understand the role of template fluctuations in

  19. Preformed template fluctuations promote fibril formation: insights from lattice and all-atom models.

    PubMed

    Kouza, Maksim; Co, Nguyen Truong; Nguyen, Phuong H; Kolinski, Andrzej; Li, Mai Suan

    2015-04-14

    Fibril formation resulting from protein misfolding and aggregation is a hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Despite the fact that the fibril formation process is very slow and thus poses a significant challenge for theoretical and experimental studies, a number of alternative pictures of molecular mechanisms of amyloid fibril formation have been recently proposed. What seems to be common for the majority of the proposed models is that fibril elongation involves the formation of pre-nucleus seeds prior to the creation of a critical nucleus. Once the size of the pre-nucleus seed reaches the critical nucleus size, its thermal fluctuations are expected to be small and the resulting nucleus provides a template for sequential (one-by-one) accommodation of added monomers. The effect of template fluctuations on fibril formation rates has not been explored either experimentally or theoretically so far. In this paper, we make the first attempt at solving this problem by two sets of simulations. To mimic small template fluctuations, in one set, monomers of the preformed template are kept fixed, while in the other set they are allowed to fluctuate. The kinetics of addition of a new peptide onto the template is explored using all-atom simulations with explicit water and the GROMOS96 43a1 force field and simple lattice models. Our result demonstrates that preformed template fluctuations can modulate protein aggregation rates and pathways. The association of a nascent monomer with the template obeys the kinetics partitioning mechanism where the intermediate state occurs in a fraction of routes to the protofibril. It was shown that template immobility greatly increases the time of incorporating a new peptide into the preformed template compared to the fluctuating template case. This observation has also been confirmed by simulation using lattice models and may be invoked to understand the role of template fluctuations in

  20. Biofuel-Promoted Polychlorinated Dibenzodioxin/furan Formation in an Iron-Catalyzed Diesel Particle Filter.

    PubMed

    Heeb, Norbert V; Rey, Maria Dolores; Zennegg, Markus; Haag, Regula; Wichser, Adrian; Schmid, Peter; Seiler, Cornelia; Honegger, Peter; Zeyer, Kerstin; Mohn, Joachim; Bürki, Samuel; Zimmerli, Yan; Czerwinski, Jan; Mayer, Andreas

    2015-08-04

    Iron-catalyzed diesel particle filters (DPFs) are widely used for particle abatement. Active catalyst particles, so-called fuel-borne catalysts (FBCs), are formed in situ, in the engine, when combusting precursors, which were premixed with the fuel. The obtained iron oxide particles catalyze soot oxidation in filters. Iron-catalyzed DPFs are considered as safe with respect to their potential to form polychlorinated dibenzodioxins/furans (PCDD/Fs). We reported that a bimetallic potassium/iron FBC supported an intense PCDD/F formation in a DPF. Here, we discuss the impact of fatty acid methyl ester (FAME) biofuel on PCDD/F emissions. The iron-catalyzed DPF indeed supported a PCDD/F formation with biofuel but remained inactive with petroleum-derived diesel fuel. PCDD/F emissions (I-TEQ) increased 23-fold when comparing biofuel and diesel data. Emissions of 2,3,7,8-TCDD, the most toxic congener [toxicity equivalence factor (TEF) = 1.0], increased 90-fold, and those of 2,3,7,8-TCDF (TEF = 0.1) increased 170-fold. Congener patterns also changed, indicating a preferential formation of tetra- and penta-chlorodibenzofurans. Thus, an inactive iron-catalyzed DPF becomes active, supporting a PCDD/F formation, when operated with biofuel containing impurities of potassium. Alkali metals are inherent constituents of biofuels. According to the current European Union (EU) legislation, levels of 5 μg/g are accepted. We conclude that risks for a secondary PCDD/F formation in iron-catalyzed DPFs increase when combusting potassium-containing biofuels.

  1. HGF expression induced by HIF-1α promote the proliferation and tube formation of endothelial progenitor cells.

    PubMed

    Yu, Feng; Lin, Yuhua; Zhan, Tao; Chen, Lili; Guo, Sufen

    2015-03-01

    Hypoxia-inducible factor-1α (HIF-1α) and hepatocyte growth factor (HGF) play important roles in postnatal neovascularization. However, the interaction of these two pathways is not fully understood. The present study utilized CoCl(2) treated-endothelial progenitor cells (EPCs) (EPCs exposure to CoCl(2) are under mimic hypoxia) to examine the expressions of HIF-1α and HGF and futher to assess whether or not the inhibitor (2-methoxyestradiol [2ME2]) of HIF-1α decrease the HGF expression. In addition, to investigate the effects of HGF on the proliferation and tube formation of EPCs under mimic hypoxia, EPCs were transfected with NK4 (HGF antagonist) plasmid and exposed to CoCl(2), then the proliferation of these EPCs was assayed by MTS and the tube formation capacity of these EPCs on Matrigel was detected. The analysis indicated that CoCl(2) treatment induced HIF-1α expression of EPCs, and futher promoted HGF expression. While after 2ME2 was used in CoCl(2) treated-EPCs, HGF expression was markedly inhibited compared with non-pretreated EPCs with 2ME2, which also showed that HGF expression in EPCs was mediated by HIF-1α. Further, the results showed that after EPCs were transfected with NK4 in spite of being exposed to CoCl(2), their proliferation activity and tube formation capacity were weakened, which in turn indicated that HGF could promote the proliferation and the tube formation of EPCs, and this process might be regulated by HIF-1α.

  2. Crmp4 deletion promotes recovery from spinal cord injury by neuroprotection and limited scar formation

    PubMed Central

    Nagai, Jun; Kitamura, Yoshiteru; Owada, Kazuki; Yamashita, Naoya; Takei, Kohtaro; Goshima, Yoshio; Ohshima, Toshio

    2015-01-01

    Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4−/− mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI. PMID:25652774

  3. Reactive oxidation products promote secondary organic aerosol formation from green leaf volatiles

    NASA Astrophysics Data System (ADS)

    Hamilton, J. F.; Lewis, A. C.; Carey, T. J.; Wenger, J. C.; Garcia, E. Borrás. I.; Muñoz, A.

    2009-02-01

    Green leaf volatiles (GLVs) are an important group of chemicals released by vegetation which have emission fluxes that can be significantly increased when plants are damaged or stressed. A series of simulation chamber experiments has been conducted at the European Photoreactor in Valencia, Spain, to investigate secondary organic aerosol (SOA) formation from the atmospheric oxidation of the major GLVs cis-3-hexenylacetate and cis-3-hexen-1-ol. Liquid chromatography-ion trap mass spectrometry was used to identify chemical species present in the SOA. Cis-3-hexen-1-ol proved to be a more efficient SOA precursor due to the high reactivity of its first generation oxidation product, 3-hydroxypropanal, which can hydrate and undergo further reactions with other aldehydes resulting in SOA dominated by higher molecular weight oligomers. The lower SOA yields produced from cis-3-hexenylacetate are attributed to the acetate functionality, which inhibits oligomer formation in the particle phase. Based on observed SOA yields and best estimates of global emissions, these compounds may be calculated to be a substantial unidentified global source of SOA, contributing 1-5 TgC yr-1, equivalent to around a third of that predicted from isoprene. Molecular characterization of the SOA, combined with organic mechanistic information, has provided evidence that the formation of organic aerosols from GLVs is closely related to the reactivity of their first generation atmospheric oxidation products, and indicates that this may be a simple parameter that could be used in assessing the aerosol formation potential for other unstudied organic compounds in the atmosphere.

  4. Reactive oxidation products promote secondary organic aerosol formation from green leaf volatiles

    NASA Astrophysics Data System (ADS)

    Hamilton, J. F.; Lewis, A. C.; Carey, T. J.; Wenger, J. C.; Garcia, E. Borrás. I.; Muñoz, A.

    2009-06-01

    Green leaf volatiles (GLVs) are an important group of chemicals released by vegetation which have emission fluxes that can be significantly increased when plants are damaged or stressed. A series of simulation chamber experiments has been conducted at the European Photoreactor in Valencia, Spain, to investigate secondary organic aerosol (SOA) formation from the atmospheric oxidation of the major GLVs cis-3-hexenylacetate and cis-3-hexen-1-ol. Liquid chromatography-ion trap mass spectrometry was used to identify chemical species present in the SOA. Cis-3-hexen-1-ol proved to be a more efficient SOA precursor due to the high reactivity of its first generation oxidation product, 3-hydroxypropanal, which can hydrate and undergo further reactions with other aldehydes resulting in SOA dominated by higher molecular weight oligomers. The lower SOA yields produced from cis-3-hexenylacetate are attributed to the acetate functionality, which inhibits oligomer formation in the particle phase. Based on observed SOA yields and best estimates of global emissions, these compounds may be calculated to be a substantial unidentified global source of SOA, contributing 1-5 TgC yr-1, equivalent to around a third of that predicted from isoprene. Molecular characterization of the SOA, combined with organic mechanistic information, has provided evidence that the formation of organic aerosols from GLVs is closely related to the reactivity of their first generation atmospheric oxidation products, and indicates that this may be a simple parameter that could be used in assessing the aerosol formation potential for other unstudied organic compounds in the atmosphere.

  5. Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization

    PubMed Central

    Blanchette, Krystle A.; Shenoy, Anukul T.; Milner, Jeffrey; Gilley, Ryan P.; McClure, Erin; Hinojosa, Cecilia A.; Kumar, Nikhil; Daugherty, Sean C.; Tallon, Luke J.; Ott, Sandra; King, Samantha J.; Ferreira, Daniela M.; Gordon, Stephen B.; Tettelin, Hervé

    2016-01-01

    Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro. Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo. Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role. PMID:27481242

  6. Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

    SciTech Connect

    Lafleur, Michel; Courtemanche, Lesley; Karlsson, Goeran; Edwards, Katarina; Schwartz, Jean-Louis; Manjunath, Puttaswamy

    2010-08-27

    Research highlights: {yields} Binder-of-sperm protein 1 (BSP1) modifies the morphology of lipidic vesicles inducing bead necklace-like and thread-like structures. {yields} In the presence of multilamellar liposomes, BSP1 leads to the formation of long nanotubes. {yields} The insertion of BSP1 in the external lipid leaflet of membranes induces local changes in bilayer curvature. -- Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.

  7. Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts.

    PubMed

    Petry, Sabine; Pugieux, Céline; Nédélec, François J; Vale, Ronald D

    2011-08-30

    Female meiotic spindles in many organisms form in the absence of centrosomes, the organelle typically associated with microtubule (MT) nucleation. Previous studies have proposed that these meiotic spindles arise from RanGTP-mediated MT nucleation in the vicinity of chromatin; however, whether this process is sufficient for spindle formation is unknown. Here, we investigated whether a recently proposed spindle-based MT nucleation pathway that involves augmin, an 8-subunit protein complex, also contributes to spindle morphogenesis. We used an assay system in which hundreds of meiotic spindles can be observed forming around chromatin-coated beads after introduction of Xenopus egg extracts. Spindles forming in augmin-depleted extracts showed reduced rates of MT formation and were predominantly multipolar, revealing a function of augmin in stabilizing the bipolar shape of the acentrosomal meiotic spindle. Our studies also have uncovered an apparent augmin-independent MT nucleation process from acentrosomal poles, which becomes increasingly active over time and appears to partially rescue the spindle defects that arise from augmin depletion. Our studies reveal that spatially and temporally distinct MT generation pathways from chromatin, spindle MTs, and acentrosomal poles all contribute to robust bipolar spindle formation in meiotic extracts.

  8. Translation suppression promotes stress granule formation and cell survival in response to cold shock

    PubMed Central

    Hofmann, Sarah; Cherkasova, Valeria; Bankhead, Peter; Bukau, Bernd; Stoecklin, Georg

    2012-01-01

    Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia. PMID:22875991

  9. Hydroxyapatite bioactivated bacterial cellulose promotes osteoblast growth and the formation of bone nodules

    PubMed Central

    2012-01-01

    The goal of this study was to investigate the feasibility of bacterial cellulose (BC) scaffold to support osteoblast growth and bone formation. BC was produced by culturing Acetobacter xylinum supplemented with hydroxyapatite (HA) to form BC membranes (without HA) and BC/HA membranes. Membranes were subjected to X-ray photoelectron spectroscopy (XPS) analysis to determine surface element composition. The membranes were further used to evaluate osteoblast growth, alkaline phosphatase activity and bone nodule formation. BC was free of calcium and phosphate. However, XPS analysis revealed the presence of both calcium (10%) and phosphate (10%) at the surface of the BC/HA membrane. Osteoblast culture showed that BC alone was non-toxic and could sustain osteoblast adhesion. Furthermore, osteoblast adhesion and growth were significantly (p ≤0.05) increased on BC/HA membranes as compared to BC alone. Both BC and BC/HA membranes improved osteoconductivity, as confirmed by the level of alkaline phosphatase (ALP) activity that increased from 2.5 mM with BC alone to 5.3 mM with BC/HA. BC/HA membranes also showed greater nodule formation and mineralization than the BC membrane alone. This was confirmed by Alizarin red staining (ARS) and energy dispersive X-ray spectroscopy (EDX). This work demonstrates that both BC and BC/HA may be useful in bone tissue engineering. PMID:23174338

  10. Abluminal stimulation of sphingosine 1-phosphate receptors 1 and 3 promotes and stabilizes endothelial sprout formation.

    PubMed

    Das, Anusuya; Lenz, Steven M; Awojoodu, Anthony O; Botchwey, Edward A

    2015-01-01

    Local delivery of lipid mediators has become a promising new approach for therapeutic angiogenesis and regenerative medicine. In this study, we investigated how gradient stimulation (either abluminal/distal or luminal/proximal) of engineered microvessels with sphingosine 1-phosphate (S1P) receptor-subtype-targeted molecules affects endothelial sprout growth using a microfluidic device. Our studies show that distal stimulation of microvessels with FTY720, an S1P1/3 selective agonist, promotes both arterial and venular sprout growth, whereas proximal stimulation does not. Using novel pharmacological antagonists of S1P receptor subtypes, we further show that S1P3 functionality is necessary for VEGF-induced sprouting, and confirmed these findings ex vivo using a murine aortic ring assay from S1P3-deficient mice. S1P3 agonist stimulation enhanced vascular stability in both cell types via upregulation of the interendothelial junction protein VE-cadherin. Lastly, S1P3 activation under flow promoted endothelial sprouting and branching while decreasing migratory cell fate in the microfluidic device. We used an in vivo murine dorsal skinfold window chamber model to confirm S1P3's role in neovascular branching. Together, these data suggest that a distal transendothelial gradient of S1P1/3-targeted drugs is an effective technique for both enhancing and stabilizing capillary morphogenesis in angiogenic applications.

  11. Institute of social justice and medicine: developing a think tank to promote policy formation.

    PubMed

    Boozary, Andrew; Dugani, Sagar B

    2011-10-01

    The World Health Organization (WHO) defines health as a "resource for everyday living, not the objective of living"; however, worldwide, there remains an unmistakable inequity in level of health and access to healthcare. The WHO has published documents on financing health systems towards universal health coverage [1], promoting healthy life [2], improving performance of health systems [3], and enriching humanity [4], highlighting our shared responsibility towards improving both national and global health and access to healthcare. These documents also recognize that, despite our local and regional priorities, there is a global desire to develop international strategies to improve healthcare. [1] WHO Report. Health systems financing and the path to universal coverage. 2010. http://www.who.int/bulletin/health_financing/en/index.html [2] WHO Report. Reducing risks, promoting healthy life. 2002. http://www.who.int/whr/2002/en/index.html [3] WHO Bulletin. Health systems: improving performance. 2000. http://www.who.int/whr/2000/en/index.html [4] WHO Bulletin. Conquering suffering, enriching humanity 1997. http://www.who.int/whr/1997/en/index.html.

  12. Vinca alkaloid drugs promote stress-induced translational repression and stress granule formation

    PubMed Central

    Szaflarski, Witold; Fay, Marta M.; Kedersha, Nancy; Zabel, Maciej; Anderson, Paul; Ivanov, Pavel

    2016-01-01

    Resistance to chemotherapy drugs is a serious therapeutic problem and its underlying molecular mechanisms are complex. Stress granules (SGs), cytoplasmic ribonucleoprotein complexes assembled in cells exposed to stress, are implicated in various aspects of cancer cell metabolism and survival. SGs promote the survival of stressed cells by reprogramming gene expression and inhibiting pro-apoptotic signaling cascades. We show that the vinca alkaloid (VA) class of anti-neoplastic agents potently activates a SG-mediated stress response program. VAs inhibit translation initiation by simultaneous activation of eIF4E-BP1 and phosphorylation of eIF2α, causing polysome disassembly and SG assembly. VA-induced SGs contain canonical SG components but lack specific signaling molecules. Blocking VA-induced SG assembly by inactivating eIF4EBP1 or inhibiting eIF2α phosphorylation decreases cancer cell viability and promotes apoptosis. Our data describe previously unappreciated effects of VAs on cellular RNA metabolism and illuminate the roles of SGs in cancer cell survival. PMID:27083003

  13. Gibberellins Promote Trichome Formation by Up-Regulating GLABROUS1 in Arabidopsis1

    PubMed Central

    Perazza, Daniel; Vachon, Gilles; Herzog, Michel

    1998-01-01

    Trichome development is dependent on gibberellin (GA) signaling in Arabidopsis thaliana. Using the GA-deficient mutant ga1–3, the GA-response mutant spy-5, and uniconazol (a GA-biosynthesis inhibitor), we show that the GA level response correlates positively with both trichome number and trichome branch number. Two genes, GL1 and TTG, are required for trichome initiation. In ga1–3, coexpression of GL1 and R, the maize TTG functional homolog, under control of the constitutive 35S promoter, restored trichome development, whereas overexpression of neither GL1 nor R alone was sufficient to significantly suppress the glabrous phenotype. We next focused on GL1 regulation by GAs. In the double mutant the gl1–1 glabrous phenotype is epistatic to the spy-5 phenotype, suggesting that GL1 acts downstream of the GA signal transduction pathway. The activity of a β-glucuronidase reporter gene driven by the GL1 promoter was decreased in the wild type grown on uniconazol and showed a clear GA-dependent activation in ga1–3. Finally, quantification of GL1 transcript levels by reverse transcriptase-polymerase chain reaction demonstrated that relative to wild type, ga1–3 plants contained less transcript. These data support the hypothesis that GAs induce trichome development through up-regulation of GL1 and possibly TTG genes. PMID:9625690

  14. Abluminal Stimulation of Sphingosine 1-Phosphate Receptors 1 and 3 Promotes and Stabilizes Endothelial Sprout Formation

    PubMed Central

    Lenz, Steven M.; Awojoodu, Anthony O.

    2015-01-01

    Local delivery of lipid mediators has become a promising new approach for therapeutic angiogenesis and regenerative medicine. In this study, we investigated how gradient stimulation (either abluminal/distal or luminal/proximal) of engineered microvessels with sphingosine 1-phosphate (S1P) receptor-subtype-targeted molecules affects endothelial sprout growth using a microfluidic device. Our studies show that distal stimulation of microvessels with FTY720, an S1P1/3 selective agonist, promotes both arterial and venular sprout growth, whereas proximal stimulation does not. Using novel pharmacological antagonists of S1P receptor subtypes, we further show that S1P3 functionality is necessary for VEGF-induced sprouting, and confirmed these findings ex vivo using a murine aortic ring assay from S1P3-deficient mice. S1P3 agonist stimulation enhanced vascular stability in both cell types via upregulation of the interendothelial junction protein VE-cadherin. Lastly, S1P3 activation under flow promoted endothelial sprouting and branching while decreasing migratory cell fate in the microfluidic device. We used an in vivo murine dorsal skinfold window chamber model to confirm S1P3's role in neovascular branching. Together, these data suggest that a distal transendothelial gradient of S1P1/3-targeted drugs is an effective technique for both enhancing and stabilizing capillary morphogenesis in angiogenic applications. PMID:25315888

  15. Protonation switching to the least-basic heteroatom of carbamate through cationic hydrogen bonding promotes the formation of isocyanate cations.

    PubMed

    Kurouchi, Hiroaki; Sumita, Akinari; Otani, Yuko; Ohwada, Tomohiko

    2014-07-07

    We found that phenethylcarbamates that bear ortho-salicylate as an ether group (carbamoyl salicylates) dramatically accelerate OC bond dissociation in strong acid to facilitate generation of isocyanate cation (N-protonated isocyanates), which undergo subsequent intramolecular aromatic electrophilic cyclization to give dihydroisoquinolones. To generate isocyanate cations from carbamates in acidic media as electrophiles for aromatic substitution, protonation at the ether oxygen, the least basic heteroatom, is essential to promote CO bond cleavage. However, the carbonyl oxygen of carbamates, the most basic site, is protonated exclusively in strong acids. We found that the protonation site can be shifted to an alternative basic atom by linking methyl salicylate to the ether oxygen of carbamate. The methyl ester oxygen ortho to the phenolic (ether) oxygen of salicylate is as basic as the carbamate carbonyl oxygen, and we found that monoprotonation at the methyl ester oxygen in strong acid resulted in the formation of an intramolecular cationic hydrogen bond (>CO(+) H⋅⋅⋅O<) with the phenolic ether oxygen. This facilitates OC bond dissociation of phenethylcarbamates, thereby promoting isocyanate cation formation. In contrast, superacid-mediated diprotonation at the methyl ester oxygen of the salicylate and the carbonyl oxygen of the carbamate afforded a rather stable dication, which did not readily undergo CO bond dissociation. This is an unprecedented and unknown case in which the monocation has greater reactivity than the dication.

  16. LGR5 promotes the proliferation and tumor formation of cervical cancer cells through the Wnt/β-catenin signaling pathway

    PubMed Central

    Chen, Qing; Cao, Hao-Zhe; Zheng, Peng-Sheng

    2014-01-01

    Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a seven transmembrane receptor known as a potential stem cell marker for intestinal crypts and hair follicles, has recently been found to be overexpressed in some types of human cancers. However, the role of LGR5 in cervical cancer remains unclear. In this study, the expression of LGR5 gradually increases from normal cervix to cervical cancer in situ and to cervical cancers as revealed by immunohistochemistry and western blot analyses. Through knocking down or overexpressing LGR5 in SiHa and HeLa cells, the expression level of LGR5 was found to be positively related to cell proliferation in vitro and to tumor formation in vivo. Further investigation indicated that LGR5 protein could significantly promote the acceleration of cell cycle. Moreover, the TOP-Flash reporter assay and western blot for β-catenin, cyclinD1, and c-myc proteins, target genes of the Wnt/β-catenin pathway, indicated that LGR5 significantly activated Wnt/β-catenin signaling. Additionally, the blockage of Wnt/β-catenin pathway resulted in a significant inhibition of cell proliferation induced by LGR5. Taken together, these results demonstrate that LGR5 can promote proliferation and tumor formation in cervical cancer cells by activating the Wnt/β-catenin pathway. PMID:25193857

  17. Appalachian mountaintop mining particulate matter induces neoplastic transformation of human bronchial epithelial cells and promotes tumor formation.

    PubMed

    Luanpitpong, Sudjit; Chen, Michael; Knuckles, Travis; Wen, Sijin; Luo, Juhua; Ellis, Emily; Hendryx, Michael; Rojanasakul, Yon

    2014-11-04

    Epidemiological studies suggest that living near mountaintop coal mining (MTM) activities is one of the contributing factors for high lung cancer incidence. The purpose of this study was to investigate the long-term carcinogenic potential of MTM particulate matter (PMMTM) exposure on human bronchial epithelial cells. Our results show that chronic exposure (3 months) to noncytotoxic, physiological relevant concentration (1 μg/mL) of PMMTM, but not control particle PMCON, induced neoplastic transformation, accelerated cell proliferation, and enhanced cell migration of the exposed lung cells. Xenograft transplantation of the PMMTM-exposed cells in mice caused no apparent tumor formation, but promoted tumor growth of human lung carcinoma H460 cells, suggesting the tumor-promoting effect of PMMTM. Chronic exposure to the main inorganic chemical constituent of PMMTM, molybdenum but not silica, similarly induced cell transformation and tumor promotion, suggesting the contribution of molybdenum, at least in part, in the PMMTM effects. These results provide new evidence for the carcinogenic potential of PMMTM and support further risk assessment and implementation of exposure control for PMMTM.

  18. Cbln family proteins promote synapse formation by regulating distinct neurexin signaling pathways in various brain regions.

    PubMed

    Matsuda, Keiko; Yuzaki, Michisuke

    2011-04-01

    Cbln1 (a.k.a. precerebellin) is a unique bidirectional synaptic organizer that plays an essential role in the formation and maintenance of excitatory synapses between granule cells and Purkinje cells in the mouse cerebellum. Cbln1 secreted from cerebellar granule cells directly induces presynaptic differentiation and indirectly serves as a postsynaptic organizer by binding to its receptor, the δ2 glutamate receptor. However, it remains unclear how Cbln1 binds to the presynaptic sites and interacts with other synaptic organizers. Furthermore, although Cbln1 and its family members Cbln2 and Cbln4 are expressed in brain regions other than the cerebellum, it is unknown whether they regulate synapse formation in these brain regions. In this study, we showed that Cbln1 and Cbln2, but not Cbln4, specifically bound to its presynaptic receptor -α and β isoforms of neurexin carrying the splice site 4 insert [NRXs(S4+)] - and induced synaptogenesis in cerebellar, hippocampal and cortical neurons in vitro. Cbln1 competed with synaptogenesis mediated by neuroligin 1, which lacks the splice sites A and B, but not leucine-rich repeat transmembrane protein 2, possibly by sharing the presynaptic receptor NRXs(S4+). However, unlike neurexins/neuroligins or neurexins/leucine-rich repeat transmembrane proteins, the interaction between NRX1β(S4+) and Cbln1 was insensitive to extracellular Ca(2+) concentrations. These findings revealed the unique and general roles of Cbln family proteins in mediating the formation and maintenance of synapses not only in the cerebellum but also in various other brain regions.

  19. Condensin II Promotes the Formation of Chromosome Territories by Inducing Axial Compaction of Polyploid Interphase Chromosomes

    PubMed Central

    Bauer, Christopher R.; Hartl, Tom A.; Bosco, Giovanni

    2012-01-01

    The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate commonalities in the mechanisms that regulate the spatial structure of the genome during mitosis and interphase. PMID:22956908

  20. Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate*

    PubMed Central

    Platen, Mitja; Junius, Meike; Diederichsen, Ulf; Schaap, Iwan A. T.; Honigmann, Alf; Jahn, Reinhard; van den Bogaart, Geert

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P2 and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca2+ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2 complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca2+ concentrations is likely to be important for Ca2+-regulated secretion. PMID:26884341

  1. Curcumin promotes fibril formation in F isomer of human serum albumin via amorphous aggregation.

    PubMed

    Mothi, Nivin; Muthu, Shivani A; Kale, Avinash; Ahmad, Basir

    2015-12-01

    We here describe the amyloid fibrils promoting behavior of curcumin, which ability to inhibit amyloid fibrillization of several globular proteins is well documented. Transmission electron microscopy (TEM), 90° light scattering (RLS), thioflavine T (ThT) and Congo red (CR) binding studies demonstrated that both F (pH3.4) and E (pH1.8) isomers of human serum albumin (HSA) in the absence and presence of curcumin initially converted into amorphous aggregates. Interestingly, only the sample containing F isomer preincubated with curcumin formed fibrils on incubation for longer period. We also found that curcumin strongly bind to the F isomer, alter its secondary, tertiary structures and thermal stability. We conclude that the conversion of intermediate states into amorphous aggregate to fibrils is dictated by its conformation. This study provides unique insights into ligand-controlled HSA aggregation pathway and should provide a useful model system to study both amorphous and the fibrillar aggregation of multidomain proteins.

  2. Natural products for treatment of osteoporosis: The effects and mechanisms on promoting osteoblast-mediated bone formation.

    PubMed

    An, Jing; Yang, Hao; Zhang, Qian; Liu, Cuicui; Zhao, Jingjing; Zhang, Lingling; Chen, Bo

    2016-02-15

    Osteoporosis is a systemic metabolic bone disease characterized by a reduction in bone mass, bone quality, and microarchitectural deterioration. An imbalance in bone remodeling that is caused by more osteoclast-mediated bone resorption than osteoblast-mediated bone formation results in such pathologic bone disorder. Traditional Chinese medicines (TCM) have long been used to prevent and treat osteoporosis and have received extensive attentions and researches at home and abroad, because they have fewer adverse reactions and are more suitable for long-term use compared with chemically synthesized medicines. Here, we put the emphasis on osteoblasts, summarized the detailed research progress on the active compounds derived from TCM with potential anti-osteoporosis effects and their molecular mechanisms on promoting osteoblast-mediated bone formation. It could be concluded that TCM with kidney-tonifying, spleen-tonifying, and stasis-removing effects all have the potential effects on treating osteoporosis. The active ingredients derived from TCM that possess effects on promoting osteoblasts proliferation and differentiation include flavonoids, glycosides, coumarins, terpenoids (sesquiterpenoids, monoterpenoids, diterpenoids), phenolic acids, phenols and others (tetrameric stilbene, anthraquinones, diarylheptanoids). And it was confirmed that the bone formation effect induced by the above natural products was regulated by the expressions of bone specific matrix proteins (ALP, BSP, OCN, OPN, COL I), transcription factor (Runx2, Cbfa1, Osx), signal pathways (MAPK, BMP), local factors (ROS, NO), OPG/RANKL system of osteoblasts and estrogen-like biological activities. All the studies provided theoretical basis for clinical application, as well as new drug research and development on treating osteoporosis.

  3. Vaccination with DKK1-derived peptides promotes bone formation and bone mass in an aged mouse osteoporosis model.

    PubMed

    Wu, Qiong; Li, Rui-Shu; Zhao, Yue; Wang, Zhi-Xia; Tang, Yan-Chun; Zhang, Jing; Liu, Jian-Ning; Tan, Xiang-Yang

    2014-08-01

    The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22 weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.

  4. Telomeres and centromeres have interchangeable roles in promoting meiotic spindle formation

    PubMed Central

    Fennell, Alex; Fernández-Álvarez, Alfonso; Tomita, Kazunori

    2015-01-01

    Telomeres and centromeres have traditionally been considered to perform distinct roles. During meiotic prophase, in a conserved chromosomal configuration called the bouquet, telomeres gather to the nuclear membrane (NM), often near centrosomes. We found previously that upon disruption of the fission yeast bouquet, centrosomes failed to insert into the NM at meiosis I and nucleate bipolar spindles. Hence, the trans-NM association of telomeres with centrosomes during prophase is crucial for efficient spindle formation. Nonetheless, in approximately half of bouquet-deficient meiocytes, spindles form properly. Here, we show that bouquet-deficient cells can successfully undergo meiosis using centromere–centrosome contact instead of telomere–centrosome contact to generate spindle formation. Accordingly, forced association between centromeres and centrosomes fully rescued the spindle defects incurred by bouquet disruption. Telomeres and centromeres both stimulate focal accumulation of the SUN domain protein Sad1 beneath the centrosome, suggesting a molecular underpinning for their shared spindle-generating ability. Our observations demonstrate an unanticipated level of interchangeability between the two most prominent chromosomal landmarks. PMID:25688135

  5. The extracellular matrix component laminin promotes gap junction formation in the rat anterior pituitary gland.

    PubMed

    Horiguchi, Kotaro; Kouki, Tom; Fujiwara, Ken; Kikuchi, Motoshi; Yashiro, Takashi

    2011-03-01

    Folliculo-stellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. FS cells connect to each other not only by mechanical means, but also by gap junctional cell-to-cell communication. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture markedly change their shape, and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. Morphological and functional changes in cells are believed to be partly modified by matricrine signaling, by which ECM components function as cellular signals. In the present study, we examined whether gap junction formation between FS cells is affected by matricrine cues. A cell sorter was used to isolate FS cells from male S100b-GFP rat anterior pituitary for primary culture. We observed that mRNA and protein levels of connexin 43 in gap junction channels were clearly higher in the presence of laminin. In addition, we confirmed the formation of gap junctions between FS cells in primary culture by electron microscopy. Interestingly, we also observed that FS cells in the presence of laminin displayed well-developed rough endoplasmic reticulum and Golgi apparatus. Our findings suggest that, in anterior pituitary gland, FS cells may facilitate functional roles such as gap junctional cell-to-cell communication by matricrine signaling.

  6. Multiple endothelial cells constitute the tip of developing blood vessels and polarize to promote lumen formation.

    PubMed

    Pelton, John C; Wright, Catherine E; Leitges, Michael; Bautch, Victoria L

    2014-11-01

    Blood vessel polarization in the apical-basal axis is important for directed secretion of proteins and lumen formation; yet, when and how polarization occurs in the context of angiogenic sprouting is not well understood. Here, we describe a novel topology for endothelial cells at the tip of angiogenic sprouts in several mammalian vascular beds. Two cells that extend filopodia and have significant overlap in space and time were present at vessel tips, both in vitro and in vivo. The cell overlap is more extensive than predicted for tip cell switching, and it sets up a longitudinal cell-cell border that is a site of apical polarization and lumen formation, presumably via a cord-hollowing mechanism. The extent of cell overlap at the tip is reduced in mice lacking aPKCζ, and this is accompanied by reduced distal extension of both the apical border and patent lumens. Thus, at least two polarized cells occupy the distal tip of blood vessel sprouts, and topology, polarization and lumenization along the longitudinal border of these cells are influenced by aPKCζ.

  7. Multiple endothelial cells constitute the tip of developing blood vessels and polarize to promote lumen formation

    PubMed Central

    Pelton, John C.; Wright, Catherine E.; Leitges, Michael; Bautch, Victoria L.

    2014-01-01

    Blood vessel polarization in the apical-basal axis is important for directed secretion of proteins and lumen formation; yet, when and how polarization occurs in the context of angiogenic sprouting is not well understood. Here, we describe a novel topology for endothelial cells at the tip of angiogenic sprouts in several mammalian vascular beds. Two cells that extend filopodia and have significant overlap in space and time were present at vessel tips, both in vitro and in vivo. The cell overlap is more extensive than predicted for tip cell switching, and it sets up a longitudinal cell-cell border that is a site of apical polarization and lumen formation, presumably via a cord-hollowing mechanism. The extent of cell overlap at the tip is reduced in mice lacking aPKCζ, and this is accompanied by reduced distal extension of both the apical border and patent lumens. Thus, at least two polarized cells occupy the distal tip of blood vessel sprouts, and topology, polarization and lumenization along the longitudinal border of these cells are influenced by aPKCζ. PMID:25336741

  8. INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

    PubMed Central

    Andrés-Delgado, Laura; Antón, Olga M.; Bartolini, Francesca; Ruiz-Sáenz, Ana; Correas, Isabel; Gundersen, Gregg G.

    2012-01-01

    T cell antigen receptor–proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor–induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells. PMID:22986496

  9. Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis.

    PubMed

    Luo, Yongting; Duan, Hongxia; Qian, Yining; Feng, Liqun; Wu, Zhenzhen; Wang, Fei; Feng, Jing; Yang, Dongling; Qin, Zhihai; Yan, Xiyun

    2017-03-01

    The persistence of cholesterol-engorged macrophages (foam cells) in the artery wall fuels the development of atherosclerosis. However, the mechanism that regulates the formation of macrophage foam cells and impedes their emigration out of inflamed plaques is still elusive. Here, we report that adhesion receptor CD146 controls the formation of macrophage foam cells and their retention within the plaque during atherosclerosis exacerbation. CD146 is expressed on the macrophages in human and mouse atheroma and can be upregulated by oxidized low-density lipoprotein (oxLDL). CD146 triggers macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake. In response to oxLDL, macrophages show reduced migratory capacity toward chemokines CCL19 and CCL21; this capacity can be restored by blocking CD146. Genetic deletion of macrophagic CD146 or targeting of CD146 with an antibody result in much less complex plaques in high-fat diet-fed ApoE(-/-) mice by causing lipid-loaded macrophages to leave plaques. Collectively, our findings identify CD146 as a novel retention signal that traps macrophages within the artery wall, and a promising therapeutic target in atherosclerosis treatment.

  10. Loss of pRB and p107 disrupts cartilage development and promotes enchondroma formation

    PubMed Central

    Landman, Allison S.; Danielian, Paul S.; Lees, Jacqueline A.

    2013-01-01

    The pocket proteins pRB, p107, and p130 have established roles in regulating the cell cycle through the control of E2F activity. The pocket proteins regulate differentiation of a number of tissues in both cell cycle-dependent and –independent manners. Prior studies showed that mutation of p107 and p130 in the mouse leads to defects in cartilage development during endochondral ossification, the process by which long bones form. Despite evidence of a role for pRB in osteoblast differentiation, it is unknown whether it functions during cartilage development. Here, we show that mutation of Rb in the early mesenchyme of p107-mutant mice results in severe cartilage defects in the growth plates of long bones. This is attributable to inappropriate chondrocyte proliferation that persists after birth and leads to the formation of enchondromas in the growth plates as early as 8 weeks of age. Genetic crosses show that development of these tumorigenic lesions is E2f3 dependent. These results reveal an overlapping role for pRB and p107 in cartilage development, endochondral ossification and enchondroma formation that reflects their coordination of cell cycle exit at appropriate developmental stages. PMID:23146901

  11. Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis

    PubMed Central

    Luo, Yongting; Duan, Hongxia; Qian, Yining; Feng, Liqun; Wu, Zhenzhen; Wang, Fei; Feng, Jing; Yang, Dongling; Qin, Zhihai; Yan, Xiyun

    2017-01-01

    The persistence of cholesterol-engorged macrophages (foam cells) in the artery wall fuels the development of atherosclerosis. However, the mechanism that regulates the formation of macrophage foam cells and impedes their emigration out of inflamed plaques is still elusive. Here, we report that adhesion receptor CD146 controls the formation of macrophage foam cells and their retention within the plaque during atherosclerosis exacerbation. CD146 is expressed on the macrophages in human and mouse atheroma and can be upregulated by oxidized low-density lipoprotein (oxLDL). CD146 triggers macrophage activation by driving the internalization of scavenger receptor CD36 during lipid uptake. In response to oxLDL, macrophages show reduced migratory capacity toward chemokines CCL19 and CCL21; this capacity can be restored by blocking CD146. Genetic deletion of macrophagic CD146 or targeting of CD146 with an antibody result in much less complex plaques in high-fat diet-fed ApoE−/− mice by causing lipid-loaded macrophages to leave plaques. Collectively, our findings identify CD146 as a novel retention signal that traps macrophages within the artery wall, and a promising therapeutic target in atherosclerosis treatment. PMID:28084332

  12. Syndapin promotes pseudocleavage furrow formation by actin organization in the syncytial Drosophila embryo

    PubMed Central

    Sherlekar, Aparna; Rikhy, Richa

    2016-01-01

    Coordinated membrane and cytoskeletal remodeling activities are required for membrane extension in processes such as cytokinesis and syncytial nuclear division cycles in Drosophila. Pseudocleavage furrow membranes in the syncytial Drosophila blastoderm embryo show rapid extension and retraction regulated by actin-remodeling proteins. The F-BAR domain protein Syndapin (Synd) is involved in membrane tubulation, endocytosis, and, uniquely, in F-actin stability. Here we report a role for Synd in actin-regulated pseudocleavage furrow formation. Synd localized to these furrows, and its loss resulted in short, disorganized furrows. Synd presence was important for the recruitment of the septin Peanut and distribution of Diaphanous and F-actin at furrows. Synd and Peanut were both absent in furrow-initiation mutants of RhoGEF2 and Diaphanous and in furrow-progression mutants of Anillin. Synd overexpression in rhogef2 mutants reversed its furrow-extension phenotypes, Peanut and Diaphanous recruitment, and F-actin organization. We conclude that Synd plays an important role in pseudocleavage furrow extension, and this role is also likely to be crucial in cleavage furrow formation during cell division. PMID:27146115

  13. Cholesterol gallstones and bile host diverse bacterial communities with potential to promote the formation of gallstones.

    PubMed

    Peng, Yuhong; Yang, Yang; Liu, Yongkang; Nie, Yuanyang; Xu, Peilun; Xia, Baixue; Tian, Fuzhou; Sun, Qun

    2015-01-01

    The prevalence of cholesterol gallstones has increased in recent years. Bacterial infection correlates with the formation of gallstones. We studied the composition and function of bacterial communities in cholesterol gallstones and bile from 22 cholesterol gallstone patients using culture-dependent and culture-independent methods. Altogether fourteen and eight bacterial genera were detected in cholesterol gallstones and bile, respectively. Pseudomonas spp. were the dominant bacteria in both cholesterol gallstones and bile. As judged by diversity indices, hierarchical clustering and principal component analysis, the bacterial communities in gallstones were different from those in bile. The gallstone microbiome was considered more stable than that of bile. The different microbial communities may be partially explained by differences in their habitats. We found that 30% of the culturable strains from cholesterol gallstones secreted β-glucuronidase and phospholipase A2. Pseudomonas aeruginosa strains showed the highest β-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones.

  14. microRNAs That Promote or Inhibit Memory Formation in Drosophila melanogaster

    PubMed Central

    Busto, Germain U.; Guven-Ozkan, Tugba; Fulga, Tudor A.; Van Vactor, David; Davis, Ronald L.

    2015-01-01

    microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Prior studies have shown that they regulate numerous physiological processes critical for normal development, cellular growth control, and organismal behavior. Here, we systematically surveyed 134 different miRNAs for roles in olfactory learning and memory formation using “sponge” technology to titrate their activity broadly in the Drosophila melanogaster central nervous system. We identified at least five different miRNAs involved in memory formation or retention from this large screen, including miR-9c, miR-31a, miR-305, miR-974, and miR-980. Surprisingly, the titration of some miRNAs increased memory, while the titration of others decreased memory. We performed more detailed experiments on two miRNAs, miR-974 and miR-31a, by mapping their roles to subpopulations of brain neurons and testing the functional involvement in memory of potential mRNA targets through bioinformatics and a RNA interference knockdown approach. This screen offers an important first step toward the comprehensive identification of all miRNAs and their potential targets that serve in gene regulatory networks important for normal learning and memory. PMID:26088433

  15. Reduction of the uncertainty of the PTB vacuum pressure scale by a new large area non-rotating piston gauge

    NASA Astrophysics Data System (ADS)

    Bock, Th; Ahrendt, H.; Jousten, K.

    2009-10-01

    This paper describes the metrological characterization of a new large area piston gauge (FRS5, Furness Rosenberg Standard) installed at the vacuum metrology laboratory of the Physikalisch-Technische Bundesanstalt (PTB). The operational procedure and the uncertainty budget for pressures between 30 Pa and 11 kPa are given. Comparisons between the FRS5 and a mercury manometer, a rotary piston gauge and a force-balanced piston gauge are described. We show that the reproducibility of the calibration values of capacitance diaphragm gauges is enhanced by a factor of 6 compared with a static expansion primary standard (SE2). Improvements of the SE2 performance by reducing the number of expansions and smaller uncertainties of expansion ratios are discussed.

  16. UV-induced Oxygen Removal for Photostable High Efficiency PTB7-Th: PC71BM Photovoltaic Cells.

    PubMed

    Liu, Quan; Mantilla-Perez, Paola; Montes Bajo, Miguel; Romero-Gomez, Pablo; Martorell, Jordi

    2016-10-03

    Solution-processed ZnO sol-gel or nanoparticles are widely used as the electron transporting layer (ETL) in optoelectronic devices. However, chemisorbed oxygen on the ZnO layer surface has been shown to be detrimental for the device performance as well as stability. Herein, we demonstrate that a chemisorbed oxygen removal based on a UV illumination of the ZnO surface layer under a nitrogen atmosphere can, simultaneously, improve power conversion efficiency and photostability of PTB7-Th: PC71BM based inverted polymer solar cells. By a systematic study of such UV illumination procedure, we obtained optimal conditions where, both, the cell efficiency and stability were improved. We fabricated cells with a power conversion efficiency higher than 9.8%, and with a T80 lifetime larger than 500 hours, corresponding to about a 2.5-fold enhancement relative to non-UV treated ZnO reference devices.

  17. Activity standardization of 131I at CENTIS-DMR and PTB within the scope of a bilateral comparison.

    PubMed

    Oropesa Verdecia, P; Kossert, K

    2009-06-01

    The activity of an (131)I solution was measured at the Cuban Institute, CENTIS-DMR, as well as at the German National Metrology Institute, PTB, within the scope of a bilateral comparison. In particular, the comparison is aimed at an investigation of the measurement capabilities of CENTIS-DMR which provides activity standards in Cuba and organizes national comparisons, placing a particular emphasis on radionuclides for nuclear medicine, such as (131)I. Both institutes applied liquid scintillation counting techniques with efficiency tracing as well as secondary standardization procedures by means of calibrated ionization chambers and gamma-ray spectrometers. The results were checked for consistency and a good agreement was found. Moreover, a virtual link of the Cuban result to the International Reference System (SIR) at the Bureau International des Poids et Mesures (BIPM) is discussed.

  18. Does radiative feedback by the first stars promote or prevent second generation star formation?

    NASA Astrophysics Data System (ADS)

    Ahn, Kyungjin; Shapiro, Paul R.

    2007-03-01

    We study the effect of starlight from the first stars on the ability of other minihaloes in their neighbourhood to form additional stars. The first stars in the Λ cold dark matter (ΛCDM) universe are believed to have formed in minihaloes of total mass ~105-6 Msolar at redshifts z >~ 20, when molecular hydrogen (H2) formed and cooled the dense gas at their centres, leading to gravitational collapse. Simulations suggest that the Population III (Pop III) stars thus formed were massive (~100 Msolar) and luminous enough in ionizing radiation to cause an ionization front (I-front) to sweep outward, through their host minihalo and beyond, into the intergalactic medium. Our previous work suggested that this I-front was trapped when it encountered other, nearby minihaloes, and that it failed to penetrate the dense gas at their centres within the lifetime of the Pop III stars (<~3 Myr). The question of what the dynamical consequences were for these target minihaloes, of their exposure to the ionizing and dissociating starlight from the Pop III star requires further study, however. Towards this end, we have performed a series of detailed, one-dimensional (1D), radiation-hydrodynamical simulations to answer the question of whether star formation in these surrounding minihaloes was triggered or suppressed by radiation from the first stars. We have varied the distance to the source (and, hence, the flux) and the mass and evolutionary stage of the target haloes to quantify this effect. We find (1) trapping of the I-front and its transformation from R-type to D-type, preceded by a shock front; (2) photoevaporation of the ionized gas (i.e. all gas originally located outside the trapping radius); (3) formation of an H2 precursor shell which leads the I-front, stimulated by partial photoionization; and (4) the shock-induced formation of H2 in the minihalo neutral core when the shock speeds up and partially ionizes the gas. The fate of the neutral core is mostly determined by the

  19. The hot-spot p53R172H mutant promotes formation of giant spermatogonia triggered by DNA damage.

    PubMed

    Xue, Y; Raharja, A; Sim, W; Wong, E S M; Rahmat, S A B; Lane, D P

    2017-04-06

    Overexpression of mutant p53 is a common finding in most cancers but testicular tumours accumulate wild-type p53 (wtp53). In contrast to the accepted concept that p53 homozygous mutant mice do not accumulate mutant p53 in normal cells, our study on a mutant p53 mouse model of Li-Fraumeni syndrome harbouring the hot-spot p53R172H mutation described an elevated level of mutant p53 in non-cancerous mouse tissues. Here we use detailed immunohistochemical analysis to document the expression of p53R172H in mouse testis. In developing and adult testes, p53R172H was expressed in gonocytes, type A, Int, B spermatogonia as well as in pre-Sertoli cells and Leydig cells but was undetectable in spermatocytes and spermatids. A similar staining pattern was demonstrated for wtp53. However, the intensity of wtp53 staining was generally weaker than that of p53R172H, which indicates that the expression of p53R172H can be a surrogate marker of p53 gene transcription. Comparing the responses of wtp53 and p53R172H to irradiation, we found persistent DNA double-strand breaks in p53R172H testes and the formation of giant spermatogonia (GSG) following persistent DNA damage in p53R172H and p53-null mice. Strikingly, we found that p53R172H promotes spontaneous formation of GSG in non-stressed p53R172H ageing mice. Two types of GSG: Viable and Degenerative GSG were defined. We elucidate the factors involved in the formation of GSG: the loss of p53 function is a requirement for the formation of GSG whereas DNA damage acts as a promoting trigger. The formation of GSG does not translate to higher efficacy of testicular tumorigenesis arising from mutant p53 cells, which might be due to the presence of delayed-onset of p53-independent apoptosis.

  20. An Exploration of Teachers' Narratives: What Are the Facilitators and Constraints Which Promote or Inhibit "Good" Formative Assessment Practices in Schools?

    ERIC Educational Resources Information Center

    Sach, Elizabeth

    2015-01-01

    This paper set out to explore teachers' narratives in order to understand some of the facilitators and constraints which promote or inhibit good formative assessment practices in schools. A "responsive interview" approach was used to probe a small sample of lower and middle school teachers' perceptions of formative assessment.…

  1. Mg(2+)/Ca(2+) promotes the adhesion of marine bacteria and algae and enhances following biofilm formation in artificial seawater.

    PubMed

    He, Xiaoyan; Wang, Jinpeng; Abdoli, Leila; Li, Hua

    2016-10-01

    Adhesion of microorganisms in the marine environment is essential for initiation and following development of biofouling. A variety of factors play roles in regulating the adhesion. Here we report the influence of Ca(2+) and Mg(2+) in artificial seawater on attachment and colonization of Bacillus sp., Chlorella and Phaeodactylum tricornutum on silicon wafer. Extra addition of the typical divalent cations in culturing solution gives rise to significantly enhanced adhesion of the microorganisms. Mg(2+) and Ca(2+) affect the adhesion of Bacillus sp. presumably by regulating aggregation and formation of extracellular polymeric substances (EPS). The ions alter quantity and types of the proteins in EPS, in turn affecting subsequent adhesion. However, it is noted that Mg(2+) promotes adhesion of Chlorella likely by regulating EPS formation and polysaccharide synthesis. Ca(2+) plays an important role in protein expression to enhance the adhesion of Chlorella. For Phaeodactylum tricornutum, Ca(2+) expedites protein synthesis for enhanced adhesion. The results shed some light on effective ways of utilizing divalent cations to mediate formation of biofilms on the marine structures for desired performances.

  2. The matrix protein CCN1 (CYR61) promotes proliferation, migration and tube formation of endothelial progenitor cells.

    PubMed

    Yu, Yang; Gao, Yu; Wang, Hong; Huang, Lan; Qin, Jun; Guo, Ruiwei; Song, Mingbao; Yu, Shiyong; Chen, Jianfei; Cui, Bin; Gao, Pan

    2008-10-15

    Neovascularization and re-endothelialization relies on circulating endothelial progenitor cells (EPCs), but their recruitment and angiogenic roles are subjected to regulation by the vascular microenvironment, which remains largely unknown. The present study was designed to investigate the effects of mature ECs and matrix protein CCN1 on the properties of EPCs. In a coculture system, effects of ECs on proliferation, migration and participation in tube-like formation of EPCs were evaluated, and functional assays were employed to identify the exact role of CCN1 in EPCs vitality and function. We demonstrated that ECs, as an indispensable part of the cellular milieu, significantly promoted the proliferation, migration and tube formation activities of EPCs, and more importantly, CCN1 was potentially involved in such effects of ECs. Expression of CCN1 in EPCs was significantly increased by serum, VEGF, ECs-cocultivation and ECs conditioned medium. Moreover, Ad-CCN1-mediated overexpression of CCN1 directly enhanced migration and tube formation of EPCs, whereas silencing of endogenous CCN1 in EPCs inhibits cell functions. Furthermore, CCN1 induced the expressions of chemokines and growth factors, such as MCP-1 and VEGF, suggesting a complex interaction between those proangiogenic factors. Our data suggest that matrix protein CCN1 may play an important role in microenvironment-mediated biological properties of EPCs.

  3. The matrix protein CCN1 (CYR61) promotes proliferation, migration and tube formation of endothelial progenitor cells

    SciTech Connect

    Yu Yang; Gao Yu; Wang, Hong; Huang Lan Qin Jun; Guo Ruiwei; Song Mingbao; Yu Shiyong; Chen Jianfei; Cui Bin; Gao Pan

    2008-10-15

    Neovascularization and re-endothelialization relies on circulating endothelial progenitor cells (EPCs), but their recruitment and angiogenic roles are subjected to regulation by the vascular microenvironment, which remains largely unknown. The present study was designed to investigate the effects of mature ECs and matrix protein CCN1 on the properties of EPCs. In a coculture system, effects of ECs on proliferation, migration and participation in tube-like formation of EPCs were evaluated, and functional assays were employed to identify the exact role of CCN1 in EPCs vitality and function. We demonstrated that ECs, as an indispensable part of the cellular milieu, significantly promoted the proliferation, migration and tube formation activities of EPCs, and more importantly, CCN1 was potentially involved in such effects of ECs. Expression of CCN1 in EPCs was significantly increased by serum, VEGF, ECs-cocultivation and ECs conditioned medium. Moreover, Ad-CCN1-mediated overexpression of CCN1 directly enhanced migration and tube formation of EPCs, whereas silencing of endogenous CCN1 in EPCs inhibits cell functions. Furthermore, CCN1 induced the expressions of chemokines and growth factors, such as MCP-1 and VEGF, suggesting a complex interaction between those proangiogenic factors. Our data suggest that matrix protein CCN1 may play an important role in microenvironment-mediated biological properties of EPCs.

  4. Wnt3a Promotes the Vasculogenic Mimicry Formation of Colon Cancer via Wnt/β-Catenin Signaling.

    PubMed

    Qi, Lisha; Song, Wangzhao; Liu, Zhiyong; Zhao, Xiulan; Cao, Wenfeng; Sun, Baocun

    2015-08-10

    Our previous study provided evidence that non-canonical Wnt signaling is involved in regulating vasculogenic mimicry (VM) formation. However, the functions of canonical Wnt signaling in VM formation have not yet been explored. In this study, we found the presence of VM was related to colon cancer histological differentiation (p < 0.001), the clinical stage (p < 0.001), and presence of metastasis and recurrence (p < 0.001). VM-positive colon cancer samples showed increased Wnt3a expression (p < 0.001) and β-catenin nuclear expression (p < 0.001) compared with the VM-negative samples. In vitro, over-regulated Wnt3a expression in HT29 colon cancer cells promoted the capacity to form tube-like structures in the three-dimensional (3-D) culture together with increased expression of endothelial phenotype-associated proteins such as VEGFR2 and VE-cadherin. The mouse xenograft model showed that Wnt3a-overexpressing cells grew into larger tumor masses and formed more VM than the control cells. In addition, the Wnt/β-catenin signaling antagonist Dickkopf-1(Dkk1) can reverse the capacity to form tube-like structures and can decrease the expressions of VEGFR2 and VE-cadherin in Wnt3a-overexpressing cells. Taken together, our results suggest that Wnt/β-catenin signaling is involved in VM formation in colon cancer and might contribute to the development of more accurate treatment modalities aimed at VM.

  5. Taxol-stabilized microtubules promote the formation of filaments from unmodified full-length Tau in vitro.

    PubMed

    Duan, Aranda R; Goodson, Holly V

    2012-12-01

    Tau is a neuronal protein that stabilizes the microtubule (MT) network, but it also forms filaments associated with Alzheimer's disease. Understanding Tau-MT and Tau-Tau interactions would help to establish Tau function in health and disease. For many years, literature reports on Tau-MT binding behavior and affinity have remained surprisingly contradictory (e.g., 10-fold variation in Tau-MT affinity). Tau-Tau interactions have also been investigated, but whether MTs might affect Tau filament formation is unknown. We have addressed these issues through binding assays and microscopy. We assessed Tau-MT interactions via cosedimentation and found that the measured affinity of Tau varies greatly, depending on the experimental design and the protein concentrations used. To investigate this dependence, we used fluorescence microscopy to examine Tau-MT binding. Strikingly, we found that Taxol-stabilized MTs promote Tau filament formation without characterized Tau-filament inducers. We propose that these novel Tau filaments account for the incongruence in Tau-MT affinity measurements. Moreover, electron microscopy reveals that these filaments appear similar to the heparin-induced Alzheimer's model. These observations suggest that the MT-induced Tau filaments provide a new model for Alzheimer's studies and that MTs might play a role in the formation of Alzheimer's-associated neurofibrillary tangles.

  6. Amelogenin Promotes the Formation of Elongated Apatite Microstructures in a Controlled Crystallization System

    PubMed Central

    Wang, Lijun; Guan, Xiangying; Du, Chang; Moradian-Oldak, Janet; Nancollas, George H.

    2009-01-01

    The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that play a pivotal role in controlling the oriented and elongated growth of highly ordered apatitic crystals during enamel biomineralization. However, the mechanisms of amelogenin-mediated mineralization have not yet been fully elucidated. Here we report that amelogenin dramatically accelerates the nucleation kinetics by decreasing the induction time in a dose-dependent manner in a controlled constant composition (CC) in vitro crystallization system. Remarkably, at very low protein concentrations, elongated microstructures which are similar in appearance to apatitic crystals in enamel were formed at relatively low supersaturations, through interfacial structural match/synergy between structured amelogenin assemblies and apatite nanocrystallites. This heterogeneous crystallization study provides experimental evidence to support the concept that templating by amelogenin very early in the crystallization process facilitates the formation of developing enamel crystals. PMID:20333260

  7. EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

    PubMed Central

    Béguelin, Wendy; Popovic, Relja; Teater, Matt; Jiang, Yanwen; Bunting, Karen L.; Rosen, Monica; Shen, Hao; Yang, Shao Ning; Wang, Ling; Ezponda, Teresa; Martinez-Garcia, Eva; Zhang, Haikuo; Zhang, Yupeng; Verma, Sharad K.; McCabe, Michael T.; Ott, Heidi M.; Van Aller, Glenn S.; Kruger, Ryan G.; Liu, Yan; McHugh, Charles F.; Scott, David W.; Chung, Young Rock; Kelleher, Neil; Shaknovich, Rita; Creasy, Caretha L.; Gascoyne, Randy D.; Wong, Kwok-Kin; Cerchietti, Leandro C.; Levine, Ross L.; Abdel-Wahab, Omar; Licht, Jonathan D.; Elemento, Olivier; Melnick, Ari M.

    2013-01-01

    The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting. PMID:23680150

  8. Zinc promotes clot stability by accelerating clot formation and modifying fibrin structure.

    PubMed

    Henderson, Sara J; Xia, Jing; Wu, Huayin; Stafford, Alan R; Leslie, Beverly A; Fredenburgh, James C; Weitz, David A; Weitz, Jeffrey I

    2016-03-01

    Zinc released from activated platelets binds fibrin(ogen) and attenuates fibrinolysis. Although zinc also affects clot formation, the mechanism and consequences are poorly understood. To address these gaps, the effect of zinc on clot formation and structure was examined in the absence or presence of factor (F) XIII. Zinc accelerated a) plasma clotting by 1.4-fold, b) fibrinogen clotting by 3.5- and 2.3-fold in the absence or presence of FXIII, respectively, c) fragment X clotting by 1.3-fold, and d) polymerisation of fibrin monomers generated with thrombin or batroxobin by 2.5- and 1.8-fold, respectively. Whereas absorbance increased up to 3.3-fold when fibrinogen was clotted in the presence of zinc, absorbance of fragment X clots was unaffected by zinc, consistent with reports that zinc binds to the αC-domain of fibrin(ogen). Scanning electron microscopic analysis revealed a two-fold increase in fibre diameter in the presence of zinc and in permeability studies, zinc increased clot porosity by 30-fold with or without FXIII. Whereas FXIII increased clot stiffness from 128 ± 19 Pa to 415 ± 27 Pa in rheological analyses, zinc reduced clot stiffness by 10- and 8.5-fold in the absence and presence of FXIII, respectively. Clots formed in the presence of zinc were more stable and resisted rupture with or without FXIII. Therefore, zinc accelerates clotting and reduces fibrin clot stiffness in a FXIII-independent manner, suggesting that zinc may work in concert with FXIII to modulate clot strength and stability.

  9. Inhibition of transforming growth factor β signaling promotes epiblast formation in mouse embryos.

    PubMed

    Ghimire, Sabitri; Heindryckx, Björn; Van der Jeught, Margot; Neupane, Jitesh; O'Leary, Thomas; Lierman, Sylvie; De Vos, Winnok H; Chuva de Sousa Lopes, Susana; Deroo, Tom; De Sutter, Petra

    2015-02-15

    Early lineage segregation in preimplantation embryos and maintenance of pluripotency in embryonic stem cells (ESCs) are both regulated by specific signaling pathways. Small molecules have been shown to modulate these signaling pathways. We examined the influence of several small molecules and growth factors on second-lineage segregation of the inner cell mass toward hypoblast and epiblast lineage during mouse embryonic preimplantation development. We found that the second-lineage segregation is influenced by activation or inhibition of the transforming growth factor (TGF)β pathway. Inhibition of the TGFβ pathway from the two-cell, four-cell, and morula stages onward up to the blastocyst stage significantly increased the epiblast cell proliferation. The epiblast formed in the embryos in which TGFβ signaling was inhibited was fully functional as demonstrated by the potential of these epiblast cells to give rise to pluripotent ESCs. Conversely, activating the TGFβ pathway reduced epiblast formation. Inhibition of the glycogen synthase kinase (GSK)3 pathway and activation of bone morphogenetic protein 4 signaling reduced the formation of both epiblast and hypoblast cells. Activation of the protein kinase A pathway and of the Janus kinase/signal transducer and activator of transcription 3 pathway did not influence the second-lineage segregation in mouse embryos. The simultaneous inhibition of three pathways--TGFβ, GSK3β, and the fibroblast growth factor (FGF)/extracellular signal-regulated kinases (Erk)--significantly enhanced the proliferation of epiblast cells than that caused by inhibition of either TGFβ pathway alone or by combined inhibition of the GSK3β and FGF/Erk pathways only.

  10. The Extract of Fructus Psoraleae Promotes Viability and Cartilaginous Formation of Rat Chondrocytes In Vitro

    PubMed Central

    Xu, Kang; Qiu, Xuefeng; Zhao, Wen; Wang, Dong

    2016-01-01

    This study aimed to investigate the extract components of FP on rat chondrocyte function and cartilaginous formation in vitro. Petroleum ether extract (P-e) of FP extract components was selected to treat Sprague-Dawley rat chondrocytes. Cell viability was tested with different concentrations (0.1, 1, 10, and 100 μg/mL) of P-e treatment. Concentrations of 0.1 and 1 μg/mL P-e conditioned culture mediums were used for treating chondrocytes in experiments. Cell proliferation was measured via DNA incorporation assay. Type II collagen, aggrecan, and Sox-9 genes expression levels were measured with RT-PCR. Additionally, cartilaginous formation was analyzed with type II collagen immunofluorescence, H&E, and alcian blue staining. Concentrations of 0.1 and 1 μg/mL P-e showed low cytotoxicity and demonstrated stimulatory effects on chondrocyte proliferation in early stages. Following 6 days of P-e culture, aggrecan and Sox-9 gene expression levels of the 1 μg/mL P-e group were upregulated by 1.82- (p < 0.05) and 2.06-fold (p < 0.05), respectively, versus controls. Moreover, 1 μg/mL P-e significantly stimulated cell aggregation and type II collagen deposits after 1 week of treatment. Noteworthy, tight cartilaginous structures formed in the 10-day 1 μg/mL P-e conditioned culture. These findings suggest that P-e has the potential to treat cartilage degeneration induced by chondrocyte failure. PMID:27994628

  11. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    SciTech Connect

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-Kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-08-26

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

  12. Promotion of wound collagen formation in normal and diabetic mice by quadrol.

    PubMed

    Bhide, M V; Dunphy, M J; Mirkopulos, N; Smith, D J

    1988-01-01

    The rate of collagen deposition in implanted polytetrafluoroethylene (PTFE) tubing in non-diabetic and streptozotocin-induced (STZ) diabetic mice was measured during 14 days post-wounding. At the time of implantation, test groups received injections of either Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine], glucan, or buffer in an area adjacent to the wound site. The accumulation of collagen in the implants of Quadrol-treated non-diabetic animals was more than 200% above control on days 8 to 11 and was 50% above control on day 14. In Quadrol-treated STZ-diabetic mice, the collagen accumulation gradually increased from 50% above control on day 8 to 200% above control on day 14. Treatment with glucan increased the collagen accumulation in normal mice 200 to 300% above control from days 8 to 11 respectively and then 30% above control on day 14. Collagen accumulation in the implants of the glucan-treated STZ-diabetic mice was similar to the control group. These results indicate that Quadrol promotes in vivo collagen synthesis and that Quadrol may be effective as a stimulator of wound healing in diabetic and non-diabetic animals.

  13. Silicon-Doped Titanium Dioxide Nanotubes Promoted Bone Formation on Titanium Implants.

    PubMed

    Zhao, Xijiang; Wang, Tao; Qian, Shi; Liu, Xuanyong; Sun, Junying; Li, Bin

    2016-02-26

    While titanium (Ti) implants have been extensively used in orthopaedic and dental applications, the intrinsic bioinertness of untreated Ti surface usually results in insufficient osseointegration irrespective of the excellent biocompatibility and mechanical properties of it. In this study, we prepared surface modified Ti substrates in which silicon (Si) was doped into the titanium dioxide (TiO₂) nanotubes on Ti surface using plasma immersion ion implantation (PIII) technology. Compared to TiO₂ nanotubes and Ti alone, Si-doped TiO₂ nanotubes significantly enhanced the expression of genes related to osteogenic differentiation, including Col-I, ALP, Runx2, OCN, and OPN, in mouse pre-osteoblastic MC3T3-E1 cells and deposition of mineral matrix. In vivo, the pull-out mechanical tests after two weeks of implantation in rat femur showed that Si-doped TiO₂ nanotubes improved implant fixation strength by 18% and 54% compared to TiO₂-NT and Ti implants, respectively. Together, findings from this study indicate that Si-doped TiO₂ nanotubes promoted the osteogenic differentiation of osteoblastic cells and improved bone-Ti integration. Therefore, they may have considerable potential for the bioactive surface modification of Ti implants.

  14. Calcium carbonate hybrid coating promotes the formation of biomimetic hydroxyapatite on titanium surfaces

    NASA Astrophysics Data System (ADS)

    Cruz, Marcos Antônio E.; Ruiz, Gilia C. M.; Faria, Amanda N.; Zancanela, Daniela C.; Pereira, Lourivaldo S.; Ciancaglini, Pietro; Ramos, Ana P.

    2016-05-01

    CaCO3 particles dispersed in liquid media have proven to be good inductors of hydroxyapatite (HAp) growth. However, the use of CaCO3 deposited as thin films for this propose is unknown. Here, we report the growth of CaCO3 continuous films on Langmuir-Blodgett (LB) modified titanium surfaces and its use as HAp growth inductor. The Ti surfaces were modified with two, four, and six layers of dihexadecylphosphate (DHP)-LB films containing Ca2+, exposed to CO2 (g) for 12 h. The modified surfaces were immersed in simulated body fluid (SBF) at 37 °C for 36 h and submitted to bioactivity studies. This procedure originates bioactive coatings composed by non-stoichiometric HAp as evidenced by Fourier-Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and X-ray Photoelectron Spectroscopy (XPS). The presence of the CaCO3 film as pre-coating diminished the time necessary to growth continuous and homogeneous HAp films using a biomimetic approach. The surface properties of the films regarding their roughness, composition, charge, wettability, and surface free energy (γs) were accessed. The presence of HAp increased the wettability and γs of the surfaces. The coatings are not toxic for osteoblasts as observed for cell viability assays obtained after 7 and 14 days of culture. Moreover, the CaCO3 thin films promote the recovery of the osteoblasts viability more than the Ti surfaces themselves.

  15. Attenuation of AMPK signaling by ROQUIN promotes T follicular helper cell formation

    PubMed Central

    Ramiscal, Roybel R; Parish, Ian A; Lee-Young, Robert S; Babon, Jeffrey J; Blagih, Julianna; Pratama, Alvin; Martin, Jaime; Hawley, Naomi; Cappello, Jean Y; Nieto, Pablo F; Ellyard, Julia I; Kershaw, Nadia J; Sweet, Rebecca A; Goodnow, Christopher C; Jones, Russell G; Febbraio, Mark A; Vinuesa, Carola G; Athanasopoulos, Vicki

    2015-01-01

    T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN–AMPK metabolic signaling nexus essential for selectively promoting Tfh responses. DOI: http://dx.doi.org/10.7554/eLife.08698.001 PMID:26496200

  16. Silicon-Doped Titanium Dioxide Nanotubes Promoted Bone Formation on Titanium Implants

    PubMed Central

    Zhao, Xijiang; Wang, Tao; Qian, Shi; Liu, Xuanyong; Sun, Junying; Li, Bin

    2016-01-01

    While titanium (Ti) implants have been extensively used in orthopaedic and dental applications, the intrinsic bioinertness of untreated Ti surface usually results in insufficient osseointegration irrespective of the excellent biocompatibility and mechanical properties of it. In this study, we prepared surface modified Ti substrates in which silicon (Si) was doped into the titanium dioxide (TiO2) nanotubes on Ti surface using plasma immersion ion implantation (PIII) technology. Compared to TiO2 nanotubes and Ti alone, Si-doped TiO2 nanotubes significantly enhanced the expression of genes related to osteogenic differentiation, including Col-I, ALP, Runx2, OCN, and OPN, in mouse pre-osteoblastic MC3T3-E1 cells and deposition of mineral matrix. In vivo, the pull-out mechanical tests after two weeks of implantation in rat femur showed that Si-doped TiO2 nanotubes improved implant fixation strength by 18% and 54% compared to TiO2-NT and Ti implants, respectively. Together, findings from this study indicate that Si-doped TiO2 nanotubes promoted the osteogenic differentiation of osteoblastic cells and improved bone-Ti integration. Therefore, they may have considerable potential for the bioactive surface modification of Ti implants. PMID:26927080

  17. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    PubMed Central

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-01-01

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism. PMID:22922757

  18. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    SciTech Connect

    Radzimanowski, Jens; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  19. Mesenchymal Wnt signaling promotes formation of sternum and thoracic body wall.

    PubMed

    Snowball, John; Ambalavanan, Manoj; Cornett, Bridget; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

    2015-05-15

    Midline defects account for approximately 5% of congenital abnormalities observed at birth. However, the molecular mechanisms underlying the formation of the ventral body wall are not well understood. Recent studies linked mutations in Porcupine-an O-acetyl transferase mediating Wnt ligand acylation-with defects in the thoracic body wall. We hypothesized that anomalous Wnt signaling is involved in the pathogenesis of defective closure of the thoracic body wall. We generated a mouse model wherein Wntless (Wls), which encodes a cargo receptor mediating secretion of Wnt ligands, was conditionally deleted from the developing mesenchyme using Dermo1Cre mice. Wls(f/f);Dermo1(Cre/+) embryos died during mid-gestation. At E13.5, skeletal defects were observed in the forelimbs, jaw, and rib cage. At E14.5, midline defects in the thoracic body wall began to emerge: the sternum failed to fuse and the heart protruded through the body wall at the midline (ectopia cordis). To determine the molecular mechanism underlying the phenotype observed in Wls(f/f);Dermo1(Cre/+) embryos, we tested whether Wnt/β-catenin signaling was operative in developing the embryonic ventral body wall using Axin2(LacZ) and BatGal reporter mice. While Wnt/β-catenin signaling activity was observed at the midline of the ventral body wall before sternal fusion, this pattern of activity was altered and scattered throughout the body wall after mesenchymal deletion of Wls. Mesenchymal cell migration was disrupted in Wls(f/f);Dermo1(Cre/+) thoracic body wall partially due to anomalous β-catenin independent Wnt signaling as determined by in vitro assays. Deletion of Lrp5 and Lrp6 receptors, which mediate Wnt/β-catenin signaling in the mesenchyme, partially recapitulated the phenotype observed in the chest midline of Wls(f/f);Dermo1(Cre/+) embryos supporting a role for Wnt/β-catenin signaling activity in the normal formation of the ventral body wall mesenchyme. We conclude that Wls-mediated secretion of Wnt

  20. SOXC Transcription Factors Induce Cartilage Growth Plate Formation in Mouse Embryos by Promoting Noncanonical WNT Signaling.

    PubMed

    Kato, Kenji; Bhattaram, Pallavi; Penzo-Méndez, Alfredo; Gadi, Abhilash; Lefebvre, Véronique

    2015-09-01

    Growth plates are specialized cartilage structures that ensure the elongation of most skeletal primordia during vertebrate development. They are made by chondrocytes that proliferate in longitudinal columns and then progress in a staggered manner towards prehypertrophic, hypertrophic and terminal maturation. Complex molecular networks control the formation and activity of growth plates, but remain incompletely understood. We investigated here the importance of the SoxC genes, which encode the SOX4, SOX11 and SOX12 transcription factors, in growth plates. We show that the three genes are expressed robustly in perichondrocytes and weakly in growth plate chondrocytes. SoxC(Prx1Cre) mice, which deleted SoxC genes in limb bud skeletogenic mesenchyme, were born with tiny appendicular cartilage primordia because of failure to form growth plates. In contrast, SoxC(Col2Cre) and SoxC(ATC) mice, which deleted SoxC genes primarily in chondrocytes, were born with mild dwarfism and fair growth plates. Chondrocytes in the latter mutants matured normally, but formed irregular columns, proliferated slowly and died ectopically. Asymmetric distribution of VANGL2 was defective in both SoxC(Prx1Cre) and SoxC(ATC) chondrocytes, indicating impairment of planar cell polarity, a noncanonical WNT signaling pathway that controls growth plate chondrocyte alignment, proliferation and survival. Accordingly, SoxC genes were necessary in perichondrocytes for expression of Wnt5a, which encodes a noncanonical WNT ligand required for growth plate formation, and in chondrocytes and perichondrocytes for expression of Fzd3 and Csnk1e, which encode a WNT receptor and casein kinase-1 subunit mediating planar cell polarity, respectively. Reflecting the differential strengths of the SOXC protein transactivation domains, SOX11 was more powerful than SOX4, and SOX12 interfered with the activity of SOX4 and SOX11. Altogether, these findings provide novel insights into the molecular regulation of skeletal

  1. Mesenchymal Wnt Signaling Promotes Formation of Sternum and Thoracic Body Wall

    PubMed Central

    Snowball, John; Ambalavanan, Manoj; Cornett, Bridget; Lang, Richard; Whitsett, Jeff; Sinner, Debora

    2015-01-01

    Midline defects account for approximately 5% of congenital abnormalities observed at birth. However, the molecular mechanisms underlying the formation of the ventral body wall are not well understood. Recent studies linked mutations in Porcupine—an O-acetyl transferase mediating Wnt ligand acylation—with defects in the thoracic body wall. We hypothesized that anomalous Wnt signaling is involved in the pathogenesis of defective closure of the thoracic body wall. We generated a mouse model wherein Wntless (Wls), which encodes a cargo receptor mediating secretion of Wnt ligands, was conditionally deleted from the developing mesenchyme using Dermo1Cre mice. Wlsf/f;Dermo1Cre/+ embryos died during mid-gestation. At E13.5, skeletal defects were observed in the forelimbs, jaw, and rib cage. At E14.5, midline defects in the thoracic body wall began to emerge: the sternum failed to fuse and the heart protruded through the body wall at the midline (ectopia cordis). To determine the molecular mechanism underlying the phenotype observed in Wlsf/f;Dermo1Cre/+ embryos, we tested whether Wnt/β-catenin signaling was operative in developing the embryonic ventral body wall using Axin2LacZ and BatGal reporter mice. While Wnt/β-catenin signaling activity was observed at the midline of the ventral body wall before sternal fusion, this pattern of activity was altered and scattered throughout the body wall after mesenchymal deletion of Wls. Mesenchymal cell migration was disrupted in Wlsf/f;Dermo1Cre/+ thoracic body wall partially due to anomalous non-canonical Wnt signaling as determined by in vitro assays. Deletion of Lrp5 and Lrp6 receptors, which mediate Wnt/β-catenin signaling in the mesenchyme, partially recapitulated the phenotype observed in the chest midline of Wlsf/f;Dermo1Cre/+ embryos supporting a role for Wnt/β-catenin signaling activity in the normal formation of the ventral body wall mesenchyme. We conclude that Wls-mediated secretion of Wnt ligands from the

  2. Pili of oral Streptococcus sanguinis bind to salivary amylase and promote the biofilm formation.

    PubMed

    Okahashi, Nobuo; Nakata, Masanobu; Terao, Yutaka; Isoda, Ryutaro; Sakurai, Atsuo; Sumitomo, Tomoko; Yamaguchi, Masaya; Kimura, Richard K; Oiki, Eiji; Kawabata, Shigetada; Ooshima, Takashi

    2011-01-01

    Streptococcus sanguinis is a member of oral streptococci and one of the most abundant species found in oral biofilm called dental plaque. Colonization of the oral streptococci on the tooth surface depends on the adhesion of bacteria to salivary components adsorbed to the tooth surface. Recently, we identified unique cell surface long filamentous structures named pili in this species. Herein, we investigated the role of S. sanguinis pili in biofilm formation. We found that pili-deficient mutant, in which the genes encoding the three pilus proteins PilA, PilB and PilC have been deleted, showed an impaired bacterial accumulation on saliva-coated surfaces. Confocal microscopic observations suggested that the mutant was incapable of producing typical three-dimensional layer of biofilm. Ligand blot analysis showed that the ancillary pilus proteins PilB and PilC bound to human whole saliva. Additional analysis demonstrated that PilC bound to multiple salivary components, and one of which was found to be salivary α-amylase. These results indicate that pilus proteins are members of saliva-binding proteins of oral S. sanguinis, and suggest the involvement of pili in its colonization on saliva-coated tooth surfaces and in the human oral cavity.

  3. Does Radiative Feedback by the First Stars Promote or Prevent Second Generation Star Formation?

    NASA Astrophysics Data System (ADS)

    Ahn, Kyungjin; Shapiro, Paul R.

    2008-03-01

    We present a self-consistent study of formation of Pop III stars in the early stage of cosmic reionization. We study the effect of starlight from the first stars on the ability of other minihalos in their neighborhood to form additional stars. We show that the ionization front (I-front) is trapped by the neighboring minihalos, after it is transformed from R-type to D-type and preceded by a shock front. The fate of the core of nearby minihalos is mostly determined by the response of the core to this shock front, which leads to molecular cooling and collapse that, when compared to the same halo without external radiation, is (a) expedited, (b) delayed, (c) unaltered, or (d) reversed and prevented, depending upon the flux and halo mass and evolutionary stage. Roughly speaking, most halos that were destined to cool, collapse and form stars in the absence of external radiation are found to do so even when exposed to the first Pop III star in their neighborhood, while those that would not have done so are still not able to.

  4. Type IV collagen aggregates promote keratinocyte proliferation and formation of epidermal layer in human skin equivalents.

    PubMed

    Matsuura-Hachiya, Yuko; Arai, Koji Y; Muraguchi, Taichi; Sasaki, Tasuku; Nishiyama, Toshio

    2017-03-07

    Type IV collagen isolated from lens capsule without enzymatic treatment is known to form a gel under physiological condition and influences cellular activities. In case of human keratinocytes, the suppression of proliferation on reconstituted type IV collagen gels was reported in monolayer culture. In this study, we examined effects of type IV collagen isolated from porcine lens capsule on epidermal formation in human skin equivalents. Type IV collagen aggregates were prepared under the culture condition and the aggregates suppressed keratinocyte proliferation in monolayer culture as well as the culture on the gels. In human skin equivalents type IV collagen aggregates were reconstituted on the surface of contracted collagen gels containing human dermal fibroblasts and the keratinocytes were then cultured on the aggregates for 14 days. Interestingly, in human skin equivalents with type IV collagen aggregates, the BrdU-positive keratinocytes were increased and the thickness of the epidermal layer was around twice than that of control culture. Epidermal differentiation markers were expressed in the upper layer of the epidermis and the defined deposition of human basement membrane components were increased at the dermal-epidermal junction. These results indicate that the type IV collagen aggregates stimulate the proliferation of basal keratinocytes and improve the stratification of epidermal layers in human skin equivalents. This article is protected by copyright. All rights reserved.

  5. Thromboxane A2 exacerbates acute lung injury via promoting edema formation

    PubMed Central

    Kobayashi, Koji; Horikami, Daiki; Omori, Keisuke; Nakamura, Tatsuro; Yamazaki, Arisa; Maeda, Shingo; Murata, Takahisa

    2016-01-01

    Thromboxane A2 (TXA2) is produced in the lungs of patients suffering from acute lung injury (ALI). We assessed its contribution in disease progression using three different ALI mouse models. The administration of hydrochloric acid (HCl) or oleic acid (OA)+ lipopolysaccharide (LPS) caused tissue edema and neutrophil infiltration with TXA2 production in the lungs of the experimental mice. The administration of LPS induced only neutrophil accumulation without TXA2 production. Pretreatment with T prostanoid receptor (TP) antagonist attenuated the tissue edema but not neutrophil infiltration in these models. Intravital imaging and immunostaining demonstrated that administration of TP agonist caused vascular hyper-permeability by disrupting the endothelial barrier formation in the mouse ear. In vitro experiments showed that TP-stimulation disrupted the endothelial adherens junction, and it was inhibited by Ca2+ channel blockade or Rho kinase inhibition. Thus endogenous TXA2 exacerbates ALI, and its blockade attenuates it by modulating the extent of lung edema. This can be explained by the endothelial hyper-permeability caused by the activation of TXA2-TP axis, via Ca2+- and Rho kinase-dependent signaling. PMID:27562142

  6. mTORC2 Signaling Promotes Skeletal Growth and Bone Formation in Mice

    PubMed Central

    Chen, Jianquan; Holguin, Nilsson; Shi, Yu; Silva, Matthew J.; Long, Fanxin

    2015-01-01

    Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase controlling many physiological processes in mammals. mTOR functions in two distinct protein complexes, namely mTORC1 and mTORC2. Compared to mTORC1, the specific roles of mTORC2 are less well understood. To investigate the potential contribution of mTORC2 to skeletal development and homeostasis, we have genetically deleted Rictor, an essential component of mTORC2, in the limb skeletogenic mesenchyme of the mouse embryo. Loss of Rictor leads to shorter and narrower skeletal elements in both embryos and postnatal mice. In the embryo, Rictor deletion reduces the width but not the length of the initial cartilage anlage. Subsequently, the embryonic skeletal elements are shortened due to a delay in chondrocyte hypertrophy, with no change in proliferation, apoptosis, cell size, or matrix production. Postnatally, Rictor-deficient mice exhibit impaired bone formation, resulting in thinner cortical bone, but the trabecular bone mass is relatively normal thanks to a concurrent decrease in bone resorption. Moreover, Rictor-deficient bones exhibit a lesser anabolic response to mechanical loading. Thus, mTORC2 signaling is necessary for optimal skeletal growth and bone anabolism. PMID:25196701

  7. CD147 promotes the formation of functional osteoclasts through NFATc1 signalling.

    PubMed

    Nishioku, Tsuyoshi; Terasawa, Mariko; Baba, Misaki; Yamauchi, Atsushi; Kataoka, Yasufumi

    2016-04-29

    CD147, a membrane glycoprotein of the immunoglobulin superfamily, is highly upregulated during dynamic cellular events including tissue remodelling. Elevated CD147 expression is present in the joint of rheumatoid arthritis patients. However, the role of CD147 in bone destruction remains unclear. To determine whether CD147 is involved in osteoclastogenesis, we studied its expression in mouse osteoclasts and its role in osteoclast differentiation and function. CD147 expression was markedly upregulated during osteoclast differentiation. To investigate the role of CD147 in receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption activity, osteoclast precursor cells were transfected with CD147 siRNA. Decreased CD147 expression inhibited osteoclast formation and bone resorption, inhibited RANKL-induced nuclear translocation of the nuclear factor of activated T cells (NFAT) c1 and decreased the expression of the d2 isoform of vacuolar ATPase Vo domain and cathepsin K. Therefore, CD147 plays a critical role in the differentiation and function of osteoclasts by upregulating NFATc1 through the autoamplification of its expression in osteoclastogenesis.

  8. The Gαo Activator Mastoparan-7 Promotes Dendritic Spine Formation in Hippocampal Neurons.

    PubMed

    Ramírez, Valerie T; Ramos-Fernández, Eva; Inestrosa, Nibaldo C

    2016-01-01

    Mastoparan-7 (Mas-7), an analogue of the peptide mastoparan, which is derived from wasp venom, is a direct activator of Pertussis toxin- (PTX-) sensitive G proteins. Mas-7 produces several biological effects in different cell types; however, little is known about how Mas-7 influences mature hippocampal neurons. We examined the specific role of Mas-7 in the development of dendritic spines, the sites of excitatory synaptic contact that are crucial for synaptic plasticity. We report here that exposure of hippocampal neurons to a low dose of Mas-7 increases dendritic spine density and spine head width in a time-dependent manner. Additionally, Mas-7 enhances postsynaptic density protein-95 (PSD-95) clustering in neurites and activates Gα(o) signaling, increasing the intracellular Ca(2+) concentration. To define the role of signaling intermediates, we measured the levels of phosphorylated protein kinase C (PKC), c-Jun N-terminal kinase (JNK), and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) after Mas-7 treatment and determined that CaMKII activation is necessary for the Mas-7-dependent increase in dendritic spine density. Our results demonstrate a critical role for Gα(o) subunit signaling in the regulation of synapse formation.

  9. The Gαo Activator Mastoparan-7 Promotes Dendritic Spine Formation in Hippocampal Neurons

    PubMed Central

    Ramírez, Valerie T.; Ramos-Fernández, Eva; Inestrosa, Nibaldo C.

    2016-01-01

    Mastoparan-7 (Mas-7), an analogue of the peptide mastoparan, which is derived from wasp venom, is a direct activator of Pertussis toxin- (PTX-) sensitive G proteins. Mas-7 produces several biological effects in different cell types; however, little is known about how Mas-7 influences mature hippocampal neurons. We examined the specific role of Mas-7 in the development of dendritic spines, the sites of excitatory synaptic contact that are crucial for synaptic plasticity. We report here that exposure of hippocampal neurons to a low dose of Mas-7 increases dendritic spine density and spine head width in a time-dependent manner. Additionally, Mas-7 enhances postsynaptic density protein-95 (PSD-95) clustering in neurites and activates Gαo signaling, increasing the intracellular Ca2+ concentration. To define the role of signaling intermediates, we measured the levels of phosphorylated protein kinase C (PKC), c-Jun N-terminal kinase (JNK), and calcium-calmodulin dependent protein kinase IIα (CaMKIIα) after Mas-7 treatment and determined that CaMKII activation is necessary for the Mas-7-dependent increase in dendritic spine density. Our results demonstrate a critical role for Gαo subunit signaling in the regulation of synapse formation. PMID:26881110

  10. Tau and spectraplakins promote synapse formation and maintenance through Jun kinase and neuronal trafficking

    PubMed Central

    Voelzmann, Andre; Okenve-Ramos, Pilar; Qu, Yue; Chojnowska-Monga, Monika; del Caño-Espinel, Manuela; Prokop, Andreas; Sanchez-Soriano, Natalia

    2016-01-01

    The mechanisms regulating synapse numbers during development and ageing are essential for normal brain function and closely linked to brain disorders including dementias. Using Drosophila, we demonstrate roles of the microtubule-associated protein Tau in regulating synapse numbers, thus unravelling an important cellular requirement of normal Tau. In this context, we find that Tau displays a strong functional overlap with microtubule-binding spectraplakins, establishing new links between two different neurodegenerative factors. Tau and the spectraplakin Short Stop act upstream of a three-step regulatory cascade ensuring adequate delivery of synaptic proteins. This cascade involves microtubule stability as the initial trigger, JNK signalling as the central mediator, and kinesin-3 mediated axonal transport as the key effector. This cascade acts during development (synapse formation) and ageing (synapse maintenance) alike. Therefore, our findings suggest novel explanations for intellectual disability in Tau deficient individuals, as well as early synapse loss in dementias including Alzheimer’s disease. DOI: http://dx.doi.org/10.7554/eLife.14694.001 PMID:27501441

  11. Studies of the structure and function of Mms6, a bacterial protein that promotes the formation of magnetic nanoparticles

    SciTech Connect

    Wang, Lijun

    2011-01-01

    Here we report structural and functional studies of Mms6, a biomineralization protein that can promote the formation in vitro of magnetic nanoparticles with sizes and morphologies similar to the magnetites synthesized by magnetotactic bacteria. We found the binding pattern of Mms6 to ferric ion to be two-phase and multivalent. We quantatively determined that Mms6 binds one Fe3+ with a very high affinity (Kd = 1016 M). The second phase of iron binding is multivalent and cooperative with respect to iron with a Kd in the μM range and a stoichiometry of about 20 ferric ion per protein molecule. We found that Mms6 exists in large particles of two sizes, one consisting of 20-40 monomeric units and the other of 200 units. From proteolytic digestion, ultracentrifugation and liposome fusion studies, we found that Mms6 forms a large micellar quaternary structure with the N-terminal domain self-assembling into a uniformly sized micelle and the C-terminal domain on the surface. The two-phase iron-binding pattern may be relevant to iron crystal formation. We propose that the first high affinity phase may stabilize a new conformation of the C-terminal domain that allows interaction with other C-terminal domains leading to a structural change in the multimeric protein complex that enables the second low affinity iron binding phase to organize iron and initiate crystal formation. We also observed a dimeric apparent molecular mass of the Mms6 C-terminal peptide (C21Mms6). We speculate that the C-terminal domain may form higher order quaternary arrangements on the surface of the micelle or when anchored to a membrane by the N-terminal domain. The change in fluorescence quenching in the N-terminal domain with iron binding suggests a structural integrity between the C- and N-terminal domains. The slow change in trp fluorescence as a function of time after adding iron suggests a very slow conformational change in the protein that involves

  12. RacGAP1-driven focal adhesion formation promotes melanoma transendothelial migration through mediating adherens junction disassembly.

    PubMed

    Zhang, Pu; Bai, Huiyuan; Fu, Changliang; Chen, Feng; Zeng, Panying; Wu, Chengxiang; Ye, Qichao; Dong, Cheng; Song, Yang; Song, Erqun

    2015-03-27

    Melanoma cell migration across vascular endothelial cells is an essential step of tumor metastasis. Here, we provide evidence that RacGAP1, a cytokinesis-related Rho GTPase-activating protein, contributed to this process. Depletion of RacGAP1 with RacGAP1-targeting siRNA or overexpression of RacGAP1 mutant (T249A) attenuated melanoma cell transendothelial migration and concomitant changes of adherens junctions. In addition, RacGAP1 promoted the activations of RhoA, FAK, paxillin and triggered focal adhesion formation and cytoskeletal rearrangement. By overexpressing FAK-related non-kinase (FRNK) in endothelium, we showed that RacGAP1 mediated endothelial barrier function loss and melanoma transmigration in a focal adhesion-dependent manner. These results suggest that endothelial RacGAP1 may play critical roles in pathogenic processes of cancer by regulating endothelial permeability.

  13. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

    PubMed

    Simsek, Deniz; Brunet, Erika; Wong, Sunnie Yan-Wai; Katyal, Sachin; Gao, Yankun; McKinnon, Peter J; Lou, Jacqueline; Zhang, Lei; Li, James; Rebar, Edward J; Gregory, Philip D; Holmes, Michael C; Jasin, Maria

    2011-06-01

    Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

  14. Fibronectin promotes proplatelet formation in the human megakaryocytic cell line UT-7/TPO.

    PubMed

    Kawaguchi, Tatsuya; Hatano, Ryo; Yamaguchi, Kyoji; Nawa, Katsuhiko; Hashimoto, Ryuji; Yokota, Hiroshi

    2012-01-01

    We investigated PPF (proplatelet formation) in the human megakaryocytic cell line UT-7/TPO in vitro and signal transduction pathways responsible for PPF. The megakaryocytic cell lines are useful for studying megakaryocyte biology, although PPF is induced only in the presence of phorbol ester. TPO (thrombopoietin) stimulates megakaryocyte proliferation and differentiation; however, no PPF occurred in the megakaryocytic cell lines, even after the addition of TPO. Therefore, factors other than TPO may play an important role in the process of PPF. As PPF occurs in the bone marrow in vivo, we noted extracellular matrix proteins and found that soluble FN (fibronectin) induced potent PPF in UT-7/TPO without phorbol ester. A Western blot analysis showed that the expression of integrins was not increased by FN treatment. Anti-β1 antibody and the RGD (arginine-glycine-aspartate) peptide inhibited FN-induced PPF. This result indicates that the signal originated from integrin β1, which is essential to inducing PPF in UT-7/TPO. Results of the experiments using several inhibitors suggest that activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]-ERK and PI3K (phosphoinositide 3-kinase) pathways are necessary for PPF. The phosphorylation of ERK gradually increased for 2 h after the addition of soluble FN, which suggests that activation of ERK is essential for the initial induction of FN-induced PPF in UT-7/TPO. UT-7/TPO is a useful cell line that enables us to study the signals of PPF without effects of chemical compounds.

  15. Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

    PubMed

    Mitchell, D C; Litman, B J

    1999-06-15

    Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.

  16. DNA Ligase III Promotes Alternative Nonhomologous End-Joining during Chromosomal Translocation Formation

    PubMed Central

    Wong, Sunnie Yan-Wai; Katyal, Sachin; Gao, Yankun; McKinnon, Peter J.; Lou, Jacqueline; Zhang, Lei; Li, James; Rebar, Edward J.; Gregory, Philip D.; Holmes, Michael C.; Jasin, Maria

    2011-01-01

    Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1. PMID:21655080

  17. Prenatal secondhand cigarette smoke promotes Th2 polarization and impairs goblet cell differentiation and airway mucus formation.

    PubMed

    Singh, Shashi P; Gundavarapu, Sravanthi; Peña-Philippides, Juan C; Rir-Sima-ah, Jules; Mishra, Neerad C; Wilder, Julie A; Langley, Raymond J; Smith, Kevin R; Sopori, Mohan L

    2011-11-01

    Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8-10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.

  18. Pyrrolidine and Piperidine Formation Via Copper(II) Carboxylate Promoted Intramolecular Carboamination of Unactivated Olefins: Diastereoselectivity and Mechanism

    PubMed Central

    Sherman, Eric S.; Fuller, Peter H.; Kasi, Dhanalakshmi; Chemler, Sherry R.

    2008-01-01

    An expanded substrate scope and in depth analysis of the reaction mechanism of the copper(II) carboxylate promoted intramolecular carboamination of unactivated alkenes is described. This method provides access to N-functionalized pyrrolidines and piperidines. Both aromatic and aliphatic γ- and δ-alkenyl N-arylsulfonamides undergo the oxidative cyclization reaction efficiently. N-Benzoyl-2-allylaniline also underwent the oxidative cyclization. The terminal olefin substrates examined were more reactive than those with internal olefins, and the latter terminated in elimination rather than carbon-carbon bond formation. The efficiency of the reaction was enhanced by the use of more organic soluble copper(II) carboxylate salts, copper(II) neodecanoate in particular. The reaction times were reduced by the use of microwave heating. High levels of diastereoselectivity were observed in the synthesis of 2,5-disubstituted pyrrolidines, wherein the cis substitution pattern predominates. The mechanism of the reaction is discussed in the context of the observed reactivity and in comparison to analogous reactions promoted by other reagents and conditions. Our evidence supports a mechanism wherein the N-C bond is formed via intramolecular syn aminocupration and the C-C bond is formed via intramolecular addition of a primary carbon radical to an aromatic ring. PMID:17428100

  19. Silicon Promotes Exodermal Casparian Band Formation in Si-Accumulating and Si-Excluding Species by Forming Phenol Complexes

    PubMed Central

    Hinrichs, Martin; Specht, André; Waßmann, Friedrich; Schreiber, Lukas; Schenk, Manfred K.

    2015-01-01

    We studied the effect of Silicon (Si) on Casparian band (CB) development, chemical composition of the exodermal CB and Si deposition across the root in the Si accumulators rice and maize and the Si non-accumulator onion. Plants were cultivated in nutrient solution with and without Si supply. The CB development was determined in stained root cross-sections. The outer part of the roots containing the exodermis was isolated after enzymatic treatment. The exodermal suberin was transesterified with MeOH/BF3 and the chemical composition was measured using gas chromatography-mass spectroscopy (GC-MS) and flame ionization detector (GC-FID). Laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) was used to determine the Si deposition across root cross sections. Si promoted CB formation in the roots of Si-accumulator and Si non-accumulator species. The exodermal suberin was decreased in rice and maize due to decreased amounts of aromatic suberin fractions. Si did not affect the concentration of lignin and lignin-like polymers in the outer part of rice, maize and onion roots. The highest Si depositions were found in the tissues containing CB. These data along with literature were used to suggest a mechanism how Si promotes the CB development by forming complexes with phenols. PMID:26383862

  20. Polygonatum sibiricum polysaccharide inhibits osteoporosis by promoting osteoblast formation and blocking osteoclastogenesis through Wnt/β-catenin signalling pathway

    PubMed Central

    Du, Li; Nong, Meng-Ni; Zhao, Jin-Min; Peng, Xiao-Ming; Zong, Shao-Hui; Zeng, Gao-Feng

    2016-01-01

    Bone homeostasis is maintained by a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteoporosis occurs when osteoclast activity surpasses osteoblast activity. Our previous studies showed the plant-derived natural polysaccharide (Polygonatum sibiricum polysaccharide or PSP) had significant anti-ovariectomy (OVX)-induced osteoporosis effects in vivo, but the mechanisms of PSP’s anti-osteoporosis effect remains unclear. In this study, we assessed PSP’s effect on the generation of osteoblast and osteoclast in vitro. This study showed that PSP promoted the osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) without affecting BMPs signaling pathway. This effect was due to the increased nuclear accumulation of β-catenin, resulting in a higher expression of osteoblast-related genes. Furthermore, the study showed PSP could inhibit the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and exert prophylatic protection against LPS-induced osteolysis in vivo. This effect was also related to the increased nuclear accumulation of β-catenin, resulting in the decreased expression of osteoclast-related genes. In conclusion, our results showed that PSP effectively promoted the osteogenic differentiation of mouse BMSCs and suppressed osteoclastogenesis; therefore, it could be used to treat osteoporosis. PMID:27554324

  1. Thermomineral water promotes axonal sprouting but does not reduce glial scar formation in a mouse model of spinal cord injury

    PubMed Central

    Aleksić, Dubravka; Aksić, Milan; Divac, Nevena; Radonjić, Vidosava; Filipović, Branislav; Jakovčevski, Igor

    2014-01-01

    Thermomineral water from the Atomic Spa Gornja Trepča has been used for a century in the treatment of neurologic disease. The thermomineral water contains microelements, including lithium and magnesium, which show neural regeneration-promoting effects after central nervous system injury. In this study, we investigated the effects of oral intake of thermomineral water from the Atomic Spa Gornja Trepča on nerve regeneration in a 3-month-old mouse model of spinal cord injury. The mice receiving oral intake of thermomineral water showed better locomotor recovery than those without administration of thermomineral water at 8 and 12 weeks after lower thoracic spinal cord compression. At 12 weeks after injury, sprouting of catecholaminergic axons was better in mice that drank thermomineral water than in those without administration of thermomineral water, but there was no difference in glial reaction to injury between mice with and without administration of thermomineral water. These findings suggest that thermomineral water can promote the nerve regeneration but cannot reduce glial scar formation in a mouse model of spinal cord injury. PMID:25657739

  2. Lewis acid promoted titanium alkylidene formation: off-cycle intermediates relevant to olefin trimerization catalysis.

    PubMed

    Sattler, Aaron; VanderVelde, David G; Labinger, Jay A; Bercaw, John E

    2014-07-30

    Two new precatalysts for ethylene and α-olefin trimerization, (FI)Ti(CH2SiMe3)2Me and (FI)Ti(CH2CMe3)2Me (FI = phenoxy-imine), have been synthesized and structurally characterized by X-ray diffraction. (FI)Ti(CH2SiMe3)2Me can be activated with 1 equiv of B(C6F5)3 at room temperature to give the solvent-separated ion pair [(FI)Ti(CH2SiMe3)2][MeB(C6F5)3], which catalytically trimerizes ethylene or 1-pentene to produce 1-hexene or C15 olefins, respectively. The neopentyl analogue (FI)Ti(CH2CMe3)2Me is unstable toward activation with B(C6F5)3 at room temperature, giving no discernible diamagnetic titanium complexes, but at -30 °C the following can be observed by NMR spectroscopy: (i) formation of the bis-neopentyl cation [(FI)Ti(CH2CMe3)2](+), (ii) α-elimination of neopentane to give the neopentylidene complex [(FI)Ti(═CHCMe3)](+), and (iii) subsequent conversion to the imido-olefin complex [(MeOAr2N═)Ti(OArHC═CHCMe3)](+) via an intramolecular metathesis reaction with the imine fragment of the (FI) ligand. If the reaction is carried out at low temperature in the presence of ethylene, catalytic production of 1-hexene is observed, in addition to the titanacyclobutane complex [(FI)Ti(CH(CMe3)CH2CH2)](+), resulting from addition of ethylene to the neopentylidene [(FI)Ti(═CHCMe3)](+). None of the complexes observed spectroscopically subsequent to [(FI)Ti(CH2CMe3)2](+) is an intermediate or precursor for ethylene trimerization, but notwithstanding these off-cycle pathways, [(FI)Ti(CH2CMe3)2](+) is a precatalyst that undergoes rapid initiation to generate a catalyst for trimerizing ethylene or 1-pentene.

  3. aPKC phosphorylates JAM-A at Ser285 to promote cell contact maturation and tight junction formation.

    PubMed

    Iden, Sandra; Misselwitz, Steve; Peddibhotla, Swetha S D; Tuncay, Hüseyin; Rehder, Daniela; Gerke, Volker; Robenek, Horst; Suzuki, Atsushi; Ebnet, Klaus

    2012-03-05

    The PAR-3-atypical protein kinase C (aPKC)-PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3-independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell-cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell-cell contact maturation, TJ formation, and single lumen specification.

  4. Collagen-nanofiber hydrogel composites promote contact guidance of human lymphatic microvascular endothelial cells and directed capillary tube formation.

    PubMed

    Laco, Filip; Grant, M Helen; Black, Richard A

    2013-06-01

    Collagen and fibronectin matrices are known to stimulate migration of microvascular endothelial cells and the process of tubulogenesis, but the physical, chemical, and topographical cues for directed vessel formation have yet to be determined. In this study, growth, migration, elongation, and tube formation of human lymphatic microvascular endothelial cells (LECs) were investigated on electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic-co-D-lactic acid) (PLDL) nanofiber-coated substrates, and correlated with fiber density and diameter. Directed migration of LECs was observed in the presence of aligned nanofibers, whereas random fiber alignment slowed down migration and growth of LECs. Cell guidance was significantly enhanced in the presence of more hydrophobic PLDL polymer nanofibers compared to PLGA (10:90). Subsequent experiments with tube-forming assays reveal the ability of resorbable hydrophobic nanofibers >300 nm in diameter to promote cell guidance in collagen gels without direct cell-fiber contact, in contrast to the previously reported contact-guidance phenomena. Our results show that endothelial cell guidance is possible within nanofiber/collagen-gel constructs that mimic the native extracellular matrix in terms of size and orientation of fibrillar components.

  5. Formation of 100S ribosomes in Staphylococcus aureus by the hibernation promoting factor homolog SaHPF.

    PubMed

    Ueta, Masami; Wada, Chieko; Wada, Akira

    2010-01-01

    In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF/MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1 : 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus.

  6. Pulsed electromagnetic fields partially preserve bone mass, microarchitecture, and strength by promoting bone formation in hindlimb-suspended rats.

    PubMed

    Jing, Da; Cai, Jing; Wu, Yan; Shen, Guanghao; Li, Feijiang; Xu, Qiaoling; Xie, Kangning; Tang, Chi; Liu, Juan; Guo, Wei; Wu, Xiaoming; Jiang, Maogang; Luo, Erping

    2014-10-01

    A large body of evidence indicates that pulsed electromagnetic fields (PEMF), as a safe and noninvasive method, could promote in vivo and in vitro osteogenesis. Thus far, the effects and underlying mechanisms of PEMF on disuse osteopenia and/or osteoporosis remain poorly understood. Herein, the efficiency of PEMF on osteoporotic bone microarchitecture, bone strength, and bone metabolism, together with its associated signaling pathway mechanism, was systematically investigated in hindlimb-unloaded (HU) rats. Thirty young mature (3-month-old), male Sprague-Dawley rats were equally assigned to control, HU, and HU + PEMF groups. The HU + PEMF group was subjected to daily 2-hour PEMF exposure at 15 Hz, 2.4 mT. After 4 weeks, micro-computed tomography (µCT) results showed that PEMF ameliorated the deterioration of trabecular and cortical bone microarchitecture. Three-point bending test showed that PEMF mitigated HU-induced reduction in femoral mechanical properties, including maximum load, stiffness, and elastic modulus. Moreover, PEMF increased serum bone formation markers, including osteocalcin (OC) and N-terminal propeptide of type 1 procollagen (P1NP); nevertheless, PEMF exerted minor inhibitory effects on bone resorption markers, including C-terminal crosslinked telopeptides of type I collagen (CTX-I) and tartrate-resistant acid phosphatase 5b (TRAcP5b). Bone histomorphometric analysis demonstrated that PEMF increased mineral apposition rate, bone formation rate, and osteoblast numbers in cancellous bone, but PEMF caused no obvious changes on osteoclast numbers. Real-time PCR showed that PEMF promoted tibial gene expressions of Wnt1, LRP5, β-catenin, OPG, and OC, but did not alter RANKL, RANK, or Sost mRNA levels. Moreover, the inhibitory effects of PEMF on disuse-induced osteopenia were further confirmed in 8-month-old mature adult HU rats. Together, these results demonstrate that PEMF alleviated disuse-induced bone loss by promoting skeletal anabolic activities

  7. The Listeria monocytogenes Hibernation-Promoting Factor Is Required for the Formation of 100S Ribosomes, Optimal Fitness, and Pathogenesis

    PubMed Central

    Kline, Benjamin C.; McKay, Susannah L.; Tang, William W.

    2014-01-01

    During exposure to certain stresses, bacteria dimerize pairs of 70S ribosomes into translationally silent 100S particles in a process called ribosome hibernation. Although the biological roles of ribosome hibernation are not completely understood, this process appears to represent a conserved and adaptive response that contributes to optimal survival during stress and post-exponential-phase growth. Hibernating ribosomes are formed by the activity of one or more highly conserved proteins; gammaproteobacteria produce two relevant proteins, ribosome modulation factor (RMF) and hibernation promoting factor (HPF), while most Gram-positive bacteria produce a single, longer HPF protein. Here, we report the formation of 100S ribosomes by an HPF homolog in Listeria monocytogenes. L. monocytogenes 100S ribosomes were observed by sucrose density gradient centrifugation of bacterial extracts during mid-logarithmic phase, peaked at the transition to stationary phase, and persisted at lower levels during post-exponential-phase growth. 100S ribosomes were undetectable in bacteria carrying an hpf::Himar1 transposon insertion, indicating that HPF is required for ribosome hibernation in L. monocytogenes. Additionally, epitope-tagged HPF cosedimented with 100S ribosomes, supporting its previously described direct role in 100S formation. We examined hpf mRNA by quantitative PCR (qPCR) and identified several conditions that upregulated its expression, including carbon starvation, heat shock, and exposure to high concentrations of salt or ethanol. Survival of HPF-deficient bacteria was impaired under certain conditions both in vitro and during animal infection, providing evidence for the biological relevance of 100S ribosome formation. PMID:25422304

  8. The Listeria monocytogenes hibernation-promoting factor is required for the formation of 100S ribosomes, optimal fitness, and pathogenesis.

    PubMed

    Kline, Benjamin C; McKay, Susannah L; Tang, William W; Portnoy, Daniel A

    2015-02-01

    During exposure to certain stresses, bacteria dimerize pairs of 70S ribosomes into translationally silent 100S particles in a process called ribosome hibernation. Although the biological roles of ribosome hibernation are not completely understood, this process appears to represent a conserved and adaptive response that contributes to optimal survival during stress and post-exponential-phase growth. Hibernating ribosomes are formed by the activity of one or more highly conserved proteins; gammaproteobacteria produce two relevant proteins, ribosome modulation factor (RMF) and hibernation promoting factor (HPF), while most Gram-positive bacteria produce a single, longer HPF protein. Here, we report the formation of 100S ribosomes by an HPF homolog in Listeria monocytogenes. L. monocytogenes 100S ribosomes were observed by sucrose density gradient centrifugation of bacterial extracts during mid-logarithmic phase, peaked at the transition to stationary phase, and persisted at lower levels during post-exponential-phase growth. 100S ribosomes were undetectable in bacteria carrying an hpf::Himar1 transposon insertion, indicating that HPF is required for ribosome hibernation in L. monocytogenes. Additionally, epitope-tagged HPF cosedimented with 100S ribosomes, supporting its previously described direct role in 100S formation. We examined hpf mRNA by quantitative PCR (qPCR) and identified several conditions that upregulated its expression, including carbon starvation, heat shock, and exposure to high concentrations of salt or ethanol. Survival of HPF-deficient bacteria was impaired under certain conditions both in vitro and during animal infection, providing evidence for the biological relevance of 100S ribosome formation.

  9. Solcoseryl, a tissue respiration stimulating agent, significantly enhances the effect of capacitively coupled electric field on the promotion of bone formation around dental implants.

    PubMed

    Ochi, Morio; Wang, Pao-Li; Ohura, Kiyoshi; Takashima, Shigenori; Kagami, Hiroyuki; Hirose, Yukito; Kaku, Tohru; Sakaguchi, Kunihiko

    2003-06-01

    In the present study we examined the combined effect of application of a capacitively coupled electric field (CCEF) and the tissue respiration stimulating agent, Solcoseryl, on the promotion of bone formation around dental implants histologically and mechanically. After a dental implant was inserted into each femur of Japanese white rabbits, Solcoseryl (2 ml/kg) was administered intravenously in the ear vein and a CCEF was applied for 4 h per day for 14 days. The degree of bone formation on microscopic observation, bone contact ratio, bone surface area ratio, and the level of removal torque of the implant in the Solcoseryl- and CCEF-treated group were significantly higher than the respective value in the control group, which had not been treated with Solcoseryl nor CCEF. Thus, the combination of CCEF stimulation and Solcoseryl effectively promoted the formation of new bone. It is suggested that the clinical use of a combination of CCEF stimulation and Solcoseryl for dental implants promotes osseointegration.

  10. Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

    PubMed Central

    Wong, Mun-Teng

    2016-01-01

    ABSTRACT Hepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication. IMPORTANCE HCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum

  11. Promoted reduction of tellurite and formation of extracellular tellurium nanorods by concerted reaction between iron and Shewanella oneidensis MR-1.

    PubMed

    Kim, Dong-Hun; Kim, Min-Gyu; Jiang, Shenghua; Lee, Ji-Hoon; Hur, Hor-Gil

    2013-08-06

    The reduction of tellurite (Te(IV)) by dissimilatory metal reducing bacterium, Shewanella oneidensis MR-1, was promoted in the presence of Fe(III) in comparison with Te(IV) bioreduction in the absence of Fe(III). Electron microscopic analyses revealed that iron promoted Te(IV) reduction led to form exclusively extracellular crystalline Te(0) nanorods, as compared to the mostly intracellular formation of Te(0) nanorods in the absence of Fe(III). The Te K-edge X-ray absorption spectrometric analyses demonstrated that S. oneidensis MR-1 in the presence of Fe(III) reduced Te(IV) to less harmful metallic Te(0) nanorods through the precipitation of tellurite (Te(IV)Ox) complex by the bacterial respiration of Fe(III) to Fe(II) under anaerobic conditions. However, Fe(II) ion itself was only able to precipitate the solid tellurite (Te(IV)Ox) complex from the Te(IV) solution, which was not further reduced to Te(0). The results clearly indicated that bacterial S. oneidensis MR-1 plays important roles in the reduction and crystallization of Te(0) nanorods by as yet undetermined biochemical mechanisms. As compared to the slow bacterial Te(IV) reduction in the absence of Fe(III), the rapid reduction of Te(IV) to Te(0) by the concerted biogeochemical reaction between Fe(II) and S. oneidensis MR-1 could be applied for the sequestration and detoxification of Te(IV) in the environments as well as for the preparation of extracellular Te(0) nanorod structures.

  12. Vascular mimicry formation is promoted by paracrine TGF-β and SDF1 of cancer-associated fibroblasts and inhibited by miR-101 in hepatocellular carcinoma.

    PubMed

    Yang, Jine; Lu, Yang; Lin, Ying-Ying; Zheng, Zhi-Yuan; Fang, Jian-Hong; He, Shuai; Zhuang, Shi-Mei

    2016-12-01

    Vascular mimicry (VM) describes the phenomenon that tumor cells but not endothelial cells form vascular-like channels, which provide blood perfusion for tumor tissues. VM is associated with tumor growth, metastasis and worse survival of different cancers. The mechanisms of VM formation remain largely unknown. We showed that the conditioned medium of cancer-associated fibroblast (CM-CAF) promoted tumor cells to form capillary-like structure in vitro. Consistently, co-implantation of CAFs with tumor cells significantly enhanced VM formation in mouse xenografts, and higher amount of CAFs was found in VM(+) human HCC tissues compared to VM(-) ones. However, the CM-CAF-promoted VM formation was attenuated when TGF-β or SDF1 signaling was abrogated. Similar to CM-CAF, recombinant TGF-β1 and SDF1 induced VM formation. We further disclosed that the CAF-secreted TGF-β and SDF1 enhanced the expression of VE-cadherin, MMP2 and laminin5γ2 via TGF-βR1 and CXCR4 in tumor cells, thereby promoted VM formation. Moreover, tumor cells with high activity of self-sustaining TGF-β signaling displayed strong capability of VM formation. Subsequent investigations showed that miR-101, which was down-regulated in both tumor cells and CAFs, suppressed the CAF-promoted VM formation in vitro and in vivo. Gain- and loss-of-function analyses revealed that miR-101 attenuated TGF-β signaling transduction by targeting TGF-βR1 and Smad2 in tumor cells, and simultaneously abrogated SDF1 signaling by suppressing SDF1 expression in CAFs and inhibiting VE-cadherin expression in tumor cells. Our findings suggest that the miR-101-TGF-β/SDF1-VE-cadherin/MMP2/LAMC2 networks regulate VM formation and represent the potential targets for cancer therapy.

  13. Design and realization of the high-precision weighing systems as the gravimetric references in PTB's national water flow standard

    NASA Astrophysics Data System (ADS)

    Engel, Rainer; Beyer, Karlheinz; Baade, Hans-Joachim

    2012-07-01

    PTB's ‘Hydrodynamic Test Field’, which represents a high-accuracy water flow calibration facility, serves as the national primary standard for liquid flow measurands. As the core reference device of this flow facility, a gravimetric standard has been incorporated, which comprises three special-design weighing systems: 300 kg, 3 tons and 30 tons. These gravimetric references were realized as a combination of a strain-gauge-based and an electromagnetic-force-compensation load-cell-based balance, each. Special emphasis had to be placed upon the dynamics design of the whole weighing system, due to the high measurement resolution and the dynamic behavior of the weighing systems, which are dynamically affected by mechanical vibrations caused by environmental impacts, flow machinery operation, flow noise in the pipework and induced wave motions in the weigh tanks. Taking into account all the above boundary conditions, the design work for the gravimetric reference resulted in a concrete foundation ‘rock’ of some 300 tons that rests on a number of vibration isolators. In addition to these passively operating vibration isolators, the vibration damping effect is enhanced by applying an electronic level regulation device.

  14. Irradiation-induced degradation of PTB7 investigated by valence band and S 2p photoelectron spectroscopy

    NASA Astrophysics Data System (ADS)

    Darlatt, Erik; Muhsin, Burhan; Roesch, Roland; Lupulescu, Cosmin; Roth, Friedrich; Kolbe, Michael; Gottwald, Alexander; Hoppe, Harald; Richter, Mathias

    2016-08-01

    Monochromatic radiation with known absolute radiant power from an undulator at the electron storage ring Metrology Light Source (MLS) was used to irradiate PTB7 (a thieno[3, 4-b]thiophene-alt-benzodithiophene polymer) thin films at wavelengths (photon energies) of 185 nm (6.70 eV), 220 nm (5.64 eV), 300 nm (4.13 eV), 320 nm (3.88 eV), 356 nm (3.48 eV) and 675 nm (1.84 eV) under ultra-high vacuum conditions for the investigation of radiation-induced degradation effects. The characterization of the thin films is focused at ultraviolet photoelectron spectroscopy (UPS) of valence bands and is complemented by S 2p x-ray photoelectron spectroscopy (S 2p XPS) before and after the irradiation procedure. The radiant exposure was determined for each irradiation by means of photodiodes traceably calibrated to the international system of units SI. The valence band spectra show the strongest changes for the shortest wavelengths and no degradation effect at 356 nm and 675 nm even with the highest radiant exposure applied. In the spectral range where the Sun appears bright on the Earth’s surface, no degradation effects are observed.

  15. Correlating high power conversion efficiency of PTB7:PC71BM inverted organic solar cells with nanoscale structures.

    PubMed

    Das, Sanjib; Keum, Jong K; Browning, James F; Gu, Gong; Yang, Bin; Dyck, Ondrej; Do, Changwoo; Chen, Wei; Chen, Jihua; Ivanov, Ilia N; Hong, Kunlun; Rondinone, Adam J; Joshi, Pooran C; Geohegan, David B; Duscher, Gerd; Xiao, Kai

    2015-10-14

    Advances in material design and device engineering led to inverted organic solar cells (i-OSCs) with superior power conversion efficiencies (PCEs) compared to their "conventional" counterparts, in addition to the well-known better ambient stability. Here, we report an in-depth morphology study of the i-OSC active and cathode modifying layers, employing a model system with a well-established bulk-heterojunction, PTB7:PC71BM as the active layer and poly-[(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene)-alt-2,7-(9,9-dioctylfluorene)] (PFN) as the cathode surface modifying layer. We have also identified the role of a processing additive, 1,8-diiodooctane (DIO), used in the spin-casting of the active layer to increase PCE. Using various characterization techniques, we demonstrate that the high PCEs of i-OSCs are due to the diffusion of electron-accepting PC71BM into the PFN layer, resulting in improved electron transport. The diffusion occurs when residual solvent molecules in the spun-cast film act as a plasticizer. Addition of DIO to the casting solution results in more PC71BM diffusion and therefore more efficient electron transport. This work provides important insight and guidance to further enhancement of i-OSC performance by materials and interface engineering.

  16. Shoot bending promotes flower bud formation by miRNA-mediated regulation in apple (Malus domestica Borkh.).

    PubMed

    Xing, Libo; Zhang, Dong; Zhao, Caiping; Li, Youmei; Ma, Juanjuan; An, Na; Han, Mingyu

    2016-02-01

    Flower induction in apple (Malus domestica Borkh.) trees plays an important life cycle role, but young trees produce fewer and inferior quality flower buds. Therefore, shoot bending has become an important cultural practice, significantly promoting the capacity to develop more flower buds during the growing seasons. Additionally, microRNAs (miRNAs) play essential roles in plant growth, flower induction and stress responses. In this study, we identified miRNAs potentially involved in the regulation of bud growth, and flower induction and development, as well as in the response to shoot bending. Of the 195 miRNAs identified, 137 were novel miRNAs. The miRNA expression profiles revealed that the expression levels of 68 and 27 known miRNAs were down-regulated and up-regulated, respectively, in response to shoot bending, and that the 31 differentially expressed novel miRNAs between them formed five major clusters. Additionally, a complex regulatory network associated with auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) plays important roles in cell division, bud growth and flower induction, in which related miRNAs and targets mediated regulation. Among them, miR396, 160, 393, and their targets associated with AUX, miR159, 319, 164, and their targets associated with ABA and GA, and flowering-related miRNAs and genes, regulate bud growth and flower bud formation in response to shoot bending. Meanwhile, the flowering genes had significantly higher expression levels during shoot bending, suggesting that they are involved in this regulatory process. This study provides a framework for the future analysis of miRNAs associated with multiple hormones and their roles in the regulation of bud growth, and flower induction and formation in response to shoot bending in apple trees.

  17. GhAGL15s, preferentially expressed during somatic embryogenesis, promote embryogenic callus formation in cotton (Gossypium hirsutum L.).

    PubMed

    Yang, Zuoren; Li, Changfeng; Wang, Ye; Zhang, Chaojun; Wu, Zhixia; Zhang, Xueyan; Liu, Chuanliang; Li, Fuguang

    2014-10-01

    Somatic embryogenesis is a useful tool for gene transfer and propagation of plants. AGAMOUS-LIKE15 (AGL15) promotes somatic embryogenesis in many plant species. In this study, three homologous AGL15 genes were isolated from Gossypium hirsutum L., namely GhAGL15-1, GhAGL15-3, and GhAGL15-4. Their putative proteins contained a highly conserved MADS-box DNA-binding domain and a less conserved K domain. Phylogenetic analysis suggested that the three GhAGL15s clustered most closely with AGL15 proteins in other plants. Subcellular location analyses revealed that three GhAGL15s were localized in the nucleus. Furthermore, their expression levels increased following embryogenic callus induction, but sharply decreased during the embryoid stage. GhAGL15-1 and GhAGL15-3 were significantly induced by 2,4-D and kinetin, whereas GhAGL15-4 was only responsive to 2,4-D treatment. Over-expression of the three GhAGL15s in cotton callus improved callus quality and significantly increased the embryogenic callus formation rate, while GhAGL15-4 had the highest positive effect on the embryogenic callus formation rate (an increase from 38.1 to 65.2%). These results suggest that over-expression of GhAGL15s enhances embryogenic potential of transgenic calli. Therefore, spatiotemporal manipulation of GhAGL15s expression may prove valuable in improving cotton transformation efficiency.

  18. Glycoprotein130 (Gp130)/interleukin-6 (IL-6) signalling in osteoclasts promotes bone formation in periosteal and trabecular bone.

    PubMed

    Johnson, Rachelle W; McGregor, Narelle E; Brennan, Holly J; Crimeen-Irwin, Blessing; Poulton, Ingrid J; Martin, T John; Sims, Natalie A

    2015-12-01

    Interleukin-6 (IL-6) and interleukin-11 (IL-11) receptors (IL-6R and IL-11R, respectively) are both expressed in osteoclasts and transduce signal via the glycoprotein130 (gp130) co-receptor, but the physiological role of this pathway is unclear. To determine the critical roles of gp130 signalling in the osteoclast, we generated mice using cathepsin K Cre (CtskCre) to disrupt gp130 signalling in osteoclasts. Bone marrow macrophages from CtskCre.gp130(f/f) mice generated more osteoclasts in vitro than cells from CtskCre.gp130(w/w) mice; these osteoclasts were also larger and had more nuclei than controls. While no increase in osteoclast numbers was observed in vivo, osteoclasts on trabecular bone surfaces of CtskCre.gp130(f/f) mice were more spread out than in control mice, but had no functional defect detectable by serum CTX1 levels or trabecular bone cartilage remnants. However, trabecular osteoblast number and mineralising surfaces were significantly lower in male CtskCre.gp130(f/f) mice compared to controls, and this was associated with a significantly lower trabecular bone volume at 12 weeks of age. Furthermore, CtskCre.gp130(f/f) mice exhibited greatly suppressed periosteal bone formation at this age, indicated by significant reductions in both double-labelled surface and mineral apposition rate. By 26 weeks of age, CtskCre.gp130(f/f) mice exhibited narrower femora, with lower periosteal and endocortical perimeters than CtskCre.gp130(w/w) controls. Since IL-6 and IL-11R global knockout mice exhibited a similar reduction in femoral width, we also assessed periosteal bone formation in those strains, and found bone forming surfaces were also reduced in male IL-6 null mice. These data suggest that IL-6/gp130 signalling in the osteoclast is not essential for normal bone resorption in vivo, but maintains both trabecular and periosteal bone formation in male mice by promoting osteoblast activity through the stimulation of osteoclast-derived "coupling factors" and

  19. An auxin-mediated shift toward growth isotropy promotes organ formation at the shoot meristem in Arabidopsis.

    PubMed

    Sassi, Massimiliano; Ali, Olivier; Boudon, Frédéric; Cloarec, Gladys; Abad, Ursula; Cellier, Coralie; Chen, Xu; Gilles, Benjamin; Milani, Pascale; Friml, Jiří; Vernoux, Teva; Godin, Christophe; Hamant, Olivier; Traas, Jan

    2014-10-06

    To control morphogenesis, molecular regulatory networks have to interfere with the mechanical properties of the individual cells of developing organs and tissues, but how this is achieved is not well known. We study this issue here in the shoot meristem of higher plants, a group of undifferentiated cells where complex changes in growth rates and directions lead to the continuous formation of new organs. Here, we show that the plant hormone auxin plays an important role in this process via a dual, local effect on the extracellular matrix, the cell wall, which determines cell shape. Our study reveals that auxin not only causes a limited reduction in wall stiffness but also directly interferes with wall anisotropy via the regulation of cortical microtubule dynamics. We further show that to induce growth isotropy and organ outgrowth, auxin somehow interferes with the cortical microtubule-ordering activity of a network of proteins, including AUXIN BINDING PROTEIN 1 and KATANIN 1. Numerical simulations further indicate that the induced isotropy is sufficient to amplify the effects of the relatively minor changes in wall stiffness to promote organogenesis and the establishment of new growth axes in a robust manner.

  20. Laminin-521 Promotes Rat Bone Marrow Mesenchymal Stem Cell Sheet Formation on Light-Induced Cell Sheet Technology

    PubMed Central

    2017-01-01

    Rat bone marrow mesenchymal stem cell sheets (rBMSC sheets) are attractive for cell-based tissue engineering. However, methods of culturing rBMSC sheets are critically limited. In order to obtain intact rBMSC sheets, a light-induced cell sheet method was used in this study. TiO2 nanodot films were coated with (TL) or without (TN) laminin-521. We investigated the effects of laminin-521 on rBMSCs during cell sheet culturing. The fabricated rBMSC sheets were subsequently assessed to study cell sheet viability, reattachment ability, cell sheet thickness, collagen type I deposition, and multilineage potential. The results showed that laminin-521 could promote the formation of rBMSC sheets with good viability under hyperconfluent conditions. Cell sheet thickness increased from an initial 26.7 ± 1.5 μm (day 5) up to 47.7 ± 3.0 μm (day 10). Moreover, rBMSC sheets maintained their potential of osteogenic, adipogenic, and chondrogenic differentiation. This study provides a new strategy to obtain rBMSC sheets using light-induced cell sheet technology. PMID:28164129

  1. Substrate-promoted formation of a catalytically competent binuclear center and regulation of reactivity in a glycerophosphodiesterase from Enterobacter aerogenes.

    PubMed

    Hadler, Kieran S; Tanifum, Eric A; Yip, Sylvia Hsu-Chen; Mitić, Natasa; Guddat, Luke W; Jackson, Colin J; Gahan, Lawrence R; Nguyen, Kelly; Carr, Paul D; Ollis, David L; Hengge, Alvan C; Larrabee, James A; Schenk, Gerhard

    2008-10-29

    The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of this versatile enzyme using a range of techniques. An improved crystal structure (1.9 A resolution) illustrates the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles, i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby the terminally bound H2O/OH(-) acts as the nucleophile, activated via hydrogen bonding by the bridging water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site. Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility in one of the two metal binding sites is essential for optimal activity.

  2. BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

    PubMed

    Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

    2014-10-01

    Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors.

  3. Top3β is an RNA topoisomerase that works with Fragile X syndrome protein to promote synapse formation

    PubMed Central

    Xu, Dongyi; Shen, Weiping; Guo, Rong; Xue, Yutong; Peng, Wei; Sima, Jian; Yang, Jay; Sharov, Alexei; Srikantan, Subramanya; Yang, Jiandong; Fox, David; Qian, Yong; Martindale, Jennifer L.; Piao, Yulan; Machamer, James; Joshi, Samit R.; Mohanty, Subhasis; Shaw, Albert C.; Lloyd, Thomas E.; Brown, Grant W.; Ko, Minoru S.H.; Gorospe, Myriam; Zou, Sige; Wang, Weidong

    2013-01-01

    Topoisomerases are crucial to solve DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3β (Top3β) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein deficient in Fragile X syndrome and known to regulate translation of mRNAs important for neuronal function and autism. Notably, the FMRP-Top3β interaction is abolished by a disease-associated FMRP mutation, suggesting that Top3β may contribute to pathogenesis of mental disorders. Top3β binds multiple mRNAs encoded by genes with neuronal functions related to schizophrenia and autism. Expression of one such gene, ptk2/FAK, is reduced in neuromuscular junctions of Top3β mutant flies. Synapse formation is defective in Top3β mutant flies and mice, as observed in FMRP mutant animals. Our findings suggest that Top3β acts as an RNA topoisomerase and works with FMRP to promote expression of mRNAs critical for neurodevelopment and mental health. PMID:23912945

  4. Multiple Ligands of von Willebrand Factor-binding Protein (vWbp) Promote Staphylococcus aureus Clot Formation in Human Plasma*

    PubMed Central

    Thomer, Lena; Schneewind, Olaf; Missiakas, Dominique

    2013-01-01

    Staphylococcus aureus secretes coagulase (Coa) and von Willebrand factor-binding protein (vWbp) to activate host prothrombin and form fibrin cables, thereby promoting the establishment of infectious lesions. The D1-D2 domains of Coa and vWbp associate with, and non-proteolytically activate prothrombin. Moreover, Coa encompasses C-terminal tandem repeats for binding to fibrinogen, whereas vWbp has been reported to associate with von Willebrand factor and fibrinogen. Here we used affinity chromatography with non-catalytic Coa and vWbp to identify the ligands for these virulence factors in human plasma. vWbp bound to prothrombin, fibrinogen, fibronectin, and factor XIII, whereas Coa co-purified with prothrombin and fibrinogen. vWbp association with fibrinogen and factor XIII, but not fibronectin, required prothrombin and triggered the non-proteolytic activation of FXIII in vitro. Staphylococcus aureus coagulation of human plasma was associated with the recruitment of prothrombin, FXIII, and fibronectin as well as the formation of cross-linked fibrin. FXIII activity in staphylococcal clots could be attributed to thrombin-dependent proteolytic activation as well as vWbp-mediated non-proteolytic activation of FXIII zymogen. PMID:23960083

  5. Smooth muscle cell-specific Tgfbr1 deficiency promotes aortic aneurysm formation by stimulating multiple signaling events

    PubMed Central

    Yang, Pu; Schmit, Bradley M.; Fu, Chunhua; DeSart, Kenneth; Oh, S. Paul; Berceli, Scott A.; Jiang, Zhihua

    2016-01-01

    Transforming growth factor (TGF)-β signaling disorder has emerged as a common molecular signature for aortic aneurysm development. The timing of postnatal maturation plays a key role in dictating the biological outcome of TGF-β signaling disorders in the aortic wall. In this study, we investigated the impact of deficiency of TGFβ receptors on the structural homeostasis of mature aortas. We used an inducible Cre-loxP system driven by a Myh11 promoter to delete Tgfbr1, Tgfbr2, or both in smooth muscle cells (SMCs) of adult mice. TGFBR1 deficiency resulted in rapid and severe aneurysmal degeneration, with 100% penetrance of ascending thoracic aortas, whereas TGFBR2 deletion only caused mild aortic pathology with low (26%) lesion prevalence. Removal of TGFBR2 attenuated the aortic pathology caused by TGFBR1 deletion and correlated with a reduction of early ERK phosphorylation. In addition, the production of angiotensin (Ang)-converting enzyme was upregulated in TGFBR1 deficient aortas at the early stage of aneurysmal degeneration. Inhibition of ERK phosphorylation or blockade of AngII type I receptor AT1R prevented aneurysmal degeneration of TGFBR1 deficient aortas. In conclusion, loss of SMC-Tgfbr1 triggers multiple deleterious pathways, including abnormal TGFBR2, ERK, and AngII/AT1R signals that disrupt aortic wall homeostasis to cause aortic aneurysm formation. PMID:27739498

  6. Correlating high power conversion efficiency of PTB7:PC71BM inverted organic solar cells with nanoscale structures

    NASA Astrophysics Data System (ADS)

    Das, Sanjib; Keum, Jong K.; Browning, James F.; Gu, Gong; Yang, Bin; Dyck, Ondrej; Do, Changwoo; Chen, Wei; Chen, Jihua; Ivanov, Ilia N.; Hong, Kunlun; Rondinone, Adam J.; Joshi, Pooran C.; Geohegan, David B.; Duscher, Gerd; Xiao, Kai

    2015-09-01

    Advances in material design and device engineering led to inverted organic solar cells (i-OSCs) with superior power conversion efficiencies (PCEs) compared to their ``conventional'' counterparts, in addition to the well-known better ambient stability. Here, we report an in-depth morphology study of the i-OSC active and cathode modifying layers, employing a model system with a well-established bulk-heterojunction, PTB7:PC71BM as the active layer and poly-[(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene)-alt-2,7-(9,9-dioctylfluorene)] (PFN) as the cathode surface modifying layer. We have also identified the role of a processing additive, 1,8-diiodooctane (DIO), used in the spin-casting of the active layer to increase PCE. Using various characterization techniques, we demonstrate that the high PCEs of i-OSCs are due to the diffusion of electron-accepting PC71BM into the PFN layer, resulting in improved electron transport. The diffusion occurs when residual solvent molecules in the spun-cast film act as a plasticizer. Addition of DIO to the casting solution results in more PC71BM diffusion and therefore more efficient electron transport. This work provides important insight and guidance to further enhancement of i-OSC performance by materials and interface engineering.Advances in material design and device engineering led to inverted organic solar cells (i-OSCs) with superior power conversion efficiencies (PCEs) compared to their ``conventional'' counterparts, in addition to the well-known better ambient stability. Here, we report an in-depth morphology study of the i-OSC active and cathode modifying layers, employing a model system with a well-established bulk-heterojunction, PTB7:PC71BM as the active layer and poly-[(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene)-alt-2,7-(9,9-dioctylfluorene)] (PFN) as the cathode surface modifying layer. We have also identified the role of a processing additive, 1,8-diiodooctane (DIO), used in the spin-casting of the active

  7. The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner.

    PubMed

    Dotto, Cristian; Lombarte Serrat, Andrea; Cattelan, Natalia; Barbagelata, María S; Yantorno, Osvaldo M; Sordelli, Daniel O; Ehling-Schulz, Monika; Grunert, Tom; Buzzola, Fernanda R

    2017-01-01

    Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphylococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2 mM SAL induced a 27% reduction in the intracellular free Fe(2+) concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe(2+) cation in culture media. These moderate iron-limited conditions promoted an intensification of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal

  8. The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner

    PubMed Central

    Dotto, Cristian; Lombarte Serrat, Andrea; Cattelan, Natalia; Barbagelata, María S.; Yantorno, Osvaldo M.; Sordelli, Daniel O.; Ehling-Schulz, Monika; Grunert, Tom; Buzzola, Fernanda R.

    2017-01-01

    Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphylococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2 mM SAL induced a 27% reduction in the intracellular free Fe2+ concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe2+ cation in culture media. These moderate iron-limited conditions promoted an intensification of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal colonization

  9. Correlating High Power Conversion Efficiency of PTB7:PC71BM Inverted Organic Solar Cells with Nanoscale Structures

    DOE PAGES

    Das, Sanjib; Keum, Jong Kahk; Browning, Jim; ...

    2015-01-01

    Advances in materials design and device engineering led to inverted organic solar cells (i-OSCs) with superior power conversion efficiencies (PCEs) to their conventional counterparts, in addition to the well-known better ambient stability. Despite the significant progress, however, it has so far been unclear how the morphologies of the photoactive layer and its interface with the cathode modifying layer impact device performance. Here, we report an in-depth morphology study of the i-OSC active and cathode modifying layers, employing a model system with the well-established bulk-heterojunction, PTB7:PC71BM as the active layer and poly-[(9,9-bis(3 -(N,N-dimethylamino)propyl)-2,7-fluorene)-alt-2,7-(9,9-dioctylfluorene)] (PFN) as the cathode surface modifying layer. Wemore » have also identified the role of a processing additive, 1,8-diiodooctane (DIO), used in the spin-casting of the active layer to increase PCE. Using a variety of characterization techniques, we demonstrate that the high PCEs of i-OSCs are due to the smearing (diffusion) of electron-accepting PC71BM into the PFN layer, resulting in improved electron transport. The PC71BM diffusion occurs after spin-casting the active layer onto the PFN layer, when residual solvent molecules act as a plasticizer. The DIO additive, with a higher boiling point than the host solvent, has a longer residence time in the spin-cast active layer, resulting in more PC71BM smearing and therefore more efficient electron transport. This work provides important insight and guidance to further enhancement of i-OSC performance by materials and interface engineering.« less

  10. New developments at PTB in 3D-AFM with tapping and torsion AFM mode and vector approach probing strategy

    NASA Astrophysics Data System (ADS)

    Dai, G.; Hässler-Grohne, W.; Hüser, D.; Wolff, H.; Fluegge, J.; Bosse, H.

    2011-06-01

    A new 3D-AFM for true 3D measurements of nano structures has been developed at Physikalisch Technische-Bundesanstalt, the national metrology institute of Germany. In its configuration, two piezo actuators are applied to drive the AFM cantilever near its vertical and torsional resonant frequencies. In such a way, the AFM tip can probe the surface with a vertical and/or a lateral oscillation, offering high 3D probing sensitivity. For enhancing measurement flexibility as well as reducing tip wear, a so called "vector approach probing" (VAP) method has been applied. The sample is measured point by point using this method. At each probing point, the tip is approached towards the surface in its normal direction until the desired tip-sample interaction is detected and then immediately withdrawn from the surface. Preliminary experimental results show promising performance of the developed system. The measurement of a line structure of 800 nm height employing a super sharp AFM tip is performed, showing a repeatability of its 3D profiles of better than 1 nm (p-v). A single crystal critical dimension reference material (SCCDRM) having features with almost vertical sidewall is measured using a flared AFM tip. Results show that the feature has averaged left and right sidewall angles of 88.64° and 88.67deg;, respectively. However, the feature width non-uniformity may reach 10 nm within the measurement range of 1 μm. The standard deviation of the averaged middle CD values of 7 repeated measurements reaches 0.35 nm. In addition, an investigation of long term measurement stability is performed on a PTB photomask. The results shows that the 3D-AFM has a drift rate of about 0.00033 nm per line, which confirms the high measurement stability and the very low tip wear.

  11. Dipeptidyl peptidase IV inhibition prevents the formation and promotes the healing of indomethacin-induced intestinal ulcers in rats

    PubMed Central

    Inoue, Takuya; Higashiyama, Masaaki; Kaji, Izumi; Rudenkyy, Sergiy; Higuchi, Kazuhide; Guth, Paul H.; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

    2014-01-01

    Backgrounds & Aims We studied the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) as a possible therapy for non-steroidal anti-inflammatory drug (NSAID)-induced intestinal ulcers. Luminal nutrients release endogenous GLP-2 from enteroendocrine L cells. Since GLP-2 is degraded by dipeptidyl peptidase IV (DPPIV), we hypothesized that DPPIV inhibition combined with luminal administration of nutrients potentiates the effects of endogenous GLP-2 on intestinal injury. Methods Intestinal injury was induced by indomethacin (10 mg/kg, sc) in fed rats. The long-acting DPPIV inhibitor K579 was intragastrically (ig) or intraperitoneally (ip) given before or after indomethacin treatment. L-alanine (L-Ala) and 5′-inosine monophosphate (IMP) were co-administered ig after the treatment. Results Indomethacin treatment induced intestinal ulcers which gradually healed after treatment. Pretreatment with ig or ip K579 given either at 1 mg/kg reduced total ulcer length, whereas K579 at 3 mg/kg had no effect. Exogenous GLP-2 also reduced intestinal ulcers. The preventive effect of K579 was dose-dependently inhibited by a GLP-2 receptor antagonist. Daily treatment with K579 (1 mg/kg), GLP-2, or L-Ala + IMP after indomethacin treatment reduced total ulcer length. Co-administration (ig) of K579 and L-Ala + IMP further accelerated intestinal ulcer healing. Conclusion DPPIV inhibition and exogenous GLP-2 prevented the formation and promoted the healing of indomethacin-induced intestinal ulcers, although high-dose DPPIV inhibition reversed the preventive effect. Umami receptor agonists also enhanced the healing effects of the DPPIV inhibitor. The combination of DPPIV inhibition and luminal nutrient-induced GLP-2 release may be a useful therapeutic tool for the treatment of NSAIDs-induced intestinal ulcers. PMID:24379150

  12. Allotropic control: How certain fluorinated carbonate electrolytes protect aluminum current collectors by promoting the formation of insoluble coordination polymers

    SciTech Connect

    Shkrob, Ilya A.; Pupek, Krzysztof Z.; Abraham, Daniel P.

    2016-07-28

    Here, there is a strong incentive for increasing the operation voltage of Li-ion cells above 4.5 V in order to increase the density of stored energy. Aluminum is an inexpensive, lightweight metal that is commonly used as a positive electrode current collector in these cells. Imide LiX salts, such as lithium bis(trifluoromethylsulfonyl)imide (X = TFSI), and lithium bis(fluorosulfonyl)imide (X = FSI), are chemically stable on the energized lithiated transition metal oxide electrodes, but their presence in the electrolyte causes rapid anodic dissolution and pitting of Al current collectors at potentials exceeding 4.0 V versus Li/Li+. For LiBF4 and LiPF6, the release of HF near the energized surfaces passivates the exposed Al metal, inhibiting this pitting corrosion, but it also causes the gradual degradation of the cathode active material, negating this important advantage. Here we report that in certain electrolytes containing fluorinated carbonate solvents and LiX salts, the threshold voltage for safe operation of Al current collectors can be increased to 5.5 V versus Li/Li+. Interestingly, the most efficient solvent also facilitates the formation of an insoluble gel when AlX3 is introduced into this solvent. We suggest that this solvent promotes the aggregation of coordination polymers of AlX3 at the exposed Al surface that isolate this surface from the electrolyte, thereby preventing further Al dissolution and corrosion. Other examples of Al collector protection may also involve this mechanism. Our study suggests that such “allotropic control” could be a way of widening the operation window of Li-ion cells without electrode deterioration, Al current collector corrosion, and electrolyte breakdown.

  13. Androgens inhibit tumor necrosis factor-α-induced cell adhesion and promote tube formation of human coronary artery endothelial cells.

    PubMed

    Liao, Chun-Hou; Lin, Feng-Yen; Wu, Yi-No; Chiang, Han-Sun

    2012-06-01

    Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens-testosterone and dihydrotestosterone-and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10-50 nM), dihydrotestosterone (5-50 nM), and estradiol (0.1-0.4 nM) on the adhesion of tumor necrosis factor-α (TNF-α)-stimulated HCAECs was determined at different time points (12-96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-α-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism.

  14. Steviol Reduces MDCK Cyst Formation and Growth by Inhibiting CFTR Channel Activity and Promoting Proteasome-Mediated CFTR Degradation

    PubMed Central

    Yuajit, Chaowalit; Homvisasevongsa, Sureeporn; Chatsudthipong, Lisa; Soodvilai, Sunhapas; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2013-01-01

    Cyst enlargement in polycystic kidney disease (PKD) involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK) cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM) reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h) with steviol (100 microM) also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h) with steviol (100 microM) markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease. PMID:23536832

  15. Allotropic control: How certain fluorinated carbonate electrolytes protect aluminum current collectors by promoting the formation of insoluble coordination polymers

    DOE PAGES

    Shkrob, Ilya A.; Pupek, Krzysztof Z.; Abraham, Daniel P.

    2016-07-28

    Here, there is a strong incentive for increasing the operation voltage of Li-ion cells above 4.5 V in order to increase the density of stored energy. Aluminum is an inexpensive, lightweight metal that is commonly used as a positive electrode current collector in these cells. Imide LiX salts, such as lithium bis(trifluoromethylsulfonyl)imide (X = TFSI), and lithium bis(fluorosulfonyl)imide (X = FSI), are chemically stable on the energized lithiated transition metal oxide electrodes, but their presence in the electrolyte causes rapid anodic dissolution and pitting of Al current collectors at potentials exceeding 4.0 V versus Li/Li+. For LiBF4 and LiPF6, themore » release of HF near the energized surfaces passivates the exposed Al metal, inhibiting this pitting corrosion, but it also causes the gradual degradation of the cathode active material, negating this important advantage. Here we report that in certain electrolytes containing fluorinated carbonate solvents and LiX salts, the threshold voltage for safe operation of Al current collectors can be increased to 5.5 V versus Li/Li+. Interestingly, the most efficient solvent also facilitates the formation of an insoluble gel when AlX3 is introduced into this solvent. We suggest that this solvent promotes the aggregation of coordination polymers of AlX3 at the exposed Al surface that isolate this surface from the electrolyte, thereby preventing further Al dissolution and corrosion. Other examples of Al collector protection may also involve this mechanism. Our study suggests that such “allotropic control” could be a way of widening the operation window of Li-ion cells without electrode deterioration, Al current collector corrosion, and electrolyte breakdown.« less

  16. Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

    PubMed

    Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

    2016-01-26

    The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology.

  17. Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

    PubMed

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

    2017-02-24

    Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  18. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

    PubMed Central

    Kozma, R; Ahmed, S; Best, A; Lim, L

    1995-01-01

    The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology. PMID:7891688

  19. Bromodomain protein Brd3 promotes Ifnb1 transcription via enhancing IRF3/p300 complex formation and recruitment to Ifnb1 promoter in macrophages

    PubMed Central

    Ren, Wenhui; Wang, Chunmei; Wang, Qinlan; Zhao, Dezhi; Zhao, Kai; Sun, Donghao; Liu, Xingguang; Han, Chaofeng; Hou, Jin; Li, Xia; Zhang, Qian; Cao, Xuetao; Li, Nan

    2017-01-01

    As members of bromodomain and extra-terminal motif protein family, bromodomain-containing proteins regulate a wide range of biological processes including protein scaffolding, mitosis, cell cycle progression and transcriptional regulation. The function of these bromodomain proteins (Brds) in innate immune response has been reported but the role of Brd3 remains unclear. Here we find that virus infection significantly downregulate Brd3 expression in macrophages and Brd3 knockout inhibits virus-triggered IFN-β production. Brd3 interacts with both IRF3 and p300, increases p300-mediated acetylation of IRF3, and enhances the association of IRF3 with p300 upon virus infection. Importantly, Brd3 promotes the recruitment of IRF3/p300 complex to the promoter of Ifnb1, and increases the acetylation of histone3/histone4 within the Ifnb1 promoter, leading to the enhancement of type I interferon production. Therefore, our work indicated that Brd3 may act as a coactivator in IRF3/p300 transcriptional activation of Ifnb1 and provided new epigenetic mechanistic insight into the efficient activation of the innate immune response. PMID:28045112

  20. The C. elegans DSB-2 protein reveals a regulatory network that controls competence for meiotic DSB formation and promotes crossover assurance.

    PubMed

    Rosu, Simona; Zawadzki, Karl A; Stamper, Ericca L; Libuda, Diana E; Reese, Angela L; Dernburg, Abby F; Villeneuve, Anne M

    2013-01-01

    For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious

  1. RNA polymerase I transcription factors in active yeast rRNA gene promoters enhance UV damage formation and inhibit repair.

    PubMed

    Meier, Andreas; Thoma, Fritz

    2005-03-01

    UV photofootprinting and repair of pyrimidine dimers by photolyase was used to investigate chromatin structure, protein-DNA interactions, and DNA repair in the spacer and promoter of Saccharomyces cerevisiae rRNA genes. Saccharomyces cerevisiae contains about 150 copies of rRNA genes separated by nontranscribed spacers. Under exponential growth conditions about half of the genes are transcribed by RNA polymerase I (RNAP-I). Initiation of transcription requires the assembly of the upstream activating factor (UAF), the core factor (CF), TATA binding protein, and RNAP-I with Rrn3p on the upstream element and core promoter. We show that UV irradiation of wild-type cells and transcription factor mutants generates photofootprints in the promoter elements. The core footprint depends on UAF, while the UAF footprint was also detected in absence of the CFs. Fractionation of active and inactive promoters showed the core footprint mainly in the active fraction and similar UAF footprints in both fractions. DNA repair by photolyase was strongly inhibited in active promoters but efficient in inactive promoters. The data suggest that UAF is present in vivo in active and inactive promoters and that recruitment of CF and RNAP-I to active promoters generates a stable complex which inhibits repair.

  2. Parent and Clinician Preferences for an Asthma App to Promote Adolescent Self-Management: A Formative Study

    PubMed Central

    Roberts, Courtney A; Sage, Adam J; Coyne-Beasley, Tamera; Sleath, Betsy L; Carpenter, Delesha M

    2016-01-01

    Background Most youth asthma apps are not designed with parent and clinician use in mind, and rarely is the app development process informed by parent or clinician input. Objective This study was conducted to generate formative data on the use, attitudes, and preferences for asthma mHealth app features among parents and clinicians, the important stakeholders who support adolescents with asthma and promote adolescent self-management skills. Methods We conducted a mixed-methods study from 2013 to 2014 employing a user-centered design philosophy to acquire feedback from a convenience sample of 20 parents and 6 clinicians. Participants were given an iPod Touch and asked to evaluate 10 features on 2 existing asthma apps. Participant experiences using the apps were collected from questionnaires and a thematic analysis of audio-recorded and transcribed (verbatim) interviews using MAXQDA. Descriptive statistics were calculated to characterize the study sample and app feature feedback. Independent samples t tests were performed to compare parent and clinician ratings of app feature usefulness (ratings: 1=not at all useful to 5=very useful). Results All parents were female (n=20), 45% were black, 20% had an income ≥US $50,000, and 45% had a bachelor’s degree or higher education. The clinician sample included 2 nurses and 4 physicians with a mean practice time of 13 years. Three main themes provided an understanding of how participants perceived their roles and use of asthma app features to support adolescent asthma self-management: monitoring and supervision, education, and communication/information sharing. Parents rated the doctor report feature highest, and clinicians rated the doctor appointment reminder highest of all evaluated app features on usefulness. The peak flow monitoring feature was the lowest ranked feature by both parents and clinicians. Parents reported higher usefulness for the doctor report (t(10)=2.7, P<.02), diary (t(10)=2.7, P<.03), and self

  3. EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis.

    PubMed

    Béguelin, Wendy; Teater, Matt; Gearhart, Micah D; Calvo Fernández, María Teresa; Goldstein, Rebecca L; Cárdenas, Mariano G; Hatzi, Katerina; Rosen, Monica; Shen, Hao; Corcoran, Connie M; Hamline, Michelle Y; Gascoyne, Randy D; Levine, Ross L; Abdel-Wahab, Omar; Licht, Jonathan D; Shaknovich, Rita; Elemento, Olivier; Bardwell, Vivian J; Melnick, Ari M

    2016-08-08

    The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

  4. Support vector regression-guided unravelling: antioxidant capacity and quantitative structure-activity relationship predict reduction and promotion effects of flavonoids on acrylamide formation

    PubMed Central

    Huang, Mengmeng; Wei, Yan; Wang, Jun; Zhang, Yu

    2016-01-01

    We used the support vector regression (SVR) approach to predict and unravel reduction/promotion effect of characteristic flavonoids on the acrylamide formation under a low-moisture Maillard reaction system. Results demonstrated the reduction/promotion effects by flavonoids at addition levels of 1–10000 μmol/L. The maximal inhibition rates (51.7%, 68.8% and 26.1%) and promote rates (57.7%, 178.8% and 27.5%) caused by flavones, flavonols and isoflavones were observed at addition levels of 100 μmol/L and 10000 μmol/L, respectively. The reduction/promotion effects were closely related to the change of trolox equivalent antioxidant capacity (ΔTEAC) and well predicted by triple ΔTEAC measurements via SVR models (R: 0.633–0.900). Flavonols exhibit stronger effects on the acrylamide formation than flavones and isoflavones as well as their O-glycosides derivatives, which may be attributed to the number and position of phenolic and 3-enolic hydroxyls. The reduction/promotion effects were well predicted by using optimized quantitative structure-activity relationship (QSAR) descriptors and SVR models (R: 0.926–0.994). Compared to artificial neural network and multi-linear regression models, SVR models exhibited better fitting performance for both TEAC-dependent and QSAR descriptor-dependent predicting work. These observations demonstrated that the SVR models are competent for predicting our understanding on the future use of natural antioxidants for decreasing the acrylamide formation. PMID:27586851

  5. Support vector regression-guided unravelling: antioxidant capacity and quantitative structure-activity relationship predict reduction and promotion effects of flavonoids on acrylamide formation

    NASA Astrophysics Data System (ADS)

    Huang, Mengmeng; Wei, Yan; Wang, Jun; Zhang, Yu

    2016-09-01

    We used the support vector regression (SVR) approach to predict and unravel reduction/promotion effect of characteristic flavonoids on the acrylamide formation under a low-moisture Maillard reaction system. Results demonstrated the reduction/promotion effects by flavonoids at addition levels of 1–10000 μmol/L. The maximal inhibition rates (51.7%, 68.8% and 26.1%) and promote rates (57.7%, 178.8% and 27.5%) caused by flavones, flavonols and isoflavones were observed at addition levels of 100 μmol/L and 10000 μmol/L, respectively. The reduction/promotion effects were closely related to the change of trolox equivalent antioxidant capacity (ΔTEAC) and well predicted by triple ΔTEAC measurements via SVR models (R: 0.633–0.900). Flavonols exhibit stronger effects on the acrylamide formation than flavones and isoflavones as well as their O-glycosides derivatives, which may be attributed to the number and position of phenolic and 3-enolic hydroxyls. The reduction/promotion effects were well predicted by using optimized quantitative structure-activity relationship (QSAR) descriptors and SVR models (R: 0.926–0.994). Compared to artificial neural network and multi-linear regression models, SVR models exhibited better fitting performance for both TEAC-dependent and QSAR descriptor-dependent predicting work. These observations demonstrated that the SVR models are competent for predicting our understanding on the future use of natural antioxidants for decreasing the acrylamide formation.

  6. Epitope-tagged yeast strains reveal promoter driven changes to 3'-end formation and convergent antisense-transcription from common 3' UTRs.

    PubMed

    Swaminathan, Angavai; Beilharz, Traude H

    2016-01-08

    Epitope-tagging by homologous recombination is ubiquitously used to study gene expression, protein localization and function in yeast. This is generally thought to insulate the regulation of gene expression to that mediated by the promoter and coding regions because native 3' UTR are replaced. Here we show that the 3' UTRs, CYC1 and ADH1, contain cryptic promoters that generate abundant convergent antisense-transcription in Saccharomyces cerevisiae. Moreover we show that aberrant, truncating 3' -end formation is often associated with regulated transcription in TAP-tagged strains. Importantly, the steady-state level of both 3' -truncated and antisense transcription products is locus dependent. Using TAP and GFP-tagged strains we show that the transcriptional state of the gene-of-interest induces changes to 3' -end formation by alternative polyadenylation and antisense transcription from a universal 3' UTR. This means that these 3' UTRs contains plastic features that can be molded to reflect the regulatory architecture of the locus rather than bringing their own regulatory paradigm to the gene-fusions as would be expected. Our work holds a cautionary note for studies utilizing tagged strains for quantitative biology, but also provides a new model for the study of promoter driven rewiring of 3' -end formation and regulatory non-coding transcription.

  7. Injectable hydrogel promotes early survival of induced pluripotent stem cell-derived oligodendrocytes and attenuates longterm teratoma formation in a spinal cord injury model.

    PubMed

    Führmann, T; Tam, R Y; Ballarin, B; Coles, B; Elliott Donaghue, I; van der Kooy, D; Nagy, A; Tator, C H; Morshead, C M; Shoichet, M S

    2016-03-01

    Transplantation of pluripotent stem cells and their differentiated progeny has the potential to preserve or regenerate functional pathways and improve function after central nervous system injury. However, their utility has been hampered by poor survival and the potential to form tumors. Peptide-modified biomaterials influence cell adhesion, survival and differentiation in vitro, but their effectiveness in vivo remains uncertain. We synthesized a peptide-modified, minimally invasive, injectable hydrogel comprised of hyaluronan and methylcellulose to enhance the survival and differentiation of human induced pluripotent stem cell-derived oligodendrocyte progenitor cells. Cells were transplanted subacutely after a moderate clip compression rat spinal cord injury. The hydrogel, modified with the RGD peptide and platelet-derived growth factor (PDGF-A), promoted early survival and integration of grafted cells. However, prolific teratoma formation was evident when cells were transplanted in media at longer survival times, indicating that either this cell line or the way in which it was cultured is unsuitable for human use. Interestingly, teratoma formation was attenuated when cells were transplanted in the hydrogel, where most cells differentiated to a glial phenotype. Thus, this hydrogel promoted cell survival and integration, and attenuated teratoma formation by promoting cell differentiation.

  8. A Model Formative Assessment Strategy to Promote Student-Centered Self-Regulated Learning in Higher Education

    ERIC Educational Resources Information Center

    Bose, Jayakumar; Rengel, Zed

    2009-01-01

    Adult learners are already involved in the process of self-regulation; hence, higher education institutions should focus on strengthening students' self-regulatory skills. Self-regulation can be facilitated through formative assessment. This paper proposes a model formative assessment strategy that would complement existing university teaching,…

  9. Synaptopodin-2 induces assembly of peripheral actin bundles and immature focal adhesions to promote lamellipodia formation and prostate cancer cell migration

    PubMed Central

    Kai, FuiBoon; Fawcett, James P.; Duncan, Roy

    2015-01-01

    Synaptopodin-2 (Synpo2), an actin-binding protein and invasive cancer biomarker, induces formation of complex stress fiber networks in the cell body and promotes PC3 prostate cancer cell migration in response to serum stimulation. The role of these actin networks in enhanced cancer cell migration is unknown. Using time-course analysis and live cell imaging of mock- and Synpo2-transduced PC3 cells, we now show that Synpo2 induces assembly of actin fibers near the cell periphery and Arp2/3-dependent lamellipodia formation. Lamellipodia formed in a non-directional manner or repeatedly changed direction, explaining the enhanced chemokinetic activity of PC3 cells in response to serum stimulation. Myosin contraction promotes retrograde flow of the Synpo2-associated actin filaments at the leading edge and their merger with actin networks in the cell body. Enhanced PC3 cell migration correlates with Synpo2-induced formation of lamellipodia and immature focal adhesions (FAs), but is not dependent on myosin contraction or FA maturation. The previously reported correlation between Synpo2-induced stress fiber assembly and enhanced PC3 cell migration therefore reflects the role of Synpo2 as a newly identified regulator of actin bundle formation and nascent FA assembly near the leading cell edge. PMID:25883213

  10. Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network.

    PubMed

    Almeida, Claudia G; Yamada, Ayako; Tenza, Danièle; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

    2011-06-12

    The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.

  11. TolC promotes ExPEC biofilm formation and curli production in response to medium osmolarity.

    PubMed

    Hou, Bo; Meng, Xian-Rong; Zhang, Li-Yuan; Tan, Chen; Jin, Hui; Zhou, Rui; Gao, Jian-Feng; Wu, Bin; Li, Zi-Li; Liu, Mei; Chen, Huan-Chun; Bi, Ding-Ren; Li, Shao-Wen

    2014-01-01

    While a high osmolarity medium activates Cpx signaling and causes CpxR to repress csgD expression, and efflux protein TolC protein plays an important role in biofilm formation in Escherichia coli, whether TolC also responds to an osmolarity change to regulate biofilm formation in extraintestinal pathogenic E. coli (ExPEC) remains unknown. In this study, we constructed ΔtolC mutant and complement ExPEC strains to investigate the role of TolC in the retention of biofilm formation and curli production capability under different osmotic conditions. The ΔtolC mutant showed significantly decreased biofilm formation and lost the ability to produce curli fimbriae compared to its parent ExPEC strain PPECC42 when cultured in M9 medium or 1/2 M9 medium of increased osmolarity with NaCl or sucrose at 28°C. However, biofilm formation and curli production levels were restored to wild-type levels in the ΔtolC mutant in 1/2 M9 medium. We propose for the first time that TolC protein is able to form biofilm even under high osmotic stress. Our findings reveal an interplay between the role of TolC in ExPEC biofilm formation and the osmolarity of the surrounding environment, thus providing guidance for the development of a treatment for ExPEC biofilm formation.

  12. Auxin regulates first leaf development and promotes the formation of protocorm trichomes and rhizome-like structures in developing seedlings of Spathoglottis plicata (Orchidaceae)

    PubMed Central

    Novak, Stacey D.; Whitehouse, Grace A.

    2013-01-01

    Auxin flows in a polar manner to target tissues and exert its morphogenic effect. Preventing auxin movement, with polar auxin transport (PAT) inhibitors, or increasing auxin levels in tissues through exogenous application can provide a means for assessing the importance of appropriate tissue distribution and concentration of this hormone during development. The formulation of culture media for micropropagation has been the primary focus of most orchid tissue culture research, a goal that unveils seedling hormone responses at a single point in development. This study was unique because it evaluated the auxin response of orchids during three stages of seedling development. Seedlings were grown on standard culture media for 10, 35 and 85 days. Each group was sub-cultured onto auxin- and/or PAT inhibitor-containing media for an additional 10, 30 and 60 days, respectively. Data were collected on first leaf initiation, trichome formation and the appearance of propagative structures. In the 20-day seedlings, auxins and PAT inhibitors promoted precocious formation and random placement of protocorm hairs rather than in tufts, as seen in older, control seedlings. The 65-day seedlings formed protocorm-like bodies, rhizome-like growths from the stem, and fleshy leaves with trichomes. Seedlings cultured for 145 days developed microshoots or callus growth in the axils of older leaves and exhibited necrosis of original seedling roots and leaves. In general, exogenously applied auxin promoted the reversion of differentiated Spathoglottis plicata seedling tissue to a morphology that had propagative properties. Additionally, auxins commonly induced hair formation, which suggests that protocorm hairs may be root hair-like in nature. This work characterized three auxin growth responses in S. plicata seedlings that have not been reported in orchids: (i) the inhibition of first leaf initiation and abnormal first leaf morphology; (ii) the promotion of trichome formation; and (iii) the

  13. The primary function of gp130 signaling in osteoblasts is to maintain bone formation and strength, rather than promote osteoclast formation.

    PubMed

    Johnson, Rachelle W; Brennan, Holly J; Vrahnas, Christina; Poulton, Ingrid J; McGregor, Narelle E; Standal, Therese; Walker, Emma C; Koh, Thuan-Tzen; Nguyen, Huynh; Walsh, Nicole C; Forwood, Mark R; Martin, T John; Sims, Natalie A

    2014-06-01

    Interleukin-6 (IL-6) family cytokines act via gp130 in the osteoblast lineage to stimulate the formation of osteoclasts (bone resorbing cells) and the activity of osteoblasts (bone forming cells), and to inhibit expression of the osteocyte protein, sclerostin. We report here that a profound reduction in trabecular bone mass occurs both when gp130 is deleted in the entire osteoblast lineage (Osx1Cre gp130 f/f) and when this deletion is restricted to osteocytes (DMP1Cre gp130 f/f). This was caused not by an alteration in osteoclastogenesis, but by a low level of bone formation specific to the trabecular compartment. In contrast, cortical diameter increased to maintain ultimate bone strength, despite a reduction in collagen type 1 production. We conclude that osteocytic gp130 signaling is required for normal trabecular bone mass and proper cortical bone composition.

  14. Key comparison BIPM.RI(I)-K4 of the absorbed dose to water standards of the PTB, Germany and the BIPM in 60Co gamma radiation

    NASA Astrophysics Data System (ADS)

    Kessler, C.; Burns, D. T.; Kapsch, R.-P.; Krauss, A.

    2016-01-01

    An indirect comparison has been made of the standards for absorbed dose to water in 60Co radiation of the Physikalisch-Technische Bundesanstalt, (PTB), Germany and of the Bureau International des Poids et Mesures (BIPM). The measurements at the BIPM were carried out in October 2015. The comparison result, based on the calibration coefficients for two transfer standards and evaluated as a ratio of the PTB and the BIPM standards for absorbed dose to water, is 0.9977 with a combined standard uncertainty of 3.8 × 10-3. The results are analysed and presented in terms of degrees of equivalence for entry in the BIPM key comparison database. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCRI, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

  15. The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation

    PubMed Central

    Schulte, Tim; Mikaelsson, Cecilia; Beaussart, Audrey; Kikhney, Alexey; Deshmukh, Maya; Wolniak, Sebastian; Pathak, Anuj; Ebel, Christine; Löfling, Jonas; Fogolari, Federico; Henriques-Normark, Birgitta; Dufrêne, Yves F.; Svergun, Dmitri; Nygren, Per-Åke; Achour, Adnane

    2016-01-01

    The major human pathogen Streptococcus pneumoniae is a leading cause of disease and death worldwide. Pneumococcal biofilm formation within the nasopharynx leads to long-term colonization and persistence within the host. We have previously demonstrated that the capsular surface-associated pneumococcal serine rich repeat protein (PsrP), key factor for biofilm formation, binds to keratin-10 (KRT10) through its microbial surface component recognizing adhesive matrix molecule (MSCRAMM)-related globular binding region domain (BR187–385). Here, we show that BR187–385 also binds to DNA, as demonstrated by electrophoretic mobility shift assays and size exclusion chromatography. Further, heterologous expression of BR187–378 or the longer BR120–378 construct on the surface of a Gram-positive model host bacterium resulted in the formation of cellular aggregates that was significantly enhanced in the presence of DNA. Crystal structure analyses revealed the formation of BR187–385 homo-dimers via an intermolecular β-sheet, resulting in a positively charged concave surface, shaped to accommodate the acidic helical DNA structure. Furthermore, small angle X-ray scattering and circular dichroism studies indicate that the aggregate-enhancing N-terminal region of BR120–166 adopts an extended, non-globular structure. Altogether, our results suggest that PsrP adheres to extracellular DNA in the biofilm matrix and thus promotes pneumococcal biofilm formation. PMID:27582320

  16. Promotion of peripheral nerve regeneration and prevention of neuroma formation by PRGD/PDLLA/β-TCP conduit: report of two cases.

    PubMed

    Yin, Yixia; Li, Binbin; Yan, Qiongjiao; Dai, Honglian; Wang, Xinyu; Huang, Jifeng; Li, Shipu

    2015-06-01

    In the field of nerve repair, one major challenge is the formation of neuroma. However, reports on both the promotion of nerve regeneration and prevention of traumatic neuroma in the clinical settings are rare in the field of nerve repair. One of the reasons could be the insufficiency in the follow-up system. We have conducted 33 cases of nerve repair using PRGD/PDLLA/β-TCP conduit without any sign of adverse reaction, especially no neuroma formation. Among them, we have selected two cases as representatives to report in this article. The first case was a patient with an upper limb nerve wound was bridged by PRGD/PDLLA/β-TCP conduit and a plate fixation was given. After nearly 3-years' follow-up, the examination results demonstrated that nerve regeneration effect was very good. When the reoperation was performed to remove the steel plate we observed a uniform structure of the regenerated nerve without the formation of neuroma, and to our delight, the implanted conduit was completely degraded 23 months after the implantation. The second case had an obsolete nerve injury with neuroma formation. After removal of the neuroma, the nerve was bridged by PRGD/PDLLA/β-TCP conduit. Follow-up examinations showed that the structure and functional recovery were improved gradually in the 10-month follow-up; no end-enlargement and any other abnormal reaction associated with the characteristic of neuroma were found. Based on our 33-case studies, we have concluded that PRGD/PDLLA/β-TCP nerve conduit could both promote nerve regeneration and prevent neuroma formation; therefore, it is a good alternative for peripheral nerve repair.

  17. Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation*

    PubMed Central

    Zucchelli, Silvia; Marcuzzi, Federica; Codrich, Marta; Agostoni, Elena; Vilotti, Sandra; Biagioli, Marta; Pinto, Milena; Carnemolla, Alisia; Santoro, Claudio; Gustincich, Stefano; Persichetti, Francesca

    2011-01-01

    Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis. PMID:21454471

  18. Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with huntingtin protein and promotes its atypical ubiquitination to enhance aggregate formation.

    PubMed

    Zucchelli, Silvia; Marcuzzi, Federica; Codrich, Marta; Agostoni, Elena; Vilotti, Sandra; Biagioli, Marta; Pinto, Milena; Carnemolla, Alisia; Santoro, Claudio; Gustincich, Stefano; Persichetti, Francesca

    2011-07-15

    Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys(6), Lys(27), and Lys(29) linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys(6), Lys(27), and Lys(29) ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

  19. Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts

    PubMed Central

    You, Jia; Wu, Di; Yu, Yang; Liu, Chang; Wang, Lei; Wang, Fei; Xu, Lu; Wang, Liqun; Wang, Nan; Tian, Xing; Wang, Falin; Liang, Hongbin; Gao, Yating; Cui, Xiaobo; Ji, Guohua; Bai, Jing; Yu, Jingcui; Meng, Xiangning; Jin, Yan; Sun, Wenjing; Guan, Xin-yuan; Zhang, Chunyu; Fu, Songbin

    2016-01-01

    Background Sei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated. Results DMs formation significantly increased during serial passage in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3. Materials and Methods We employed NIH-3T3 cell line with Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure. Conclusions Met is an important promotor of DMs formation. PMID:27494853

  20. Cooperativity between Al Sites Promotes Hydrogen Transfer and Carbon–Carbon Bond Formation upon Dimethyl Ether Activation on Alumina

    PubMed Central

    2015-01-01

    The methanol-to-olefin (MTO) process allows the conversion of methanol/dimethyl ether into olefins on acidic zeolites via the so-called hydrocarbon pool mechanism. However, the site and mechanism of formation of the first carbon–carbon bond are still a matter of debate. Here, we show that the Lewis acidic Al sites on the 110 facet of γ-Al2O3 can readily activate dimethyl ether to yield CH4, alkenes, and surface formate species according to spectroscopic studies combined with a computational approach. The carbon–carbon forming step as well as the formation of methane and surface formate involves a transient oxonium ion intermediate, generated by a hydrogen transfer between surface methoxy species and coordinated methanol on adjacent Al sites. These results indicate that extra framework Al centers in acidic zeolites, which are associated with alumina, can play a key role in the formation of the first carbon–carbon bond, the initiation step of the industrial MTO process. PMID:27162986

  1. Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-β after vascular damage

    PubMed Central

    Schachtrup, Christian; Ryu, Jae K.; Helmrick, Matthew; Vagena, Eirini; Galanakis, Dennis K.; Degen, Jay L.; Margolis, Richard U.; Akassoglou, Katerina

    2010-01-01

    Scar formation in the nervous system begins within hours after traumatic injury and is characterized primarily by reactive astrocytes depositing proteoglycans that inhibit regeneration. A fundamental question in CNS repair has been the identity of the initial molecular mediator that triggers glial scar formation. Here we show that the blood protein fibrinogen, which leaks into the CNS immediately after blood-brain barrier (BBB) disruption or vascular damage, serves as an early signal for the induction of glial scar formation via the TGF-β/Smad signaling pathway. Our studies revealed that fibrinogen is a carrier of latent TGF-β and induces phosphorylation of Smad2 in astrocytes that leads to inhibition of neurite outgrowth. Consistent with these findings, genetic or pharmacologic depletion of fibrinogen in mice reduces active TGF-β, Smad2 phosphorylation, glial cell activation and neurocan deposition following cortical injury. Furthermore, stereotactic injection of fibrinogen into the mouse cortex is sufficient to induce astrogliosis. Inhibition of the TGF-β receptor pathway abolishes the fibrinogen-induced effects on glial scar formation in vivo and in vitro. These results identify fibrinogen as a primary astrocyte activation signal, provide evidence that deposition of inhibitory proteoglycans is induced by a blood protein that leaks in the CNS after vasculature rupture, and point to TGF-β as a molecular link between vascular permeability and scar formation. PMID:20427645

  2. In Vitro Oxidation of Collagen Promotes the Formation of Advanced Oxidation Protein Products and the Activation of Human Neutrophils.

    PubMed

    Bochi, Guilherme Vargas; Torbitz, Vanessa Dorneles; de Campos, Luízi Prestes; Sangoi, Manuela Borges; Fernandes, Natieli Flores; Gomes, Patrícia; Moretto, Maria Beatriz; Barbisan, Fernanda; da Cruz, Ivana Beatrice Mânica; Moresco, Rafael Noal

    2016-04-01

    The accumulation of advanced oxidation protein products (AOPPs) has been linked to several pathological conditions. Here, we investigated collagen as a potential source for AOPP formation and determined the effects of hypochlorous acid (HOCl)-treated collagen (collagen-AOPPs) on human neutrophil activity. We also assessed whether alpha-tocopherol could counteract these effects. Exposure to HOCl increased the levels of collagen-AOPPs. Collagen-AOPPs also stimulated the production of AOPPs, nitric oxide (NO), superoxide radicals (O2(-)), and HOCl by neutrophils. Collagen-AOPPs induced apoptosis and decreased the number of viable cells. Alpha-tocopherol prevented the formation of collagen-AOPPs, strongly inhibited the collagen-AOPP-induced production of O2(-) and HOCl, and increased the viability of neutrophils. Our results suggest that collagen is an important protein that interacts with HOCl to form AOPPs, and consequently, collagen-AOPP formation is related to human neutrophil activation and cell death.

  3. Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

    PubMed Central

    Bencsik, Norbert; Szíber, Zsófia; Liliom, Hanna; Tárnok, Krisztián; Borbély, Sándor; Gulyás, Márton; Rátkai, Anikó; Szűcs, Attila; Hazai-Novák, Diána; Ellwanger, Kornelia; Rácz, Bence; Pfizenmaier, Klaus; Hausser, Angelika

    2015-01-01

    Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines. PMID:26304723

  4. Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

    PubMed Central

    Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F.; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon

    2015-01-01

    Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections. PMID:26092919

  5. Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis.

    PubMed

    Yasukawa, K; Takido, M; Matsumoto, T; Takeuchi, M; Nakagawa, S

    1991-01-01

    A single topical application of 1 microgram of 12-O-tetradecanoylphorbol- 13-acetate (TPA) to the ears of mice was shown to induce edema, and this TPA-induced inflammation was inhibited by 4-methylsterol and triterpene derivatives. The ED50 of these compounds against TPA-induced inflammation was 0.1-3 mumol. Phytosterols had only slight inhibitory effects. Furthermore, application of 5 micrograms TPA to mouse skin rapidly caused accumulation of ornithine decarboxylase (ODC). Similarly, sitosterol and lupane-type triterpene derivatives markedly inhibited this TPA-induced ODC accumulation. In addition, 5 mumol betulinic acid markedly inhibited the promoting effect of 2.5 micrograms TPA applied twice weekly on skin tumor formation in mice initiated with 50 micrograms of 7,12-dimethylbenz[a]anthracene, and 5 mumol of sitosterol caused slight suppression. Thus, the inhibitory effects of sterol and triterpene derivatives on TPA-induced inflammation roughly parallelled their inhibitory activities against tumor promotion.

  6. Mechanistic insights on iodine(III) promoted metal-free dual C-H activation involved in the formation of a spirocyclic bis-oxindole.

    PubMed

    Sreenithya, A; Sunoj, Raghavan B

    2014-12-05

    The mechanism of a metal-free, phenyliodine(III) bis(trifluoroacetate) promoted, dual aryl C-H activation of an anilide to a spirocyclic bis-oxindole is examined using density functional theory (M06-2X). The most preferred pathway proceeds through the involvement of a novel iodonium ion intermediate and a pivotal trifluoroacetate counterion. The two sequential aryl C-H activations, assisted by trifluoroacetate as well as the superior leaving group ability of PhI, facilitate the formation of spirocyclic bis-oxindole.

  7. Task-Appropriate Visualizations: Can the Very Same Visualization Format Either Promote or Hinder Learning Depending on the Task Requirements?

    ERIC Educational Resources Information Center

    Soemer, Alexander; Schwan, Stephan

    2016-01-01

    In a series of experiments, we tested a recently proposed hypothesis stating that the degree of alignment between the form of a mental representation resulting from learning with a particular visualization format and the specific requirements of a learning task determines learning performance (task-appropriateness). Groups of participants were…

  8. An Exploratory Study of a Novel Online Formative Assessment and Instructional Tool to Promote Students' Circuit Problem Solving

    ERIC Educational Resources Information Center

    Chung, Gregory K. W. K.; Shel, Tammy; Kaiser, William J.

    2006-01-01

    We examined a novel formative assessment and instructional approach with 89 students in three electrical engineering classes in special computer-based discussion sections. The technique involved students individually solving circuit problems online, with their real-time responses observed by the instructor. While exploratory, survey and…

  9. A Formative Evaluation of Customized Pamphlets to Promote Physical Activity and Symptom Self-Management in Women with Multiple Sclerosis

    ERIC Educational Resources Information Center

    Plow, Matthew; Bethoux, Francois; Mai, Kimloan; Marcus, Bess

    2014-01-01

    Inactivity is a prevalent problem in the population affected with multiple sclerosis (MS). Thus, there is a need to develop and test physical activity (PA) interventions that can be widely disseminated. We conducted a formative evaluation as part of a randomized controlled trial of a pamphlet-based PA intervention among 30 women with MS. Pamphlets…

  10. Ammonia Catalyzed Formation of Sulfuric Acid in Troposphere: The Curious Case of A Base Promoting Acid Rain.

    PubMed

    Bandyopadhyay, Biman; Kumar, Pradeep; Biswas, Partha

    2017-04-03

    Electronic structure calculations have been performed to investigate the role of ammonia in catalyzing the formation of sulfuric acid through hydrolysis of SO3 in Earth's atmosphere. The uncatalyzed process involves a high activation barrier and, till date, is mainly known to occur in Earth's atmosphere only when catalyzed by water and acids. Here we show that hydrolysis of SO3 can be very efficiently catalyzed by ammonia, the most abundant basic component in Earth's atmosphere. It was found, based on magnitude of relative potential energies as well as rate coefficients, that ammonia is the best among all the catalysts studied until now (water and acids) and could be a considerable factor in formation of sulfuric acid in troposphere. The calculated rate coefficient (at 298 K) of ammonia catalyzed reaction has been found to be ~10^5 - 10^7 times greater than that for water catalyzed ones. It was found, based on relative rates of ammonia and water catalyzed processes that in troposphere ammonia, together with water, could be the key factor in determining the rate of formation of sulfuric acid. In fact ammonia could surpass water in catalyzing formation of sulfuric acid via hydrolysis of SO3 at various altitudes in troposphere depending upon their relative concentrations.

  11. Formative evaluation for promoting adoption of the DGA, 2005 among African American parents and children in the Lower Mississippi Delta

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Formative research was conducted to increase adherence to the healthful food and physical activity patterns set forth in the Dietary Guidelines for Americans, 2005 (DGA, 2005) and thereby reduce weight gain and risk factors for obesity-related chronic diseases in African American parents and their c...

  12. TGF-β1 promotes linear invadosome formation in hepatocellular carcinoma cells, through DDR1 up-regulation and collagen I cross-linking.

    PubMed

    Ezzoukhry, Zakaria; Henriet, Elodie; Piquet, Léo; Boyé, Kevin; Bioulac-Sage, Paulette; Balabaud, Charles; Couchy, Gabrielle; Zucman-Rossi, Jessica; Moreau, Violaine; Saltel, Frédéric

    2016-11-01

    Transforming growth factor-β1 (TGF-β1) is an important player in chronic liver diseases inducing fibrogenesis and hepatocellular carcinoma (HCC) development. TGF-β1 promotes pleiotropic modifications at the cellular and matrix microenvironment levels. TGF-β1 was described to enhance production of type I collagen and its associated cross-linking enzyme, the lysyl oxidase-like2 (LOXL2). In addition, TGF-β1 and type I collagen are potent inducers of invadosomes. Indeed, type I collagen fibers induce the formation of active linear invadosomes through the discoidin domain receptor 1 (DDR1). The goal of our study was to address the role of TGF-β1 in collagen cross-linking and its impact on the formation of linear invadosomes in liver cancer cells. We first report a significant correlation between expressions of TGF-β1, and type I collagen, LOXL2, DDR1 and MT1-MMP in human HCCs. We demonstrate that TGF-β1 promotes a Smad4-dependent up-regulation of DDR1, together with LOXL2, in cultured HCC cells. Moreover, we show that LOXL2-induced collagen cross-linking enhances linear invadosome formation. Altogether, our data demonstrate that TGF-β1 favors linear invadosome formation through the expressions of both the inducers, such as collagen and LOXL2, and the components such as DDR1 and MT1-MMP of linear invadosomes in cancer cells. Meanwhile, our data uncover a new TGF-β1-dependent regulation of DDR1 expression.

  13. A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

    PubMed

    Lohmer, Lauren L; Clay, Matthew R; Naegeli, Kaleb M; Chi, Qiuyi; Ziel, Joshua W; Hagedorn, Elliott J; Park, Jieun E; Jayadev, Ranjay; Sherwood, David R

    2016-01-01

    Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

  14. Effect of carbodiimide-derivatized hyaluronic acid gelatin on preventing postsurgical intra-abdominal adhesion formation and promoting healing in a rat model.

    PubMed

    Yuan, Fang; Lin, Long-Xiang; Zhang, Hui-Hui; Huang, Dan; Sun, Yu-Long

    2016-05-01

    Adhesions often occur after abdominal surgery. It could cause chronic pelvic pain, intestinal obstruction, and infertility. A hydrogel biomaterial, carbodiimide-derivatized hyaluronic acid gelatin (cd-HA gelatin), has been successfully used to reduce adhesion formation after flexor tendon grafting. This study investigated the efficacy of cd-HA gelatin in preventing postsurgical peritoneal adhesions in a rat model. The surgical traumas were created on the underlying muscle of the abdominal wall and the serosal layer of the cecum. The wounds were covered with or without cd-HA gelatin. Animals were euthanized at day 14 after surgery. Adhesion formation was assessed with adhesion degree and adhesion breaking strength. The healing of abdominal wall was evaluated with biomechanical testing and histological analysis. The adhesions occurred in all rats (n = 12) without cd-HA gelatin treatment. The application of cd-HA gelatin significantly reduced the adhesion rate from 100% to 58%. The decrease of adhesion breaking strength also manifested that cd-HA gelatin could reduce postsurgical intra-abdominal adhesion formation. Moreover, it was found that cd-HA gelatin was a safe material and could promote tissue healing. The cd-HA gelatin hydrogel could reduce the formation of intra-abdominal adhesions without adversely effects on wound healing.

  15. miR-29b promotes skin wound healing and reduces excessive scar formation by inhibition of the TGF-β1/Smad/CTGF signaling pathway.

    PubMed

    Guo, Jingdong; Lin, Quan; Shao, Ying; Rong, Li; Zhang, Duo

    2017-04-01

    The hypertrophic scar is a medical difficulty of humans, which has caused great pain to patients. Here, we investigated the inhibitory effect of miR-29b on scar formation. The scalded model was established in mice and miR-29b mimics or a negative control was subcutaneously injected into the injury skin. Then various molecular biological experiments were performed to assess the effect of miR-29b on scar formation. According to our present study, first, the results demonstrated that miR-29b was down-regulated in thermal injury tissue and miR-29b treatment could promote wound healing, inhibit scar formation, and alleviate histopathological morphologic alteration in scald tissues. Additionally, miR-29b treatment suppressed collagen deposition and fibrotic gene expression in scar tissues. Finally, we found that miR-29b treatment inhibited the TGF-β1/Smad/CTGF signaling pathway. Taken together, our data suggest that miR-29b treatment has an inhibitory effect against scar formation via inhibition of the TGF-β1/Smad/CTGF signaling pathway and may provide a potential molecular basis for future treatments for hypertrophic scars.

  16. A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation.

    PubMed Central

    Tuschl, T; Sharp, P A; Bartel, D P

    2001-01-01

    In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 5' exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation. PMID:11214178

  17. Ficolin-1-PTX3 complex formation promotes clearance of altered self-cells and modulates IL-8 production.

    PubMed

    Ma, Ying Jie; Doni, Andrea; Romani, Luigina; Jürgensen, Henrik Jessen; Behrendt, Niels; Mantovani, Alberto; Garred, Peter

    2013-08-01

    The long pentraxin 3 (PTX3) has been shown to be important in maintaining internal tissue homeostasis and in protecting against fungal Aspergillus fumigatus infection. However, the molecular mechanisms of how these functions are elicited are poorly delineated. Ficolin-1 is a soluble pattern recognition molecule that interacts with PTX3. We hypothesized that heterocomplexes between ficolin-1 and PTX3 might mediate the signals necessary for sequestration of altered self-cells and A. fumigatus. We were able to show that ficolin-1 interacts with PTX3 via its fibrinogen-like domain. The interaction was affected in a pH- and divalent cation-sensitive manner. The primary binding site for ficolin-1 on PTX3 was located in the N-terminal domain portion of PTX3. Ficolin-1 and PTX3 heterocomplex formation occurred on dying host cells, but not on A. fumigatus. The heterocomplex formation was a prerequisite for enhancement of phagocytosis by human monocyte-derived macrophages and downregulation of IL-8 production during phagocytosis. On A. fumigatus, PTX3 exposed the C-terminal portion of the molecule, probably resulting in steric hindrance of ficolin-1 interaction with PTX3. These results demonstrate that ficolin-1 and PTX3 heterocomplex formation acts as a noninflammatory "find me and eat me" signal to sequester altered-host cells. The fact that the ficolin-1-PTX3 complex formation did not occur on A. fumigatus shows that PTX3 uses different molecular effector mechanisms, depending on which domains it exposes during ligand interaction.

  18. Phosphoinositide 3-kinase p110δ promotes lumen formation through enhancement of apico-basal polarity and basal membrane organization

    PubMed Central

    Sar, Sokhavuth; Komaiha, Ola Hamze; Moyano, Romina; Rayal, Amel; Samuel, Didier; Shewan, Annette; Vanhaesebroeck, Bart; Mostov, Keith; Gassama-Diagne, Ama

    2016-01-01

    Signaling triggered by adhesion to the extracellular matrix plays a key role in the spatial orientation of epithelial polarity and formation of lumens in glandular tissues. Phosphoinositide 3-kinase signaling in particular is known to influence the polarization process during epithelial cell morphogenesis. Here, using Madin-Darby canine kidney epithelial cells grown in 3D culture, we show that the p110δ isoform of phosphoinositide 3-kinase colocalizes with focal adhesion proteins at the basal surface of polarized cells. Pharmacological, siRNA- or kinase-dead mediated inhibition of p110δ impair the early stages of lumen formation, resulting in inverted polarized cysts, with no laminin or type IV collagen assembly at cell/extracellular matrix contacts. p110δ also regulates the organization of focal adhesions and membrane localization of dystroglycan. Thus, we uncover a previously unrecognized role for p110δ in epithelial cells in the orientation of the apico-basal axis and lumen formation. PMID:25583025

  19. Capsicum annuum S (CaS) promotes reproductive transition and is required for flower formation in pepper (Capsicum annuum).

    PubMed

    Cohen, Oded; Borovsky, Yelena; David-Schwartz, Rakefet; Paran, Ilan

    2014-05-01

    The genetic control of the transition to flowering has mainly been studied in model species, while few data are available in crop species such as pepper (Capsicum spp.). To elucidate the genetic control of the transition to flowering in pepper, mutants that lack flowers were isolated and characterized. Genetic mapping and sequencing allowed the identification of the gene disrupted in the mutants. Double mutants and expression analyses were used to characterize the relationships between the mutated gene and other genes controlling the transition to flowering and flower differentiation. The mutants were characterized by a delay in the initiation of sympodial growth, a delay in the termination of sympodial meristems and complete inhibition of flower formation. Capsicum annuum S (CaS), the pepper (Capsicum annuum) ortholog of tomato (Solanum lycopersicum) COMPOUND INFLORESCENCE and petunia (Petunia hybrida) EVERGREEN, was found to govern the mutant phenotype. CaS is required for the activity of the flower meristem identity gene Ca-ANANTHA and does not affect the expression of CaLEAFY. CaS is epistatic over other genes controlling the transition to flowering with respect to flower formation. Comparative homologous mutants in the Solanaceae indicate that CaS has uniquely evolved to have a critical role in flower formation, while its role in meristem maturation is conserved in pepper, tomato and petunia.

  20. Chromosomal gain promotes formation of a steep RanGTP gradient that drives mitosis in aneuploid cells

    PubMed Central

    Hasegawa, Keisuke; Ryu, Sung Jin

    2013-01-01

    Many mitotic factors were shown to be activated by Ran guanosine triphosphatase. Previous studies in Xenopus laevis egg extracts and in highly proliferative cells showed that mitotic chromosomes were surrounded by steep Ran guanosine triphosphate (GTP) concentration gradients, indicating that RanGTP-activated factors promote spindle assembly around chromosomes. However, the mitotic role of Ran in normal differentiated cells is not known. In this paper, we show that although the steep mitotic RanGTP gradients were present in rapidly growing cell lines and were required for chromosome congression in mitotic HeLa cells, the gradients were strongly reduced in slow-growing primary cells, such as HFF-1 fibroblasts. The overexpression of RCC1, the guanine nucleotide exchange factor for Ran, induced steeper mitotic RanGTP gradients in HFF-1 cells, showing the critical role of RCC1 levels in the regulation of mitosis by Ran. Remarkably, in vitro fusion of HFF-1 cells produced cells with steep mitotic RanGTP gradients comparable to HeLa cells, indicating that chromosomal gain can promote mitosis in aneuploid cancer cells via Ran. PMID:23319601

  1. The Arabidopsis Transcription Factor MYB112 Promotes Anthocyanin Formation during Salinity and under High Light Stress1[OPEN

    PubMed Central

    Lotkowska, Magda E.; Tohge, Takayuki; Fernie, Alisdair R.; Xue, Gang-Ping; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-01-01

    MYB transcription factors (TFs) are important regulators of flavonoid biosynthesis in plants. Here, we report MYB112 as a formerly unknown regulator of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana). Expression profiling after chemically induced overexpression of MYB112 identified 28 up- and 28 down-regulated genes 5 h after inducer treatment, including MYB7 and MYB32, which are both induced. In addition, upon extended induction, MYB112 also positively affects the expression of PRODUCTION OF ANTHOCYANIN PIGMENT1, a key TF of anthocyanin biosynthesis, but acts negatively toward MYB12 and MYB111, which both control flavonol biosynthesis. MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative polymerase chain reaction, we show that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters, revealing them as direct downstream target genes. We further show that MYB112 expression is up-regulated by salinity and high light stress, environmental parameters that both require the MYB112 TF for anthocyanin accumulation under these stresses. In contrast to several other MYB TFs affecting anthocyanin biosynthesis, MYB112 expression is not controlled by nitrogen limitation or an excess of carbon. Thus, MYB112 constitutes a regulator that promotes anthocyanin accumulation under abiotic stress conditions. PMID:26378103

  2. Translocations of Chromosome End-Segments and Facultative Heterochromatin Promote Meiotic Ring Formation in Evening Primroses[W][OPEN

    PubMed Central

    Golczyk, Hieronim; Massouh, Amid; Greiner, Stephan

    2014-01-01

    Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled end-segments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories. PMID:24681616

  3. Transplantation of hUC-MSCs seeded collagen scaffolds reduces scar formation and promotes functional recovery in canines with chronic spinal cord injury

    PubMed Central

    Li, Xing; Tan, Jun; Xiao, Zhifeng; Zhao, Yannan; Han, Sufang; Liu, Dingyang; Yin, Wen; Li, Jing; Li, Juan; Wanggou, Siyi; Chen, Bing; Ren, Caiping; Jiang, Xingjun; Dai, Jianwu

    2017-01-01

    Spinal cord injury (SCI) can lead to locomotor deficits, and the repair of chronic SCI is considered one of the most challenging clinical problems. Although extensive studies have evaluated treatments for acute SCI in small animals, comparatively fewer studies have been conducted on large-animal SCI in the chronic phase, which is more clinically relevant. Here, we used a collagen-based biomaterial, named the NeuroRegen scaffold, loaded with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in a canine chronic SCI model. To generate chronic SCI, the T8 spinal cord segment was removed by complete transection of the spinal cord. Two months later, glial scar tissue was removed and a NeuroRegen scaffold was transplanted into the lesion area. Functionalized NeuroRegen scaffold implantation promoted both locomotor recovery and endogenous neurogenesis in the lesion area. Moreover, some newly generated neurons successfully matured into 5-HT-positive neurons at 1 year post-injury. In addition, many regenerated axon fibers in the lesion area exhibited remyelination and synapse formation at 1 year post-injury in the functionalized NeuroRegen scaffold group. In conclusion, the NeuroRegen scaffold functionalized with hUC-MSCs is a promising potential therapeutic approach to chronic SCI that promotes neuronal regeneration, reduces glial scar formation, and ultimately improves locomotor recovery. PMID:28262732

  4. The sex-determining factors SRY and SOX9 regulate similar target genes and promote testis cord formation during testicular differentiation.

    PubMed

    Li, Yunmin; Zheng, Ming; Lau, Yun-Fai Chris

    2014-08-07

    Male sex determination is mediated sequentially by sex-determining region Y (SRY) and related SRY-box 9 (SOX9) transcription factors. To understand the gene regulatory hierarchy for SRY and SOX9, a series of chromatin immunoprecipitation and whole-genome promoter tiling microarray (ChIP-Chip) experiments were conducted with mouse gonadal cells at the time of sex determination. SRY and SOX9 bind to the promoters of many common targets involved in testis differentiation and regulate their expression in Sertoli cells. SRY binds to various ovarian differentiation genes and represses their activation through WNT/β-catenin signaling. Sertoli cell-Sertoli cell junction signaling, important for testis cord formation, is the top canonical pathway among the SRY and SOX9 targets. Hence, SRY determines Sertoli cell fate by repressing ovarian and activating testicular differentiation genes, promotes early Sertoli cells to form testis cord, and then passes on its functions to SOX9, which regulates common targets and activates its own gene regulatory program, beyond SRY actions, in sex determination.

  5. A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

    PubMed Central

    Suda, T; Mishima, Y; Takayanagi, K; Asakura, H; Odani, S; Kominami, R

    1996-01-01

    The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin. PMID:8972860

  6. Influence of Blend Ratio and Processing Additive on Free Carrier Yield and Mobility in PTB7:PC71BM Photovoltaic Solar Cells.

    PubMed

    Pranculis, Vytenis; Ruseckas, Arvydas; Vithanage, Dimali A; Hedley, Gordon J; Samuel, Ifor D W; Gulbinas, Vidmantas

    2016-05-12

    Charge separation and extraction dynamics were investigated in high-performance bulk heterojunction solar cells made from the polymer PTB7 and the soluble fullerene PC71BM on a broad time scale from subpicosecond to microseconds using ultrafast optical probing of carrier drift and the integral-mode photocurrent measurements. We show that the short circuit current is determined by the separation of charge pairs into free carriers, which is strongly influenced by blend composition. This separation is found to be efficient in fullerene-rich blends where a high electron mobility of >0.1 cm(2) V(-1) s(-1) is observed in the first 10 ps after excitation. Morphology optimization using the solvent additive 1,8-diiodooctane (DIO) doubles the charge pair separation efficiency and the short-circuit current. Carrier extraction at low internal electric field is slightly faster from the cells prepared with DIO, which can reduce recombination losses and enhance a fill factor.

  7. Insights on co-catalyst-promoted enamine formation between dimethylamine and propanal through ab initio and density functional theory study.

    PubMed

    Patil, Mahendra P; Sunoj, Raghavan B

    2007-10-26

    The mechanistic details on enamine formation between dimethylamine and propanal are unraveled using the ab initio and density functional theory methods. The addition of secondary amine to the electrophile and simultaneous proton transfer results in a carbinolamine intermediate, which subsequently undergoes dehydration to form enamine. The direct addition of amine as well as the dehydration of the resulting carbinolamine intermediate is predicted to possess fairly high activation barrier implying that a unimolecular process is unlikely to be responsible for enamine formation. Different models are therefore proposed which could explain the relative ease of enamine formation under neat condition as well as under the influence of methanol as the co-catalyst. The explicit inclusion of either the reagent or the co-catalyst is considered in the transition states as stabilizing agents. The participation of the reagent or the co-catalyst as a monofunctional ancillary species is found to stabilize the transition states relative to the unassisted or the direct addition/dehydration pathways. The reduction in enthalpy of activation is found to be much more dramatic when two co-catalysts participate in an active bifunctional mode in the rate-determining dehydration step. The transition structures exhibited characteristic features of a relay proton transfer mechanism. The free energy of activation associated with the two methanol-assisted pathway is found to be 16.7 kcal/mol lower than that of the unassisted pathway. The results are found to be in concurrence with the available reports on the rate acceleration by co-catalysts in the Michael reaction between enamine and methyl vinyl ketone under neat conditions.

  8. Loss of nuclear receptor RXRα in epidermal keratinocytes promotes the formation of Cdk4-activated invasive melanomas.

    PubMed

    Hyter, Stephen; Bajaj, Gaurav; Liang, Xiaobo; Barbacid, Mariano; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2010-10-01

    Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.

  9. Precise Formation of a Hollow Carbon Nitride Structure with a Janus Surface To Promote Water Splitting by Photoredox Catalysis

    PubMed Central

    Zheng, Dandan; Cao, Xu‐Ning

    2016-01-01

    Abstract The precise modification of redox species on the inner and outer surfaces of hollow nanostructures is relevant in catalysis, surface science, and nanotechnology, but has proven difficult to achieve. Herein, we develop a facile approach to specifically fabricate Pt and Co3O4 nanoparticles (NPs) onto the interior and exterior surface of hollow carbon nitride spheres (HCNS), respectively, to promote the surface redox functions of the polymer semiconductors. The photocatalytic water splitting activities of HCNS with spatially separated oxidation and reduction centers at their nanodomains were enhanced. The origin of the enhanced activity was attributed to the spatially separated reactive sites for the evolution of H2 and O2 and also to the unidirectional migration of the electron and hole on the Janus surfaces, thereby preventing the unwanted reverse reaction of water splitting and decreasing charge recombination. PMID:27533739

  10. FGFR3 promotes angiogenesis-dependent metastasis of hepatocellular carcinoma via facilitating MCP-1-mediated vascular formation.

    PubMed

    Liu, Xinyu; Jing, Xiaoqian; Cheng, Xi; Ma, Ding; Jin, Zhijian; Yang, Weiping; Qiu, Weihua

    2016-05-01

    The biological role of fibroblast growth factor receptor 3 (FGFR3) in tumor angiogenesis of hepatocellular carcinoma (HCC) has not been discussed before. Our previous work had indicated FGFR3 was overexpressed in HCC, and silencing FGFR3 in Hu7 cells could regulate tumorigenesis via down-regulating the phosphorylation level of key members of classic signaling pathways including ERK and AKT. In the present work, we explored the role of FGFR3 in angiogenesis-dependent metastasis by using SMMC-7721 and QGY-7703 stable cell lines. Our results indicated FGFR3 could regulate in vitro cell migration ability and in vivo lung metastasis ability of HCC, which was in accordance with increased angiogenesis ability in vitro and in vivo. Using the supernatant from SMMC-7721/FGFR3 cells, we conducted a human angiogenesis protein microarray including 43 angiogenesis factors and found that FGFR3 modulated angiogenesis and metastasis of HCC mainly by promoting the protein level of monocyte chemotactic protein 1 (MCP-1). Silencing FGFR3 by short hairpin RNA (shRNA) could reduce MCP-1 level in lysates and supernatant of QGY-7703 cells and SMMC-7721 cells. Silencing MCP-1 in QGY-7703 or SMMC-7721 cells could induce similar phenotypes compared with silencing FGFR3. Our results suggested FGFR3 promoted metastasis potential of HCC, at least partially if not all, via facilitating MCP-1-mediated angiogenesis, in addition to previously found cell growth and metastasis. MCP-1, a key medium between HCC cells and HUVECs, might be a novel anti-vascular target in HCC.

  11. KISS1R signaling promotes invadopodia formation in human breast cancer cell via β-arrestin2/ERK.

    PubMed

    Goertzen, Cameron G; Dragan, Magdalena; Turley, Eva; Babwah, Andy V; Bhattacharya, Moshmi

    2016-03-01

    Kisspeptins (KPs), peptide products of the KISS1 gene are endogenous ligands for the kisspeptin receptor (KISS1R), a G protein-coupled receptor. In numerous cancers, KISS1R signaling plays anti-metastatic roles. However, we have previously shown that in breast cancer cells lacking the estrogen receptor (ERα), kisspeptin-10 stimulates cell migration and invasion by cross-talking with the epidermal growth factor receptor (EGFR), via a β-arrestin-2-dependent mechanism. To further define the mechanisms by which KISS1R stimulates invasion, we determined the effect of down-regulating KISS1R expression in triple negative breast cancer cells. We found that depletion of KISS1R reduced their mesenchymal phenotype and invasiveness. We show for the first time that KISS1R signaling induces invadopodia formation and activation of key invadopodia proteins, cortactin, cofilin and membrane type I matrix metalloproteases (MT1-MMP). Moreover, KISS1R stimulated invadopodia formation occurs via a new pathway involving a β-arrestin2 and ERK1/2-dependent mechanism, independent of Src. Taken together, our findings suggest that targeting the KISS1R signaling axis might be a promising strategy to inhibit invasiveness and metastasis.

  12. Developmental Control of a parAB Promoter Leads to Formation of Sporulation-Associated ParB Complexes in Streptomyces coelicolor

    PubMed Central

    Jakimowicz, Dagmara; Mouz, Sebastien; Zakrzewska-Czerwińska, Jolanta; Chater, Keith F.

    2006-01-01

    The Streptomyces coelicolor partitioning protein ParB binds to numerous parS sites in the oriC-proximal part of the linear chromosome. ParB binding results in the formation of large complexes, which behave differentially during the complex life cycle (D. Jakimowicz, B. Gust, J. Zakrzewska-Czerwinska, and K. F. Chater, J. Bacteriol. 187:3572-3580, 2005). Here we have analyzed the transcriptional regulation that underpins this developmentally specific behavior. Analysis of promoter mutations showed that the irregularly spaced complexes present in vegetative hyphae are dependent on the constitutive parABp1 promoter, while sporulation-specific induction of the promoter parABp2 is required for the assembly of arrays of ParB complexes in aerial hyphae and thus is necessary for efficient chromosome segregation. Expression from parABp2 depended absolutely on two sporulation regulatory genes, whiA and whiB, and partially on two others, whiH and whiI, all four of which are needed for sporulation septation. Because of this pattern of dependence, we investigated the transcription of these four whi genes in whiA and whiB mutants, revealing significant regulatory interplay between whiA and whiB. A strain in which sporulation septation (but not vegetative septation) was blocked by mutation of a sporulation-specific promoter of ftsZ showed close to wild-type induction of parABp2 and formed fairly regular ParB-enhanced green fluorescent protein foci in aerial hyphae, ruling out strong morphological coupling or checkpoint regulation between septation and DNA partitioning during sporulation. A model for developmental regulation of parABp2 expression is presented. PMID:16484182

  13. Using Formal Embedded Formative Assessments Aligned with a Short-Term Learning Progression to Promote Conceptual Change and Achievement in Science

    NASA Astrophysics Data System (ADS)

    Yin, Yue; Tomita, Miki K.; Shavelson, Richard J.

    2014-03-01

    This study examined the effect of learning progression-aligned formal embedded formative assessment on conceptual change and achievement in middle-school science. Fifty-two sixth graders were randomly assigned to either an experimental group or a control group. Both groups were taught about sinking and floating by the same teacher with identical curriculum materials and activities. The experimental group received, in addition, three sets of formal embedded formative assessments with qualitative feedback as to how to improve their understandings aligned with an expected learning progression during instruction. The control group spent the corresponding time between new curriculum activities conducting curriculum-specific extension activities. Overall, the experimental group experienced on average greater conceptual change than the control group. The experimental group also scored higher on average than the control group on general achievement tests, especially the performance assessment. This study supported the contention, then, that embedding formal formative assessments within a curricular sequence built around an expected learning progression is a useful way to promote conceptual change along that learning progression in science classrooms.

  14. Promoting oxygen vacancy formation and p-type conductivity in SrTiO3via alkali metal doping: a first principles study.

    PubMed

    Triggiani, Leonardo; Muñoz-García, Ana B; Agostiano, Angela; Pavone, Michele

    2016-10-19

    Strontium titanate (SrTiO3, STO) is a prototypical perovskite oxide, widely exploited in many technological applications, from catalysis to energy conversion devices. In the context of solid-oxide fuel cells, STO has been recently applied as an epitaxial substrate for nano-sized layers of mixed ion-electron conductive catalysts with enhanced electrochemical performances. To extend the applications of such heterogeneous nano-cathodes in real devices, also the STO support should be active for both electron transport and oxide diffusion. To this end, we explored using first-principles calculations the strategy of doping of STO at the Sr site with sodium and potassium. These two ions fit in the perovskite structure and induce holes in the STO valence band, so as to obtain the desired p-type electronic conduction. At the same time, the doping with alkali ions also promotes the formation of oxygen vacancies in STO, a prerequisite for effective oxide diffusion. Analysis of electron density rearrangements upon defect formation allows relating the favorable vacancy formation energies to an improved electronic delocalization over the oxide sub-lattice, as observed in closely related materials (e.g. Sr2Fe1.5Mo0.5O6). Overall, our results suggest the alkali-doped STO as a new potential substrate material in nanoscale heterogeneous electrodes for solid oxide electrochemical cells.

  15. Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration

    PubMed Central

    Ko, Hyo Rim; Kwon, Il-Sun; Hwang, Inwoo; Jin, Eun-Ju; Shin, Joo-Ho; Brennan-Minnella, Angela M; Swanson, Raymond; Cho, Sung-Woo; Lee, Kyung-Hoon; Ahn, Jee-Yin

    2016-01-01

    Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration. DOI: http://dx.doi.org/10.7554/eLife.20799.001 PMID:27938661

  16. Extracorporeal shock waves alone or combined with raloxifene promote bone formation and suppress resorption in ovariectomized rats

    PubMed Central

    Corrado, Bruno; Pirozzi, Claudio; Paciello, Orlando; Pagano, Teresa Bruna; Russo, Sergio; Calignano, Antonio; Mattace Raso, Giuseppina; Meli, Rosaria

    2017-01-01

    Osteoporosis is a metabolic skeletal disease characterized by an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. We examined the beneficial effect of shock waves (SW) alone or in combination with raloxifene (RAL) on bone loss in ovariectomized rats (OVX). Sixteen weeks after surgery, OVX were treated for five weeks with SW at the antero-lateral side of the right hind leg, one session weekly, at 3 Hz (EFD of 0.33 mJ/mm2), or with RAL (5 mg/kg/die, per os) or with SW+RAL. Sera, femurs, tibiae and vertebrae were sampled for following biochemical and histological analysis. SW, alone or combined with RAL, prevented femur weight reduction and the deterioration of trabecular microarchitecture both in femur and vertebrae. All treatments increased Speed of Sound (SoS) values, improving bone mineral density, altered by OVX. Serum parameters involved in bone remodeling (alkaline phosphatase, receptor activator of nuclear factor kappa-B ligand, osteoprotegerin) and osteoblast proliferation (PTH), altered by ovariectomy, were restored by SW and RAL alone or in combination. In tibiae, SW+RAL significantly reduced cathepsin k and TNF-α levels, indicating the inhibition of osteoclast activity, while all treatments significantly increased runt-related transcription factor 2 and bone morphogenetic-2 expression, suggesting an increase in osteoblastogenic activity. Finally, in bone marrow from tibiae, SW or RAL reduced PPARγ and adiponectin transcription, indicating a shift of mesenchymal cells toward osteoblastogenesis, without showing a synergistic effect. Our data indicate SW therapy, alone and in combination with raloxifene, as an innovative strategy to limit the hypoestrogenic bone loss, restoring the balance between bone formation and resorption. PMID:28158228

  17. Geochemical factors promoting die-back gap formation in colonizing patches of Spartina densiflora in an irregularly flooded marsh

    NASA Astrophysics Data System (ADS)

    Mirlean, Nicolai; Costa, Cesar S. B.

    2017-04-01

    Circular (RP) and ring-shape (RP) patches of vegetation in intertidal flats have been associated with the radial expansion of tussock growth forms and die-back gap in older central stands, respectively. RP formation has not yet been sufficiently explained. We accomplished a comparative geochemical study of CP and RP structures of Spartina densiflora within a single saltmarsh in a microtidal estuary (<0.5 m). The pore water under these structures demonstrated distinctive physical-chemical properties by marked seasonal changing in water level and salinity. During high-water period dissolved H2S was frequently low in pore waters of S. densiflora structures due to reactive-Fe, which scavenge the sulfide from solution and form solid sulfides. During less flooded-brackish water period, pore water pH goes down below 4 inside the vegetated bordering areas of RP. In these locations the concentration of soluble sulfides dramatically increases up to 140 μM L-1. The high concentration of protons in pore water is the result of solid sulfides atmospheric oxidation to sulfuric acid. High dissolution of H2S, along with the low pH, creates a toxic environment for S. densiflora and die-back central gap formation in RP. CP structure was 5 cm higher in the intertidal than RP but shows frequent presence of a water layer, less severe oxidation of sulfides and limited building-up of toxic condition to plants. Development of S. densiflora RP probably indicates the uplift of sediment by this bioengineer grass and/or periodic lowering of the water surface below a certain critical level.

  18. Is Active Management the Key to the Conservation of Saproxylic Biodiversity? Pollarding Promotes the Formation of Tree Hollows

    PubMed Central

    Sebek, Pavel; Altman, Jan; Platek, Michal; Cizek, Lukas

    2013-01-01

    Trees with hollows are key features sustaining biodiversity in wooded landscapes. They host rich assemblages of often highly specialised organisms. Hollow trees, however, have become rare and localised in Europe. Many of the associated biota is thus declining or endangered. The challenge of its conservation, therefore, is to safeguard the presence of hollow trees in sufficient numbers. Populations of numerous species associated with tree hollows and dead wood are often found in habitats that were formed by formerly common traditional silvicultural practices such as coppicing, pollarding or pasture. Although it has been occasionally mentioned that such practices increase the formation of hollows and the availability of often sun-exposed dead wood, their effect has never been quantified. Our study examined the hollow incidence in pollard and non-pollard (unmanaged) willows and the effect of pollarding on incremental growth rate by tree ring analysis. The probability of hollow occurrence was substantially higher in pollard than in non-pollard trees. Young pollards, especially, form hollows much more often than non-pollards; for instance, in trees of 50 cm DBH, the probability of hollow ocurrence was ∼0.75 in pollards, but only ∼0.3 in non-pollards. No difference in growth rate was found. Pollarding thus leads to the rapid formation of tree hollows, a habitat usually associated with old trees. It is therefore potentially a very important tool in the restoration of saproxylic habitats and conservation of hollow-dependent fauna. If applied along e.g. roads and watercourses, pollarding could also be used to increase landscape connectivity for saproxylic organisms. In reserves where pollarding was formerly practiced, its restoration would be necessary to prevent loss of saproxylic biodiversity. Our results point to the importance of active management measures for maintaining availability, and spatial and temporal continuity of deadwood microhabitats. PMID:23544142

  19. Progesterone promotes focal adhesion formation and migration in breast cancer cells through induction of protease-activated receptor-1.

    PubMed

    Diaz, Jorge; Aranda, Evelyn; Henriquez, Soledad; Quezada, Marisol; Espinoza, Estefanía; Bravo, Maria Loreto; Oliva, Bárbara; Lange, Soledad; Villalon, Manuel; Jones, Marius; Brosens, Jan J; Kato, Sumie; Cuello, Mauricio A; Knutson, Todd P; Lange, Carol A; Leyton, Lisette; Owen, Gareth I

    2012-08-01

    Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3  h and returning to basal levels at 18  h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.

  20. SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation

    PubMed Central

    Farrawell, Natalie; Shearer, Robert F.; Constantinescu, Patrick; Hatters, Danny M.; Schroder, Wayne A.; Suhrbier, Andreas; Wilson, Mark R.; Saunders, Darren N.; Ranson, Marie

    2015-01-01

    SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS. PMID:26083412

  1. Strain differences in urinary factors that promote calcium oxalate crystal formation in the kidneys of ethylene glycol-treated rats.

    PubMed

    Li, Yan; McMartin, Kenneth E

    2009-05-01

    Ethylene glycol (EG)-induced hyperoxaluria is the most commonly employed experimental regimen as an animal model of calcium oxalate (CaOx) stone formation. The variant sensitivity to CaOx among different rat strains has not been fully explored, although the Wistar rat is known to accumulate more CaOx in kidney tissue after low-dose EG exposure than in the Fischer 344 (F344) rats. Supersaturation of CaOx in tubular fluid contributes to the amount of CaOx crystal formation in the kidney. We hypothesized that the urinary supersaturation of CaOx in Wistar rats is higher than that of F344 rats, thereby allowing for greater CaOx crystal deposition in the Wistar rat. Age-matched male Wistar and F344 rats were treated with 0.75% EG or drinking water for 8 wk. Twenty-four-hour urine was collected at 0, 2, 4, 6, and 8 wk for analysis of key electrolytes to calculate the CaOx supersaturation. Plasma oxalate level was also measured. Our data confirmed the different sensitivity to renal toxicity from EG between the two rat strains (Wistar > F344). After EG treatment, the plasma oxalate level and urine oxalate excretion were markedly greater in the Wistar rats than in the F344 rats, while urine calcium was slightly decreased in Wistars. Thus, the CaOx supersaturation in urine of Wistar rats was higher, which led to a greater crystal deposition in kidney in Wistar rats. These studies suggest that during EG treatment, changes in urine electrolytes and in CaOx supersaturation occur to a greater extent in the Wistar rat, in agreement with its greater sensitivity to EG toxicity.

  2. Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils

    PubMed Central

    Funchal, Giselle A.; Jaeger, Natália; Czepielewski, Rafael S.; Machado, Mileni S.; Muraro, Stéfanie P.; Stein, Renato T.; Bonorino, Cristina B. C.; Porto, Bárbara N.

    2015-01-01

    Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis. PMID:25856628

  3. Bacterial genotoxins promote inside-out integrin β1 activation, formation of focal adhesion complexes and cell spreading.

    PubMed

    Levi, Laura; Toyooka, Tatsushi; Patarroyo, Manuel; Frisan, Teresa

    2015-01-01

    Integrins are membrane bound receptors that regulate several cellular processes, such as cell adhesion, migration, survival and proliferation, and may contribute to tumor initiation/progression in cells exposed to genotoxic stress. The extent of integrin activation and its role in cell survival upon intoxication with bacterial genotoxins are still poorly characterized. These toxins induce DNA strand breaks in the target cells and activate the DNA damage response (DDR), coordinated by the Ataxia Telangectasia Mutated (ATM) kinase. In the present study, we demonstrate that induction of DNA damage by two bacterial genotoxins promotes activation of integrin β1, leading to enhanced assembly of focal adhesions and cell spreading on fibronectin, but not on vitronectin. This phenotype is mediated by an ATM-dependent inside-out integrin signaling, and requires the actin cytoskeleton remodeler NET1. The toxin-mediated cell spreading and anchorage-independent survival further relies on ALIX and TSG101, two components of the endosomal sorting complex required for transport (ESCRT), known to regulate integrin intracellular trafficking. These data reveal a novel aspect of the cellular response to bacterial genotoxins, and provide new tools to understand the carcinogenic potential of these effectors in the context of chronic intoxication and infection.

  4. α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

    PubMed Central

    Burré, Jacqueline; Sharma, Manu; Südhof, Thomas C.

    2014-01-01

    Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration. PMID:25246573

  5. TGF-β induces p53/Smads complex formation in the PAI-1 promoter to activate transcription

    PubMed Central

    Kawarada, Yuki; Inoue, Yasumichi; Kawasaki, Fumihiro; Fukuura, Keishi; Sato, Koichi; Tanaka, Takahito; Itoh, Yuka; Hayashi, Hidetoshi

    2016-01-01

    Transforming growth factor β (TGF-β) signaling facilitates tumor development during the advanced stages of tumorigenesis, but induces cell-cycle arrest for tumor suppression during the early stages. However, the mechanism of functional switching of TGF-β is still unknown, and it is unclear whether inhibition of TGF-β signaling results amelioration or exacerbation of cancers. Here we show that the tumor suppressor p53 cooperates with Smad proteins, which are TGF-β signal transducers, to selectively activate plasminogen activator inhibitor type-1 (PAI-1) transcription. p53 forms a complex with Smad2/3 in the PAI-1 promoter to recruit histone acetyltransferase CREB-binding protein (CBP) and enhance histone H3 acetylation, resulting in transcriptional activation of the PAI-1 gene. Importantly, p53 is required for TGF-β-induced cytostasis and PAI-1 is involved in the cytostatic activity of TGF-β in several cell lines. Our results suggest that p53 enhances TGF-β-induced cytostatic effects by activating PAI-1 transcription, and the functional switching of TGF-β is partially caused by p53 mutation or p53 inactivation during cancer progression. It is expected that these findings will contribute to optimization of TGF-β-targeting therapies for cancer. PMID:27759037

  6. Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body.

    PubMed

    Terzo, Esteban A; Lyons, Shawn M; Poulton, John S; Temple, Brenda R S; Marzluff, William F; Duronio, Robert J

    2015-04-15

    Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.

  7. JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein.

    PubMed

    Zhao, L; Zhang, Y; Gao, Y; Geng, P; Lu, Y; Liu, X; Yao, R; Hou, P; Liu, D; Lu, J; Huang, B

    2015-10-01

    Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which can stabilize the state of senescence. The retinoblastoma (RB) protein has an important role in SAHF; cells that lack active RB pathway fail to form SAHF. It has been known that the posttranslational modifications of RB, for example, phosphorylation, regulate its function. To date, whether methylation of RB impacts on the SAHF formation is unknown. Here we report that JMJD3, a histone demethylase catalyzing the tri-methylation of H3K27 (H3K27me3), can demethylate the non-histone protein RB at the lysine810 residue (K810), which is a target of the methyltransferase Set7/9. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV(12), and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with RB through its demethylase domain JmjC. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4 and resulted in reduced level of phosphorylation of RB at Serine807/811 (S807/811), implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly. This study highlights the role of JMJD3 as a novel inducer of SAHF formation through demethylating RB and provides new insights into the mechanisms of cellular senescence and SAHF assembly.

  8. The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

    PubMed Central

    Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

    2016-01-01

    The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

  9. cis-Cinnamic Acid Is a Novel, Natural Auxin Efflux Inhibitor That Promotes Lateral Root Formation1[OPEN

    PubMed Central

    Steenackers, Ward; Corneillie, Sander; Araújo, Pedro; Viaene, Tom; Nowack, Moritz K.; Blakeslee, Joshua J.; Novák, Ondřej; Zažímalová, Eva

    2017-01-01

    Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis. PMID:27837086

  10. Formation of single domain magnetite by green rust oxidation promoted by microbial anaerobic nitrate-dependent iron oxidation

    NASA Astrophysics Data System (ADS)

    Miot, Jennyfer; Li, Jinhua; Benzerara, Karim; Sougrati, Moulay Tahar; Ona-Nguema, Georges; Bernard, Sylvain; Jumas, Jean-Claude; Guyot, François

    2014-08-01

    Biomineralization of magnetite is a central geomicrobiological process that might have played a primordial role over Earth’s history, possibly leaving traces of life in the geological record or controlling trace metal(loid)s and organic pollutants mobility in modern environments. Magnetite biomineralization has been attributed to two main microbial pathways to date (namely magnetotactic bacteria and dissimilatory iron-reducing bacteria). Here, we uncover a new route of magnetite biomineralization involving the anaerobic nitrate-reducing iron(II) oxidizing bacterium Acidovorax sp. strain BoFeN1. Using transmission electron microscopy, scanning transmission X-ray microscopy, transmission Mössbauer spectroscopy and rock magnetic analyses, this strain is shown to promote the transformation of hydroxychloride green rust in equilibrium with dissolved Fe(II) to (1) periplasmic lepidocrocite (γ-FeOOH) and (2) extracellular magnetite, thus leading to strong redox heterogeneities at the nanometer scale. On the one hand, lepidocrocite was associated with protein moieties and exhibited an anisotropic texture, with the elongated axis parallel to the cell wall. On the other hand, magnetite crystals exhibited grain sizes and magnetic properties consistent with stable single domain particles. By comparison, abiotic controls led to a very slow (4 months vs. 2 days in BoFeN1 cultures) and incomplete oxidation of hydroxychloride green rust towards magnetite. As this abiotic magnetite exhibited the same size and magnetic properties (stable single domain particles) as magnetite produced in BoFeN1 cultures, only the co-occurrence of textured Fe(III)-oxides and magnetite, associated with the persistence of organic carbon molecules, might constitute valuable biosignatures to be looked for in the geological record. Our results furthermore contribute to a more complex picture of Fe redox cycling in the environment, providing an additional process of Fe(II)-bearing phase

  11. RecBCD-Dependent Joint Molecule Formation Promoted by the Escherichia coli RecA and SSB Proteins

    NASA Astrophysics Data System (ADS)

    Roman, Linda J.; Dixon, Dan A.; Kowalczykowski, Stephen C.

    1991-04-01

    We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB), RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.

  12. Multinucleation and Polykaryon Formation is Promoted by the EhPC4 Transcription Factor in Entamoeba histolytica

    PubMed Central

    Cruz, Olga Hernández de la; Marchat, Laurence A.; Guillén, Nancy; Weber, Christian; Rosas, Itzel López; Díaz-Chávez, José; Herrera, Luis; Rojo-Domínguez, Arturo; Orozco, Esther; López-Camarillo, César

    2016-01-01

    Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica. PMID:26792358

  13. Surface Topography and Mechanical Strain Promote Keratocyte Phenotype and Extracellular Matrix Formation in a Biomimetic 3D Corneal Model.

    PubMed

    Zhang, Wei; Chen, Jialin; Backman, Ludvig J; Malm, Adam D; Danielson, Patrik

    2017-03-01

    The optimal functionality of the native corneal stroma is mainly dependent on the well-ordered arrangement of extracellular matrix (ECM) and the pressurized structure. In order to develop an in vitro corneal model, it is crucial to mimic the in vivo microenvironment of the cornea. In this study, the influence of surface topography and mechanical strain on keratocyte phenotype and ECM formation within a biomimetic 3D corneal model is studied. By modifying the surface topography of materials, it is found that patterned silk fibroin film with 600 grooves mm(-1) optimally supports cell alignment and ECM arrangement. Furthermore, treatment with 3% dome-shaped mechanical strain, which resembles the shape and mechanics of native cornea, significantly enhances the expression of keratocyte markers as compared to flat-shaped strain. Accordingly, a biomimetic 3D corneal model, in the form of a collagen-modified, silk fibroin-patterned construct subjected to 3% dome-shaped strain, is created. Compared to traditional 2D cultures, it supports a significantly higher expression of keratocyte and ECM markers, and in conclusion better maintains keratocyte phenotype, alignment, and fusiform cell shape. Therefore, the novel biomimetic 3D corneal model developed in this study serves as a useful in vitro 3D culture model to improve current 2D cultures for corneal studies.

  14. Multinucleation and Polykaryon Formation is Promoted by the EhPC4 Transcription Factor in Entamoeba histolytica.

    PubMed

    Hernández de la Cruz, Olga; Marchat, Laurence A; Guillén, Nancy; Weber, Christian; López Rosas, Itzel; Díaz-Chávez, José; Herrera, Luis; Rojo-Domínguez, Arturo; Orozco, Esther; López-Camarillo, César

    2016-01-21

    Entamoeba histolytica is the intestinal parasite responsible for human amoebiasis that is a leading cause of death in developing countries. In this protozoan, heterogeneity in DNA content, polyploidy and genome plasticity have been associated to alterations in mechanisms controlling DNA replication and cell division. Studying the function of the transcription factor EhPC4, we unexpectedly found that it is functionally related to DNA replication, and multinucleation. Site-directed mutagenesis on the FRFPKG motif revealed that the K127 residue is required for efficient EhPC4 DNA-binding activity. Remarkably, overexpression of EhPC4 significantly increased cell proliferation, DNA replication and DNA content of trophozoites. A dramatically increase in cell size resulting in the formation of giant multinucleated trophozoites (polykaryon) was also found. Multinucleation event was associated to cytokinesis failure leading to abortion of ongoing cell division. Consistently, genome-wide profiling of EhPC4 overexpressing trophozoites revealed the up-regulation of genes involved in carbohydrates and nucleic acids metabolism, chromosome segregation and cytokinesis. Forced overexpression of one of these genes, EhNUDC (nuclear movement protein), led to alterations in cytokinesis and partially recapitulated the multinucleation phenotype. These data indicate for the first time that EhPC4 is associated with events related to polyploidy and genome stability in E. histolytica.

  15. Rhodium Complexes Promoting C-O Bond Formation in Reactions with Oxygen: The Role of Superoxo Species.

    PubMed

    Vilella-Arribas, Laia; García-Melchor, Max; Balcells, David; Lledós, Agustí; López, José A; Sancho, Sofía; Villarroya, B Eva; Del Río, M Pilar; Ciriano, Miguel A; Tejel, Cristina

    2017-01-28

    C-O bond formation in reactions of olefins with oxygen is a long standing challenge in chemistry for which the very complicated-sometimes controversial-mechanistic panorama slows down the design of catalysts for oxygenations. In this regard, the mechanistic details of the oxidation of the complex [Rh(cod)(Ph2 N3 )] (1) (cod=1,5-cyclooctadiene) with oxygen to the unique 2-rhodaoxetane compound [{Rh(OC8 H12 )(Ph2 N3 )}2 ] (2) has been investigated by DFT calculations. The results of this study provide evidences for a novel bimetallic mechanism in which two rhodium atoms redistribute the four electrons involved in the cleavage of the O=O bond. Furthermore, both oxygen atoms are used to create two new C-O bonds in a controlled fashion with 100 % atom economy. The key intermediates that we have found in this process are a mononuclear open-shell triplet superoxo compound, an open-shell singlet "μ-(peroxo)" derivative, and a closed-shell singlet "bis(μ-oxo)" complex. Some of the findings are used to predict the reactions of Rh(I) complexes with oxygen, exemplified by that of the complex [Rh(cod)(OnapyMe2 )] (3). Starting from 3, [{Rh(OC8 H12 )(OnapyMe2 )}2 ] (4) has been prepared and characterized, which represents the second example of a 2-rhodaoxetane compound coming from an oxygenation reaction with oxygen.

  16. Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins

    PubMed Central

    Cogoi, Susanna; Paramasivam, Manikandan; Spolaore, Barbara; Xodo, Luigi E.

    2008-01-01

    The human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with Tm of ∼55°C (Q1) and ∼72°C (Q2). DMS-footprinting and CD suggest that Q1 can be a parallel and Q2 a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA–protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS. PMID:18490377

  17. Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue.

    PubMed

    Clark, Abbot F; Brotchie, Daniel; Read, A Thomas; Hellberg, Peggy; English-Wright, Sherry; Pang, Iok-Hou; Ethier, C Ross; Grierson, Ian

    2005-02-01

    Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.

  18. Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro.

    PubMed

    Hafiane, Anouar; Bielicki, John K; Johansson, Jan O; Genest, Jacques

    2015-01-01

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are

  19. Novel apo E-derived ABCA1 agonist peptide (CS-6253) promotes reverse cholesterol transport and induces formation of preβ-1 HDL in vitro

    DOE PAGES

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; ...

    2015-07-24

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from themore » carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are

  20. Potassium Ions Promote Solution-Route Li2O2 Formation in the Positive Electrode Reaction of Li-O2 Batteries.

    PubMed

    Matsuda, Shoichi; Kubo, Yoshimi; Uosaki, Kohei; Nakanishi, Shuji

    2017-03-16

    Lithium-oxygen system has attracted much attention as a battery with high energy density that could satisfy the demands for electric vehicles. However, because lithium peroxide (Li2O2) is formed as an insoluble and insulative discharge product at the positive electrode, Li-O2 batteries have poor energy capacities. Although Li2O2 deposition on the positive electrode can be avoided by inducing solution-route pathway using electrolytes composed of high donor number (DN) solvents, such systems generally have poor stability. Herein we report that potassium ions promote the solution-route formation of Li2O2. The present findings suggest that potassium or other monovalent ions have the potential to increase the volumetric energy density and life cycles of Li-O2 batteries.

  1. Using poly(lactic-co-glycolic acid) microspheres to encapsulate plasmid of bone morphogenetic protein 2/polyethylenimine nanoparticles to promote bone formation in vitro and in vivo.

    PubMed

    Qiao, Chunyan; Zhang, Kai; Jin, Han; Miao, Leiying; Shi, Ce; Liu, Xia; Yuan, Anliang; Liu, Jinzhong; Li, Daowei; Zheng, Changyu; Zhang, Guirong; Li, Xiangwei; Yang, Bai; Sun, Hongchen

    2013-01-01

    Repair of large bone defects is a major challenge, requiring sustained stimulation to continually promote bone formation locally. Bone morphogenetic protein 2 (BMP-2) plays an important role in bone development. In an attempt to overcome this difficulty of bone repair, we created a delivery system to slowly release human BMP-2 cDNA plasmid locally, efficiently transfecting local target cells and secreting functional human BMP-2 protein. For transfection, we used polyethylenimine (PEI) to create pBMP-2/PEI nanoparticles, and to ensure slow release we used poly(lactic-co-glycolic acid) (PLGA) to create microsphere encapsulated pBMP-2/PEI nanoparticles, PLGA@pBMP-2/PEI. We demonstrated that pBMP-2/PEI nanoparticles could slowly release from the PLGA@pBMP-2/PEI microspheres for a long period of time. The 3-15 μm diameter of the PLGA@pBMP-2/PEI further supported this slow release ability of the PLGA@pBMP-2/PEI. In vitro transfection assays demonstrated that pBMP-2/PEI released from PLGA@pBMP-2/PEI could efficiently transfect MC3T3-E1 cells, causing MC3T3-E1 cells to secrete human BMP-2 protein, increase calcium deposition and gene expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), SP7 and I type collagen (COLL I), and finally induce MC3T3-E1 cell differentiation. Importantly, in vivo data from micro-computed tomography (micro-CT) and histological staining demonstrated that the human BMP-2 released from PLGA@pBMP-2/PEI had a long-term effect locally and efficiently promoted bone formation in the bone defect area compared to control animals. All our data suggest that our PLGA-nanoparticle delivery system efficiently and functionally delivers the human BMP-2 cDNA and has potential clinical application in the future after further modification.

  2. HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

    PubMed Central

    Visser, Mijke; Kayser, Manfred; Palstra, Robert-Jan

    2012-01-01

    Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases. PMID:22234890

  3. HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter.

    PubMed

    Visser, Mijke; Kayser, Manfred; Palstra, Robert-Jan

    2012-03-01

    Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases.

  4. UV-B Induced Generation of Reactive Oxygen Species Promotes Formation of BFA-Induced Compartments in Cells of Arabidopsis Root Apices

    PubMed Central

    Yokawa, Ken; Kagenishi, Tomoko; Baluška, František

    2016-01-01

    UV-B radiation is an important part of the electromagnetic spectrum emitted by the sun. For much of the period of biological evolution organisms have been exposed to UV radiation, and have developed diverse mechanisms to cope with this potential stress factor. Roots are usually shielded from exposure to UV by the surrounding soil, but may nevertheless be exposed to high energy radiation on the soil surface. Due to their high sensitivity to UV-B radiation, plant roots need to respond rapidly in order to minimize exposure on the surface. In addition to root gravitropism, effective light perception by roots has recently been discovered to be essential for triggering negative root phototropism in Arabidopsis. However, it is not fully understood how UV-B affects root growth and phototropism. Here, we report that UV-B induces rapid generation of reactive oxygen species which in turn promotes the formation of BFA-induced compartments in the Arabidopsis root apex. During unilateral UV-B irradiation of roots changes in auxin concentration on the illuminated side have been recorded. In conclusion, UV-B-induced and ROS-mediated stimulation of vesicle recycling promotes root growth and induces negative phototropism. PMID:26793199

  5. Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1–induced collective directional migration and angiogenic sprouting

    PubMed Central

    Oubaha, Malika; Lin, Michelle I.; Margaron, Yoran; Filion, Dominic; Price, Emily N.; Zon, Leonard I.; Côté, Jean-François

    2012-01-01

    Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis. PMID:22936663

  6. PDGF activation in PGDS-positive arachnoid cells induces meningioma formation in mice promoting tumor progression in combination with Nf2 and Cdkn2ab loss.

    PubMed

    Peyre, Matthieu; Salaud, Céline; Clermont-Taranchon, Estelle; Niwa-Kawakita, Michiko; Goutagny, Stephane; Mawrin, Christian; Giovannini, Marco; Kalamarides, Michel

    2015-10-20

    The role of PDGF-B and its receptor in meningeal tumorigenesis is not clear. We investigated the role of PDGF-B in mouse meningioma development by generating autocrine stimulation of the arachnoid through the platelet-derived growth factor receptor (PDGFR) using the RCAStv-a system. To specifically target arachnoid cells, the cells of origin of meningioma, we generated the PGDStv-a mouse (Prostaglandin D synthase). Forced expression of PDGF-B in arachnoid cells in vivo induced the formation of Grade I meningiomas in 27% of mice by 8 months of age. In vitro, PDGF-B overexpression in PGDS-positive arachnoid cells lead to increased proliferation.We found a correlation of PDGFR-B expression and NF2 inactivation in a cohort of human meningiomas, and we showed that, in mice, Nf2 loss and PDGF over-expression in arachnoid cells induced meningioma malignant transformation, with 40% of Grade II meningiomas. In these mice, additional loss of Cdkn2ab resulted in a higher incidence of malignant meningiomas with 60% of Grade II and 30% of Grade III meningiomas. These data suggest that chronic autocrine PDGF signaling can promote proliferation of arachnoid cells and is potentially sufficient to induce meningiomagenesis. Loss of Nf2 and Cdkn2ab have synergistic effects with PDGF-B overexpression promoting meningioma malignant transformation.

  7. Tau Pathology Promotes the Reorganization of the Extracellular Matrix and Inhibits the Formation of Perineuronal Nets by Regulating the Expression and the Distribution of Hyaluronic Acid Synthases

    PubMed Central

    Li, Yin; Li, Ze-Xu; Jin, Tan; Wang, Zhan-You; Zhao, Pu

    2017-01-01

    Hyaluronic acid (HA) is the backbone of the extracellular matrix (ECM) and provides biochemical and physical support to aggrecan-based perineuronal nets (PNNs), which are associated with the selective vulnerability of neurons in Alzheimer’s disease (AD). Here, we showed that HA synthases (HASs), including Has1, Has2, and Has3, were widely expressed in murine central nervous system. All types of HASs were localized to cell bodies of neurons; only Has1 existed in the membranes of neural axons. By using TauP301S transgenic (Tg) mouse model, we found that the axonal-localization of Has1 was abolished in TauP301S overexpressed mouse brain, and the redistribution of Has1 was also observed in human AD brains, suggesting that the localization of Has1 is dependent on intact microtubules which are regulated partially by the phosphorylation and dephosphorylation cycles of tau proteins. Furthermore, Has1 was reduced and Has3 was increased in TauP301S Tg mouse brain, resulting in the upregulation of shorter-chain HA in the ECM. These findings suggest that by abolishing the axonal-localization of Has1 and promoting the expression of Has3 and the synthesis of shorter-chain HA, the tau pathology breaks the balance of ECM components, promotes the reorganization of the ECM, and inhibits the formation of PNNs in the hippocampus, and then regulates neuronal plasticity during the progression of AD. PMID:28234253

  8. Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

    PubMed Central

    Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

    2014-01-01

    Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

  9. Increase in Bacterial Colony Formation from a Permafrost Ice Wedge Dosed with a Tomitella biformata Recombinant Resuscitation-Promoting Factor Protein.

    PubMed

    Puspita, Indun Dewi; Kitagawa, Wataru; Kamagata, Yoichi; Tanaka, Michiko; Nakatsu, Cindy H

    2015-01-01

    Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821(T), an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p <0.05) higher number of CFUs on agar plates after 8 d, approximately 14-fold higher than that on control plates without rRpf. 16S rRNA gene sequences revealed that all the colonies on plates were mainly related to Brevibacterium antiquum strain VKM Ac-2118 (AY243344), with 98-99% sequence identity. This species is also a member of the phylum Actinobacteria and was originally isolated from Siberian permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample.

  10. Increase in Bacterial Colony Formation from a Permafrost Ice Wedge Dosed with a Tomitella biformata Recombinant Resuscitation-Promoting Factor Protein

    PubMed Central

    Puspita, Indun Dewi; Kitagawa, Wataru; Kamagata, Yoichi; Tanaka, Michiko; Nakatsu, Cindy H.

    2015-01-01

    Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821T, an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p <0.05) higher number of CFUs on agar plates after 8 d, approximately 14-fold higher than that on control plates without rRpf. 16S rRNA gene sequences revealed that all the colonies on plates were mainly related to Brevibacterium antiquum strain VKM Ac-2118 (AY243344), with 98–99% sequence identity. This species is also a member of the phylum Actinobacteria and was originally isolated from Siberian permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample. PMID:25843055

  11. The Transcriptional Modulator Interferon-Related Developmental Regulator 1 in Osteoblasts Suppresses Bone Formation and Promotes Bone Resorption.

    PubMed

    Iezaki, Takashi; Onishi, Yuki; Ozaki, Kakeru; Fukasawa, Kazuya; Takahata, Yoshifumi; Nakamura, Yukari; Fujikawa, Koichi; Takarada, Takeshi; Yoneda, Yukio; Yamashita, Yui; Shioi, Go; Hinoi, Eiichi

    2016-03-01

    Bone homeostasis is maintained by the synergistic actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Although interferon-related developmental regulator 1 (Ifrd1) has been identified as a transcriptional coactivator/repressor in various cells, little attention has been paid to its role in osteoblastogenesis and bone homeostasis thus far. Here, we show that Ifrd1 is a critical mediator of both the cell-autonomous regulation of osteoblastogenesis and osteoblast-dependent regulation of osteoclastogenesis. Osteoblast-specific deletion of murine Ifrd1 increased bone formation and decreased bone resorption, causing high bone mass. Ifrd1 deficiency enhanced osteoblast differentiation and maturation along with increased expression of Runx2 and osterix (Osx). Mechanistically, Ifrd1 deficiency increased the acetylation status of p65, a component of NF-κB, at residues K122 and K123 via the attenuation of the interaction between p65 and histone deacetylase (HDAC). This led to the nuclear export of p65 and a decrease in NF-κB-dependent Smad7 expression and the subsequent enhancement of Smad1/Smad5/Smad8-dependent transcription. Moreover, a high bone mass phenotype in the osteoblast-specific deletion of Ifrd1 was markedly rescued by the introduction of one Osx-floxed allele but not of Runx2-floxed allele. Coculture experiments revealed that Ifrd1-deficient osteoblasts have a higher osteoprotegerin (OPG) expression and a lower ability to support osteoclastogenesis. Ifrd1 deficiency attenuated the interaction between β-catenin and HDAC, subsequently increasing the acetylation of β-catenin at K49, leading to its nuclear accumulation and the activation of the β-catenin-dependent transcription of OPG. Collectively, the expression of Ifrd1 in osteoblasts repressed osteoblastogenesis and activated osteoclastogenesis through modulating the NF-κB/Smad/Osx and β-catenin/OPG pathways, respectively. These findings suggest that Ifrd1 has a pivotal role in bone

  12. The Hha-TomB Toxin-Antitoxin System Shows Conditional Toxicity and Promotes Persister Cell Formation by Inhibiting Apoptosis-Like Death in S. Typhimurium

    PubMed Central

    Jaiswal, Sangeeta; Paul, Prajita; Padhi, Chandrashekhar; Ray, Shilpa; Ryan, Daniel; Dash, Shantoshini; Suar, Mrutyunjay

    2016-01-01

    Toxin-antitoxin (TA) modules are two component “addictive” genetic elements found on either plasmid or bacterial chromosome, sometimes on both. TA systems perform a wide range of functions like biofilm formation, persistence, programmed cell death, phage abortive infection etc. Salmonella has been reported to contain several such TA systems. However, the hemolysin expression modulating protein (Hha) and its adjacent uncharacterized hypothetical protein TomB (previously known as YbaJ), have not been listed as a TA module in Salmonella. In this study we established that Hha and TomB form a bonafide TA system where Hha serves as a toxin while TomB functions as an antitoxin. Interestingly, the toxicity of Hha was conditional causing cell death under acid stress. The antitoxin attenuated the toxicity of Hha by forming a TA complex through stable interactions. The Hha-TomB TA system was found to increase persistence and inhibit programmed cell death under antibiotic stress where a phenotypically diverse population expressing differential level of TA components was observed. Therefore we propose that Hha and TomB prevent cells from committing suicide thereby promoting persister cell formation. PMID:27910884

  13. Intramolecular Oxyl Radical Coupling Promotes O-O Bond Formation in a Homogeneous Mononuclear Mn-based Water Oxidation Catalyst: A Computational Mechanistic Investigation.

    PubMed

    Crandell, Douglas W; Xu, Song; Smith, Jeremy M; Baik, Mu-Hyun

    2017-04-04

    The mechanism of water oxidation performed by a recently discovered manganese pyridinophane catalyst [Mn(Py2N(t)Bu2)(H2O)2](2+) is studied using density functional theory methods. A complete catalytic cycle is constructed and the catalytically active species is identified to consist of a Mn(V)-bis(oxo) moiety that is generated from the resting state by a series of proton-coupled electron transfer reactions. Whereas the electronic ground state of this key intermediate is found to be a triplet, the most favorable pathway for O-O bond formation is found on the quintet potential energy surface and involves an intramolecular coupling of two oxyl radicals with opposite spins bound to the Mn-center that adopts an electronic structure most consistent formally with a high-spin Mn(III) ion. Therefore, the thermally accessible high-spin quintet state that constitutes a typical and innate property of a first-row transition metal center plays a critical role for catalysis. It enables facile electron transfer between the oxo moieties and the Mn-center and promotes O-O bond formation via a radical coupling reaction with a calculated reaction barrier of only 14.7 kcal mol(-1). This mechanism of O-O coupling is unprecedented and provides a novel possible pathway to coupling two oxygen atoms bound to a single metal site.

  14. Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK-3β Phosphorylation.

    PubMed

    Cheng, Yuanyuan; Xia, Zhengyuan; Han, Yifan; Rong, Jianhui

    2016-01-01

    The opening of mitochondrial permeability transition pore (mPTP) is a major cause of cell death in ischemia reperfusion injury. Based on our pilot experiments, plant natural product formononetin enhanced the survival of rat cardiomyocyte H9c2 cells during oxygen glucose deprivation (OGD) and reoxygenation. For mechanistic studies, we focused on two major cellular factors, namely, reactive oxygen species (ROS) and glycogen synthase kinase 3β (GSK-3β), in the regulation of mPTP opening. We found that formononetin suppressed the formation of ROS and superoxide in a concentration-dependent manner. Formononetin also rescued OGD/reoxygenation-induced loss of mitochondrial membrane integrity. Further studies suggested that formononetin induced Akt activation and GSK-3β (Ser9) phosphorylation, thereby reducing GSK-3β activity towards mPTP opening. PI3K and PKC inhibitors abolished the effects of formononetin on mPTP opening and GSK-3β phosphorylation. Immunoprecipitation experiments further revealed that formononetin increased the binding of phosphor-GSK-3β to adenine nucleotide translocase (ANT) while it disrupted the complex of ANT with cyclophilin D. Moreover, immunofluorescence revealed that phospho-GSK-3β (Ser9) was mainly deposited in the space between mitochondria and cell nucleus. Collectively, these results indicated that formononetin protected cardiomyocytes from OGD/reoxygenation injury via inhibiting ROS formation and promoting GSK-3β phosphorylation.

  15. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

    PubMed Central

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C.

    2014-01-01

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved. PMID:25414342

  16. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination.

    PubMed

    Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C

    2014-12-16

    During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.

  17. [Overexpression of miR-125b promotes apoptosis of macrophages].

    PubMed

    Yu, Guangyuan; Zhan, Xue; Zhang, Zhenzhen; Li, Yasha

    2016-07-01

    Objective To investigate the expressions of miR-125b and target gene Raf1 proto-oncogene serine/threonine protein kinase (RAF1) in peripheral blood mononuclear cells (PBMCs) of pediatric patients with pulmonary tuberculosis (PTB), and observe the regulation of miR-125b on macrophage apoptosis and activity. Methods PBMCs of patients with PTB and healthy children were collected and separated. Real-time fluorescence quantitative PCR was used to detect mRNA expression level of miR-125b and RAF1, and Western blotting was used to detect the protein level of RAF1. THP-1 macrophages were transfected into miR-125b mimic, negative control mimic (NC-mimic), miR-125b inhibitor and negative control inhibitor (NC-inhibitor), which were cultured for 48 hours. Western blotting was performed to observe the expression of RAF1 in THP-1 macrophages, annexin V-FITC/PI double staining combined with flow cytometry was used to test cell apoptosis, and CCK-8 assay was used to detect cell proliferation. Results The expression of miR-125b in PBMCs in pediatric patients with PTB was downregulated, and mRNA and protein levels of RAF1 were upregulated. When miR-125b was over-expressed in THP-1 macrophages, the expression of RAF1 was reduced to promote the apoptosis of macrophages and decrease cell activity; when the expression of miR-125b was inhibited in THP-1 macrophages, the expression of RAF1 was elevatedand the apoptosis of macrophages was inhibited, the cell activity was promoted. Conclusion In PBMCs of children with PTB, miR-125b level is low. Upregulation of miR-125b in THP-1 macrophages, the apoptosis of THP-1 macrophages is promoted and cell activity is inhibited.

  18. MiR-1908 promotes scar formation post-burn wound healing by suppressing Ski-mediated inflammation and fibroblast proliferation.

    PubMed

    Xie, Chunhui; Shi, Kai; Zhang, Xi; Zhao, Jingchun; Yu, Jiaao

    2016-11-01

    The cell biological basis for scar formation is mainly via excessive fibroblast proliferation accompanied by hypernomic Col I accumulation and inflammation. The role of miR-1908 in scar formation has not been investigated. In this study, we found that miR-1908 expression was inversely associated with the scar suppressor Ski in normal, burn-wounded, healing and scar dermal tissues in humans. Bioinformatics and luciferase reporter gene assays confirmed that miR-1908 targeted the 3'UTR region of Ski mRNA and suppressed Ski expression. Next, human scar epidermal fibroblasts were isolated and the miR-1908 oligonucleotide mimic and inhibitor were respectively transfected into the cells. Western blot analysis proved that Ski expression was sharply reduced by the miR-1908 mimic. MTT and Cell Counting Kit-8 analyses showed that miR-1908 mimic transfection promoted cell proliferation. Simultaneously, data on real-time qPCR analysis indicated that expression of the fibrotic master gene TGF-β1, Ski-suppressing gene Meox2, Col I and proinflammatory markers IL-1α and TNF-α, were all significantly upregulated. In contrast, the miR-1908 inhibitor had a completely opposite effect on cell proliferation and gene expression. The mimic and inhibitor were locally injected into rats with abdominal burn-wounded scars during a 180-day, post-healing experiment. The miR-1908 mimic injection significantly reduced Ski expression, as well as the area, volume and fibrosis of scars in vivo. And, in contrast, the miR-1908 inhibitor injection had an opposite effect to that of the miR-1908 mimic injection. In conclusion, miR-1908 had a positive role in scar formation by suppressing Ski-mediated inflammation and fibroblast proliferation in vitro and in vivo.

  19. Unexpected promotion of PCDD/F formation by enzyme-aided Cl2 bleaching in non-wood pulp and paper mill.

    PubMed

    Fang, Liping; Zheng, Minghui; Liu, Guorui; Zhao, Yuyang; Liu, Wenbin; Huang, Linyan; Guo, Li

    2017-02-01

    Enzyme-aided Cl2 bleaching is widely considered as promising replacements for conventional Cl2 bleaching in wood pulp and paper mills. However, the effects of using enzyme-aided bleaching on the formation of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in the non-wood pulp and paper mills are unclear. A field study was performed to investigate PCDD/F formation when enzyme-aided Cl2 bleaching was used to replace conventional Cl2 bleaching in non-wood pulp and paper mills. Unexpectedly, the PCDD/F toxic equivalents (TEQs) in solid samples were higher when using enzyme-aided bleaching (0.49-5.4 pg TEQ/g) than that using conventional Cl2 bleaching (0.15-2.44 pg TEQ/g). Large amounts of octachlorodibenzo-p-dioxin were formed during the enzyme-aided bleaching process. This could have been because enzyme strongly promoted the release of organic molecules bound to lignin and thus accelerated the formation of octachlorodibenzo-p-dioxin through organic molecular precursors. Although enzyme-aided Cl2 bleaching was previously considered to be efficient for reducing PCDD/F releases and to be the best available technologies and best environmental practices for wood pulp and paper mills, the results obtained in this study suggested the necessity and urgency to evaluate the suitability of enzyme-aided Cl2 bleaching for non-wood pulp and paper mills that intensively practiced in developing countries.

  20. Polysaccharides and proteins added to flowing drinking water at microgram-per-liter levels promote the formation of biofilms predominated by bacteroidetes and proteobacteria.

    PubMed

    Sack, Eveline L W; van der Wielen, Paul W J J; van der Kooij, Dick

    2014-04-01

    Biopolymers are important substrates for heterotrophic bacteria in (ultra)oligotrophic freshwater environments, but information about their utilization at microgram-per-liter levels by attached freshwater bacteria is lacking. This study aimed at characterizing biopolymer utilization in drinking-water-related biofilms by exposing such biofilms to added carbohydrates or proteins at 10 μg C liter(-1) in flowing tap water for up to 3 months. Individually added amylopectin was not utilized by the biofilms, whereas laminarin, gelatin, and caseinate were. Amylopectin was utilized during steady-state biofilm growth with simultaneously added maltose but not with simultaneously added acetate. Biofilm formation rates (BFR) at 10 μg C liter(-1) per substrate were ranked as follows, from lowest to highest: blank or amylopectin (≤6 pg ATP cm(-2) day(-1)), gelatin or caseinate, laminarin, maltose, acetate alone or acetate plus amylopectin, and maltose plus amylopectin (980 pg ATP cm(-2) day(-1)). Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequence analyses revealed that the predominant maltose-utilizing bacteria also dominated subsequent amylopectin utilization, indicating catabolic repression and (extracellular) enzyme induction. The accelerated BFR with amylopectin in the presence of maltose probably resulted from efficient amylopectin binding to and hydrolysis by inductive enzymes attached to the bacterial cells. Cytophagia, Flavobacteriia, Gammaproteobacteria, and Sphingobacteriia grew during polysaccharide addition, and Alpha-, Beta-, and Gammaproteobacteria, Cytophagia, Flavobacteriia, and Sphingobacteriia grew during protein addition. The succession of bacterial populations in the biofilms coincided with the decrease in the specific growth rate during biofilm formation. Biopolymers can clearly promote biofilm formation at microgram-per-liter levels in drinking water distribution systems and, depending on their concentrations, might

  1. KEY COMPARISON: Update of the BIPM comparison BIPM.RI(II)-K1.F-18 of activity measurements of the radionuclide 18F to include the PTB

    NASA Astrophysics Data System (ADS)

    Ratel, G.; Michotte, C.; Kossert, K.; Janßen, H.

    2006-01-01

    Since 2001, six national metrology institutes (NMIs) have submitted six samples of known activity of 18F to the International Reference System (SIR) for activity comparison at the Bureau International des Poids et Mesures (BIPM), the most recent being that of the PTB (Germany). The activities ranged from about 1 MBq to 18 MBq. The key comparison reference value (KCRV) has been recalculated to include the latest value, with the agreement of the CCRI(II). The degrees of equivalence between each equivalent activity measured in the SIR have been recalculated and the results are given in the form of a matrix. A graphical presentation is also given for this key comparison with identifier BIPM.RI(II)-K1.F-18. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by Section II of the Consultative Committee for Ionizing Radiation (CCRI(II)), according to the provisions of the Mutual Recognition Arrangement (MRA).

  2. The C-terminal fragment of parathyroid hormone-related peptide promotes bone formation in diabetic mice with low-turnover osteopaenia

    PubMed Central

    Lozano, D; Fernández-de-Castro, L; Portal-Núñez, S; López-Herradón, A; Dapía, S; Gómez-Barrena, E; Esbrit, P

    2011-01-01

    BACKGROUND AND PURPOSE Current data suggest that parathyroid hormone (PTH)-related peptide (PTHrP) domains other than the N-terminal PTH-like domain contribute to its role as an endogenous bone anabolic factor. PTHrP-107-139 inhibits bone resorption, a fact which has precluded an unequivocal demonstration of its possible anabolic action in vivo. We thus sought to characterize the osteogenic effects of this peptide using a mouse model of diabetic low-turnover osteopaenia. EXPERIMENTAL APPROACH PTHrP-107-139 was administered to streptozotocin-induced diabetic mice, with or without bone marrow ablation, for 13 days. Osteopaenia was confirmed by dual-energy X-ray absorptiometry and microcomputed tomography analysis. Histological analysis was performed on paraffin-embedded bone tissue sections by haematoxylin/eosin and Masson's staining, and tartrate-resistent acid phosphatase immunohistochemistry. Mouse bone marrow stromal cells and osteoblastic MC3T3-E1 cells were cultured in normal and/or high glucose (HG) medium. Osteogenic and adipogenic markers were assessed by real-time PCR, and PTHrP and the PTH1 receptor protein expression by Western blot analysis. KEY RESULTS PTHrP-107-139 reversed the alterations in bone structure and osteoblast function, and also promoted bone healing after marrow ablation without affecting the number of osteoclast-like cells in diabetic mice. This peptide also reversed the high-glucose-induced changes in osteogenic differentiation in both bone marrow stromal cells and the more differentiated MC3T3-E1 cells. CONCLUSIONS AND IMPLICATIONS These findings demonstrate that PTHrP-107-139 promotes bone formation in diabetic mice. This mouse model and in vitro cell cultures allowed us to identify various anabolic effects of this peptide in this scenario. PMID:21175568

  3. MicroRNA-15b/16 attenuates vascular neointima formation by promoting the contractile phenotype of vascular smooth muscle through targeting YAP

    PubMed Central

    Xu, Fei; Ahmed, Abu Shufian Ishtiaq; Kang, Xiuhua; Hu, Guoqing; Liu, Fang; Zhang, Wei; Zhou, Jiliang

    2015-01-01

    Objective To investigate the functional role of the miR-15b/16 in vascular smooth muscle phenotypic modulation. Approach and Results We found that miR-15b/16 is the one of most abundant microRNAs expressed in contractile vascular smooth muscle cells (VSMCs). However, when contractile VSMCs convert to a synthetic phenotype miR-15b/16 expression is significantly reduced. Knocking-down endogenous miR-15b/16 in VSMCs attenuates smooth muscle-specific gene expression but promotes VSMC proliferation and migration. Conversely, over-expression of miR-15b/16 promotes smooth muscle contractile gene expression while attenuating VSMC migration and proliferation. Consistent with this, over-expression of miR-15b/16 in a rat carotid balloon injury model markedly attenuates injury-induced smooth muscle de-differentiation and neointima formation. Mechanistically, we identified the potent oncoprotein yes-associated protein (YAP) as a downstream target of miR-15b/16 in VSMCs. Reporter assays validated that miR-15b/16 targets YAP’s 3′-untranslated region. Moreover, overexpression of miR-15b/16 significantly represses YAP expression, whereas conversely, depletion of endogenous miR-15b/16 results in up-regulation of YAP expression. Conclusions These results indicate that miR-15b/16 plays a critical role in smooth muscle phenotypic modulation at least partly through targeting YAP. Restoring expression of miR-15b/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases. PMID:26293467

  4. The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation.

    PubMed

    Gstaiger, M; Georgiev, O; van Leeuwen, H; van der Vliet, P; Schaffner, W

    1996-06-03

    We have shown previously that both octamer binding transcription factors, namely the ubiquitous Oct-1 and the B cell-specific Oct-2A protein, can be enhanced in transcriptional activity by their association with the B cell-specific coactivator protein Bob1, also called OBF-1 or OCA-B. Here we study the structural requirements for ternary complex formation of DNA-Oct-Bob1 and coactivation function of Bob1. In analogy to DNA-bound transcription factors, Bob1 has a modular structure that includes an interaction domain (amino acids 1-65) and a C-terminal domain (amino acids 65-256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26-32) in the N-terminus of Bob1 that are important for contacting the DNA binding POU domain of Oct-1 or Oct-2. In contrast to the viral coactivator VP16 (vmw65), which interacts with Oct-1 via the POU homeosubdomain, Bob1 association with Oct factors requires residues located in the POU-specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct-Bob1 complex compared with free Oct factors. While Oct-1 or Oct-2 bind to a large variety of octamer sequences, Bob1 ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer-containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type-specific gene expression.

  5. Bone marrow stromal cell sheets may promote axonal regeneration and functional recovery with suppression of glial scar formation after spinal cord transection injury in rats.

    PubMed

    Okuda, Akinori; Horii-Hayashi, Noriko; Sasagawa, Takayo; Shimizu, Takamasa; Shigematsu, Hideki; Iwata, Eiichiro; Morimoto, Yasuhiko; Masuda, Keisuke; Koizumi, Munehisa; Akahane, Manabu; Nishi, Mayumi; Tanaka, Yasuhito

    2017-03-01

    OBJECTIVE Transplantation of bone marrow stromal cells (BMSCs) is a theoretical potential as a therapeutic strategy in the treatment of spinal cord injury (SCI). Although a scaffold is sometimes used for retaining transplanted cells in damaged tissue, it is also known to induce redundant immunoreactions during the degradation processes. In this study, the authors prepared cell sheets made of BMSCs, which are transplantable without a scaffold, and investigated their effects on axonal regeneration, glial scar formation, and functional recovery in a completely transected SCI model in rats. METHODS BMSC sheets were prepared from the bone marrow of female Fischer 344 rats using ascorbic acid and were cryopreserved until the day of transplantation. A gelatin sponge (GS), as a control, or BMSC sheet was transplanted into a 2-mm-sized defect of the spinal cord at the T-8 level. Axonal regeneration and glial scar formation were assessed 2 and 8 weeks after transplantation by immunohistochemical analyses using anti-Tuj1 and glial fibrillary acidic protein (GFAP) antibodies, respectively. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan scale. RESULTS The BMSC sheets promoted axonal regeneration at 2 weeks after transplantation, but there was no significant difference in the number of Tuj1-positive axons between the sheet- and GS-transplanted groups. At 8 weeks after transplantation, Tuj1-positive axons elongated across the sheet, and their numbers were significantly greater in the sheet group than in the GS group. The areas of GFAP-positive glial scars in the sheet group were significantly reduced compared with those of the GS group at both time points. Finally, hindlimb locomotor function was ameliorated in the sheet group at 4 and 8 weeks after transplantation. CONCLUSIONS The results of the present study indicate that an ascorbic acid-induced BMSC sheet is effective in the treatment of SCI and enables autologous transplantation without requiring a

  6. GmEXPB2, a Cell Wall β-Expansin, Affects Soybean Nodulation through Modifying Root Architecture and Promoting Nodule Formation and Development1[OPEN

    PubMed Central

    Li, Xinxin; Zhao, Jing; Tan, Zhiyuan; Liao, Hong

    2015-01-01

    Nodulation is an essential process for biological nitrogen (N2) fixation in legumes, but its regulation remains poorly understood. Here, a β-expansin gene, GmEXPB2, was found to be critical for soybean (Glycine max) nodulation. GmEXPB2 was preferentially expressed at the early stage of nodule development. β-Glucuronidase staining further showed that GmEXPB2 was mainly localized to the nodule vascular trace and nodule vascular bundles, as well as nodule cortical and parenchyma cells, suggesting that GmEXPB2 might be involved in cell wall modification and extension during nodule formation and development. Overexpression of GmEXPB2 dramatically modified soybean root architecture, increasing the size and number of cortical cells in the root meristematic and elongation zones and expanding root hair density and size of the root hair zone. Confocal microscopy with green fluorescent protein-labeled rhizobium USDA110 cells showed that the infection events were significantly enhanced in the GmEXPB2-overexpressing lines. Moreover, nodule primordium development was earlier in overexpressing lines compared with wild-type plants. Thereby, overexpression of GmEXPB2 in either transgenic soybean hairy roots or whole plants resulted in increased nodule number, nodule mass, and nitrogenase activity and thus elevated plant N and phosphorus content as well as biomass. In contrast, suppression of GmEXPB2 in soybean transgenic composite plants led to smaller infected cells and thus reduced number of big nodules, nodule mass, and nitrogenase activity, thereby inhibiting soybean growth. Taken together, we conclude that GmEXPB2 critically affects soybean nodulation through modifying root architecture and promoting nodule formation and development and subsequently impacts biological N2 fixation and growth of soybean. PMID:26432877

  7. GmEXPB2, a Cell Wall β-Expansin, Affects Soybean Nodulation through Modifying Root Architecture and Promoting Nodule Formation and Development.

    PubMed

    Li, Xinxin; Zhao, Jing; Tan, Zhiyuan; Zeng, Rensen; Liao, Hong

    2015-12-01

    Nodulation is an essential process for biological nitrogen (N2) fixation in legumes, but its regulation remains poorly understood. Here, a β-expansin gene, GmEXPB2, was found to be critical for soybean (Glycine max) nodulation. GmEXPB2 was preferentially expressed at the early stage of nodule development. β-Glucuronidase staining further showed that GmEXPB2 was mainly localized to the nodule vascular trace and nodule vascular bundles, as well as nodule cortical and parenchyma cells, suggesting that GmEXPB2 might be involved in cell wall modification and extension during nodule formation and development. Overexpression of GmEXPB2 dramatically modified soybean root architecture, increasing the size and number of cortical cells in the root meristematic and elongation zones and expanding root hair density and size of the root hair zone. Confocal microscopy with green fluorescent protein-labeled rhizobium USDA110 cells showed that the infection events were significantly enhanced in the GmEXPB2-overexpressing lines. Moreover, nodule primordium development was earlier in overexpressing lines compared with wild-type plants. Thereby, overexpression of GmEXPB2 in either transgenic soybean hairy roots or whole plants resulted in increased nodule number, nodule mass, and nitrogenase activity and thus elevated plant N and phosphorus content as well as biomass. In contrast, suppression of GmEXPB2 in soybean transgenic composite plants led to smaller infected cells and thus reduced number of big nodules, nodule mass, and nitrogenase activity, thereby inhibiting soybean growth. Taken together, we conclude that GmEXPB2 critically affects soybean nodulation through modifying root architecture and promoting nodule formation and development and subsequently impacts biological N2 fixation and growth of soybean.

  8. Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide.

    PubMed

    Taffe, B G; Takahashi, N; Kensler, T W; Mason, R P

    1987-09-05

    The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.

  9. Glucose Metabolism via the Entner-Doudoroff Pathway in Campylobacter: A Rare Trait that Enhances Survival and Promotes Biofilm Formation in Some Isolates

    PubMed Central

    Vegge, Christina S.; Jansen van Rensburg, Melissa J.; Rasmussen, Janus J.; Maiden, Martin C. J.; Johnsen, Lea G.; Danielsen, Morten; MacIntyre, Sheila; Ingmer, Hanne; Kelly, David J.

    2016-01-01

    Isolates of the zoonotic pathogen Campylobacter are generally considered to be unable to metabolize glucose due to lack of key glycolytic enzymes. However, the Entner-Doudoroff (ED) pathway has been identified in Campylobacter jejuni subsp. doylei and a few C. coli isolates. A systematic search for ED pathway genes in a wide range of Campylobacter isolates and in the C. jejuni/coli PubMLST database revealed that 1.7% of >6,000 genomes encoded a complete ED pathway, including both C. jejuni and C. coli from diverse clinical, environmental and animal sources. In rich media, glucose significantly enhanced stationary phase survival of a set of ED-positive C. coli isolates. Unexpectedly, glucose massively promoted floating biofilm formation in some of these ED-positive isolates. Metabolic profiling by gas chromatography–mass spectrometry revealed distinct responses to glucose in a low biofilm strain (CV1257) compared to a high biofilm strain (B13117), consistent with preferential diversion of hexose-6-phosphate to polysaccharide in B13117. We conclude that while the ED pathway is rare amongst Campylobacter isolates causing human disease (the majority of which would be of agricultural origin), some glucose-utilizing isolates exhibit specific fitness advantages, including stationary-phase survival and biofilm production, highlighting key physiological benefits of this pathway in addition to energy conservation. PMID:27920773

  10. Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation.

    PubMed

    Sarode, Bhushan; Nowell, Craig S; Ihm, JongEun; Kostic, Corinne; Arsenijevic, Yvan; Moulin, Alexandre P; Schorderet, Daniel F; Beermann, Friedrich; Radtke, Freddy

    2014-07-01

    The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intra-ocular pressure and regulate exposure of the internal eye structures to light, respectively. The cellular and molecular factors that mediate the development of the ciliary body and iris from the ocular pigmented epithelium remain to be fully elucidated. Here, we have investigated the role of Notch signaling during the development of the anterior pigmented epithelium by using genetic loss- and gain-of-function approaches. Loss of canonical Notch signaling results in normal iris development but absence of the ciliary body. This causes progressive hypotony and over time leads to phthisis bulbi, a condition characterized by shrinkage of the eye and loss of structure/function. Conversely, Notch gain-of-function results in aniridia and profound ciliary body hyperplasia, which causes ocular hypertension and glaucoma-like disease. Collectively, these data indicate that Notch signaling promotes ciliary body development at the expense of iris formation and reveals novel animal models of human ocular pathologies.

  11. An enantioselective strategy for the total synthesis of (S)-tylophorine via catalytic asymmetric allylation and a one-pot DMAP-promoted isocyanate formation/Lewis acid catalyzed cyclization sequence.

    PubMed

    Su, Bo; Zhang, Hui; Deng, Meng; Wang, Qingmin

    2014-06-14

    A new asymmetric total synthesis of a phenanthroindolizidine alkaloid (S)-tylophorine is reported, which features a catalytic asymmetric allylation of aldehydes and an unexpected one-pot DMAP promoted isocyanate formation and Lewis acid catalyzed intramolecular cyclization reaction. In addition, White's direct C-H oxidation catalyst system converting monosubstituted olefins to linear allylic acetates was also employed for late-stage transformation.

  12. Hybrid use of combined and sequential delivery of growth factors and ultrasound stimulation in porous multilayer composite scaffolds to promote both vascularization and bone formation in bone tissue engineering.

    PubMed

    Yan, Haoran; Liu, Xia; Zhu, Minghua; Luo, Guilin; Sun, Tao; Peng, Qiang; Zeng, Yi; Chen, Taijun; Wang, Yingying; Liu, Keliang; Feng, Bo; Weng, Jie; Wang, Jianxin

    2016-01-01

    In this study, a multilayer coating technology would be adopted to prepare a porous composite scaffold and the growth factor release and ultrasound techniques were introduced into bone tissue engineering to finally solve the problems of vascularization and bone formation in the scaffold whilst the designed multilayer composite with gradient degradation characteristics in the space was used to match the new bone growth process better. The results of animal experiments showed that the use of low intensity pulsed ultrasound (LIPUS) combined with growth factors demonstrated excellent capabilities and advantages in both vascularization and new bone formation in bone tissue engineering. The degradation of the used scaffold materials could match new bone formation very well. The results also showed that only RGD-promoted cell adhesion was insufficient to satisfy the needs of new bone formation while growth factors and LIPUS stimulation were the key factors in new bone formation.

  13. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    SciTech Connect

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  14. Correlating High Power Conversion Efficiency of PTB7:PC71BM Inverted Organic Solar Cells with Nanoscale Structures

    SciTech Connect

    Das, Sanjib; Keum, Jong Kahk; Browning, Jim; Gu, Gong; Yang, Bin; Do, Changwoo; Chen, Wei; Chen, Jihua; Ivanov, Ilia N; Hong, Kunlun; Rondinone, Adam J.; Joshi, Pooran C.; Geohegan, David B.; Xiao, Kai

    2015-01-01

    Advances in materials design and device engineering led to inverted organic solar cells (i-OSCs) with superior power conversion efficiencies (PCEs) to their conventional counterparts, in addition to the well-known better ambient stability. Despite the significant progress, however, it has so far been unclear how the morphologies of the photoactive layer and its interface with the cathode modifying layer impact device performance. Here, we report an in-depth morphology study of the i-OSC active and cathode modifying layers, employing a model system with the well-established bulk-heterojunction, PTB7:PC71BM as the active layer and poly-[(9,9-bis(3 -(N,N-dimethylamino)propyl)-2,7-fluorene)-alt-2,7-(9,9-dioctylfluorene)] (PFN) as the cathode surface modifying layer. We have also identified the role of a processing additive, 1,8-diiodooctane (DIO), used in the spin-casting of the active layer to increase PCE. Using a variety of characterization techniques, we demonstrate that the high PCEs of i-OSCs are due to the smearing (diffusion) of electron-accepting PC71BM into the PFN layer, resulting in improved electron transport. The PC71BM diffusion occurs after spin-casting the active layer onto the PFN layer, when residual solvent molecules act as a plasticizer. The DIO additive, with a higher boiling point than the host solvent, has a longer residence time in the spin-cast active layer, resulting in more PC71BM smearing and therefore more efficient electron transport. This work provides important insight and guidance to further enhancement of i-OSC performance by materials and interface engineering.

  15. Mechanical Vibration Mitigates the Decrease of Bone Quantity and Bone Quality of Leptin Receptor-Deficient Db/Db Mice by Promoting Bone Formation and Inhibiting Bone Resorption.

    PubMed

    Jing, Da; Luo, Erping; Cai, Jing; Tong, Shichao; Zhai, Mingming; Shen, Guanghao; Wang, Xin; Luo, Zhuojing

    2016-09-01

    Leptin, a major hormonal product of adipocytes, is involved in regulating appetite and energy metabolism. Substantial studies have revealed the anabolic actions of leptin on skeletons and bone cells both in vivo and in vitro. Growing evidence has substantiated that leptin receptor-deficient db/db mice exhibit decreased bone mass and impaired bone microstructure despite several conflicting results previously reported. We herein systematically investigated bone microarchitecture, mechanical strength, bone turnover and its potential molecular mechanisms in db/db mice. More importantly, we also explored an effective approach for increasing bone mass in leptin receptor-deficient animals in an easy and noninvasive manner. Our results show that deterioration of trabecular and cortical bone microarchitecture and decreases of skeletal mechanical strength-including maximum load, yield load, stiffness, energy, tissue-level modulus and hardness-in db/db mice were significantly ameliorated by 12-week, whole-body vibration (WBV) with 0.5 g, 45 Hz via micro-computed tomography (μCT), three-point bending, and nanoindentation examinations. Serum biochemical analysis shows that WBV significantly decreased serum tartrate-resistant acid phosphatase 5b (TRACP5b) and CTx-1 levels and also mitigated the reduction of serum osteocalcin (OCN) in db/db mice. Bone histomorphometric analysis confirmed that decreased bone formation-lower mineral apposition rate, bone formation rate, and osteoblast numbers in cancellous bone-in db/db mice were suppressed by WBV. Real-time PCR assays show that WBV mitigated the reductions of tibial alkaline phosphatase (ALP), OCN, Runt-related transcription factor 2 (RUNX2), type I collagen (COL1), BMP2, Wnt3a, Lrp6, and β-catenin mRNA expression, and prevented the increases of tibial sclerostin (SOST), RANK, RANKL, RANL/osteoprotegerin (OPG) gene levels in db/db mice. Our results show that WBV promoted bone quantity and quality in db/db mice with obvious

  16. Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial–mesenchymal transition-induced angiogenesis

    PubMed Central

    Li, C; Li, Q; Cai, Y; He, Y; Lan, X; Wang, W; Liu, J; Wang, S; Zhu, G; Fan, J; Zhou, Y; Sun, R

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck and is associated with a high rate of lymph node metastasis. The initial step in the metastasis and transition of tumors is epithelial–mesenchymal transition (EMT)-induced angiogenesis, which can be mediated by angiopoietin 2 (ANG2), a key regulatory factor in angiogenesis. In the present study, immunohistochemistry and real-time quantitative reverse transcriptase (qRT-PCR) were used to measure the expression of ANG2 in OSCC tissues. Plasmids encoding ANG2 mRNA were used for increased ANG2 expression in the OSCC cell line TCA8113. The short interfering RNA (siRNA)-targeting ANG2 mRNA sequences were used to inhibit ANG2 expression in TCA8113 cells. Subsequently, transwell assays were performed to examine the effects of ANG2 on TCA8113 cell migration and invasion. Furthermore, in vivo assays were performed to assess the effect of ANG2 on tumor growth. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemistry were used to examine cell apoptosis and angiogenesis in tumor tissues, respectively. Finally, western blot analysis was performed to evaluate tumor formation-related proteins in OSCC tissues. We found that protein expression of ANG2 was remarkably upregulated in OSCC tissues. Overexpression of ANG2 increased the migration and invasion of TCA8113 cells by regulating EMT. Further investigations showed that overexpression of ANG2 increased tumor growth in nude mice, and angiogenesis of OSCC tissues increased in the presence of ANG2 overexpression. Overexpression of ANG2 also reduced cell apoptosis in tumor tissue cells. Finally, we found that overexpression of ANG2 resulted in changes in the expression of tumor formation-related proteins including vimentin, E-cadherin, Bim, PUMA, Bcl-2, Bax, Cyclin D1, PCNA and CD31. Our findings show that ANG2 has an important role in the migration and invasion of OSCC. More importantly, further

  17. Translating formative research findings into a behaviour change strategy to promote antenatal calcium and iron and folic acid supplementation in western Kenya.

    PubMed

    Martin, Stephanie L; Seim, Gretchen L; Wawire, Salome; Chapleau, Gina M; Young, Sera L; Dickin, Katherine L

    2017-01-01

    The World Health Organization now recommends integrating calcium supplements into antenatal micronutrient supplementation programmes to prevent pre-eclampsia, a leading cause of maternal mortality. As countries consider integrating calcium supplementation into antenatal care (ANC), it is important to identify context-specific barriers and facilitators to delivery and adherence. Such insights can be gained from women's and health workers' experiences with iron and folic acid (IFA) supplements. We conducted in-depth interviews with 22 pregnant and post-partum women and 20 community-based and facility-based health workers in Kenya to inform a calcium and IFA supplementation programme. Interviews assessed awareness of anaemia, pre-eclampsia and eclampsia; ANC attendance; and barriers and facilitators to IFA supplement delivery and adherence. We analyzed interviews inductively using the constant comparative method. Women and health workers identified poor diet quality in pregnancy as a major health concern. Neither women nor health workers identified pre-eclampsia, eclampsia, anaemia or related symptoms as serious health threats. Women and community-based health workers were unfamiliar with pre-eclampsia and eclampsia and considered anaemia symptoms normal. Most women had not received IFA supplements, and those who had received insufficient amounts and little information about supplement benefits. We then developed a multi-level (health facility, community, household and individual) behaviour change strategy to promote antenatal calcium and IFA supplementation. Formative research is an essential first step in guiding implementation of antenatal calcium supplementation programmes to reduce pre-eclampsia. Because evidence on how to implement successful calcium supplementation programmes is limited, experiences with antenatal IFA supplementation can be used to guide programme development.

  18. Promotion of the halide effect in the formation of shaped metal nanocrystals via a hybrid cationic, polymeric stabilizer: Octahedra, cubes, and anisotropic growth

    NASA Astrophysics Data System (ADS)

    Sneed, Brian T.; Golden, Matthew C.; Liu, Yejing; Lee, Hiang K.; Andoni, Ilektra; Young, Allison P.; McMahon, Greg; Erdman, Natasha; Shibata, Masateru; Ling, Xing Yi; Tsung, Chia-Kuang

    2016-06-01

    To promote the effect of halide ions (Cl-, Br-, and I-) in facet-selective growth of {111} and {100} of shaped metal nanocrystals, we utilize PDADMAC, a hybrid cationic, polymeric stabilizer. SERS and synthesis experiments provide evidence supporting that the higher amount of PDADMA+ at surfaces promotes the local adsorption of halides, allowing the creation of Pd cubes, octahedra, and cuboctopods.

  19. Mammary gland specific expression of Brk/PTK6 promotes delayed involution and tumor formation associated with activation of p38 MAPK

    PubMed Central

    2011-01-01

    ; ductal and lobular carcinomas expressing Brk were significantly more likely to express elevated phospho-p38 MAPK. Conclusions These studies illustrate that forced expression of Brk/PTK6 in non-transformed mammary epithelial cells mediates p38 MAPK phosphorylation and promotes increased cellular survival, delayed involution, and latent tumor formation. Brk expression in human breast tumors may contribute to progression by inducing p38-driven pro-survival signaling pathways. PMID:21923922

  20. Strengthening health services to deliver nutrition education to promote complementary feeding and healthy growth of infants and young children: formative research for a successful intervention in peri-urban Trujillo, Peru.

    PubMed

    Robert, Rebecca C; Creed-Kanashiro, Hilary M; Villasante, Ruben; Narro, M Rocio; Penny, Mary E

    2017-04-01

    Formative research is critical for developing effective nutrition-specific interventions to improve infant and young child (IYC) feeding practices and promote healthy growth. Health workers interact with caregivers during health facility visits, yet there is limited research about how to optimize delivery of such interventions during these visits. The extensive reach of IYC health services globally calls for research to address this gap. In Trujillo, Peru, formative research was conducted to explore complementary feeding practices with caregivers as well as health worker routines and interactions with caregivers related to feeding and healthy growth; results informed the development and delivery of an educational intervention. Multiple qualitative methods were used to collect data on a purposive sample of health workers and caregivers from three health facilities and communities: household trials followed. Complementary feeding messages with doable behaviours were developed, and three were selected as key to promote based on their nutritional impact and cultural acceptability. In the health facilities, medical consultation, well-child visits and nutrition consultation all dealt with aspects of IYC nutrition/growth during their interactions with caregivers but were independent and inconsistent in approach. A nutrition education strategy was developed based on consistency, quality and coverage in the IYC health services. We conclude that formative research undertaken in the community and IYC health services was critical to developing a successful and culturally relevant intervention to promote optimal complementary feeding practices and healthy growth during interactions between health workers and caregivers at routine health facility visits. © 2016 John Wiley & Sons Ltd.

  1. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

    SciTech Connect

    Zhang, Bingyu; Luo, Qing; Mao, Xinjian; Xu, Baiyao; Yang, Li; Ju, Yang; Song, Guanbin

    2014-03-10

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the FAK-ERK1

  2. The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae.

    PubMed

    Krishnamurthy, Ajay; Kyd, Jennelle

    2014-08-01

    Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) often share a common niche within the nasopharynx, both associated with infections such as bronchitis and otitis media. This study investigated how the association between NTHi and S. pneumoniae and the host affects their propensity to form biofilms. We investigated a selection of bacterial strain and serotype combinations on biofilm formation, and the effect of contact with respiratory epithelial cells. Measurement of biofilm showed that co-infection with NTHi and S. pneumoniae increased biofilm formation following contact with epithelial cells compared to no contact demonstrating the role of epithelial cells in biofilm formation. Additionally, the influence of phosphorylcholine (ChoP) on biofilm production was investigated using the licD mutant strain of NTHi 2019 and found that ChoP had a role in mixed biofilm formation but was not the only requirement. The study highlights the complex interactions between microbes and the host epithelium during biofilm production, suggesting the importance of understanding why certain strains and serotypes differentially influence biofilm formation. A key contributor to increased biofilm formation was the upregulation of biofilm formation by epithelial cell factors.

  3. Human plasma enhances the expression of Staphylococcal microbial surface components recognizing adhesive matrix molecules promoting biofilm formation and increases antimicrobial tolerance In Vitro

    PubMed Central

    2014-01-01

    Background Microbial biofilms have been associated with the development of chronic human infections and represent a clinical challenge given their increased antimicrobial tolerance. Staphylococcus aureus is a major human pathogen causing a diverse range of diseases, of which biofilms are often involved. Staphylococcal attachment and the formation of biofilms have been shown to be facilitated by host factors that accumulate on surfaces. To better understand how host factors enhance staphylococcal biofilm formation, we evaluated the effect of whole human plasma on biofilm formation in clinical isolates of S. aureus and the expression of seven microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) known to be involved in biofilm formation by quantitative real-time PCR. We also evaluated whether plasma augmented changes in S. aureus biofilm morphology and antimicrobial resistance. Results Exposure of clinical isolates of S. aureus to human plasma (10%) within media, and to a lesser extent when coated onto plates, significantly enhanced biofilm formation in all of the clinical isolates tested. Compared to biofilms grown under non-supplemented conditions, plasma-augmented biofilms displayed significant changes in both the biofilm phenotype and cell morphology as determined by confocal scanning laser microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Exposure of bacteria to plasma resulted in a significant fold-increase in MSCRAMM expression in both a time and isolate-dependent manner. Additionally, plasma-augmented biofilms displayed an increased tolerance to vancomycin compared to biofilms grown in non-supplemented media. Conclusions Collectively, these studies support previous findings demonstrating a role for host factors in biofilm formation and provide further insight into how plasma, a preferred growth medium for staphylococcal biofilm formation enhances as well as augments other intrinsic properties of S. aureus biofilms

  4. One-electron oxidation pathway of thiols by peroxynitrite in biological fluids: bicarbonate and ascorbate promote the formation of albumin disulphide dimers in human blood plasma.

    PubMed Central

    Scorza, G; Minetti, M

    1998-01-01

    Recent studies have shown that peroxynitrite oxidizes thiol groups through competing one- and two-electron pathways. The two-electron pathway is mediated by the peroxynitrite anion and prevails quantitatively over the one-electron pathway, which is mediated by peroxynitrous acid or a reactive species derived from it. In CO2-containing fluids the oxidation of thiols might follow a different mechanism owing to the rapid formation of a different oxidant, the nitrosoperoxycarbonate anion (ONOOCO2(-)). Here we present evidence that in blood plasma peroxynitrite induces the formation of a disulphide cross-linked protein identified by immunological (anti-albumin antibodies) and biochemical criteria (peptide mapping) as a dimer of serum albumin. The albumin dimer did not form in plasma devoid of CO2 and its formation was enhanced by ascorbate. However, analysis of thiol groups showed that reconstituting dialysed plasma with NaHCO3 protected protein thiols against the oxidation mediated by peroxynitrite and that the simultaneouspresence of ascorbate provided further protection. Ascorbate alone did not protect thiol groups from peroxynitrite-mediated oxidation. ESR spin-trapping studies with N-t-butyl-alpha-phenylnitrone (PBN) revealed that peroxynitrite induced the formation of protein thiyl radicals and their intensity was markedly decreased by plasma dialysis and restored by reconstitution with NaHCO3. PBN completely inhibited the formation of albumin dimer. Moreover, the addition of iron-diethyldithiocarbamate to plasma demonstrated that peroxynitrite induced the formation of protein S-nitrosothiols and/or S-nitrothiols. Our results are consistent with the hypothesis that NaHCO3 favours the one-electron oxidation of thiols by peroxynitrite with formation of thiyl radicals, ;NO2, and RSNOx. Thiyl radicals, in turn, are involved in chain reactions by which thiols are oxidized to disulphides. PMID:9425126

  5. Poly(dA).poly(dT) rich sequences are not sufficient to exclude nucleosome formation in a constitutive yeast promoter.

    PubMed Central

    Losa, R; Omari, S; Thoma, F

    1990-01-01

    It was suggested that poly(dA).poly(dT) rich sequences in yeast Saccharomyces cerevisiae act as elements of constitutive promoters by exclusion of nucleosomes (Struhl, K. (1985). Proc. Natl. Acad. Sci. USA 82, 8419-8423). We have mapped the chromatin structure of the pet56-his3-ded1 region in minichromosomes and show that the poly(dA).poly(dT) sequences are located in nuclease sensitive regions. DNA fragments from the nuclease sensitive promoter region of DED1 were used for nucleosome reconstitution in vitro. We show that all sequences can form nucleosome cores and that the poly(dA).poly(dT) sequence can be incorporated in nucleosome cores. The results suggest that the nuclease sensitivity found in vivo is not established by poly(dA).poly(dT) mediated exclusion of nucleosomes. Images PMID:2194162

  6. Recent developments in alkene hydro-functionalisation promoted by homogeneous catalysts based on earth abundant elements: formation of C-N, C-O and C-P bond.

    PubMed

    Rodriguez-Ruiz, Violeta; Carlino, Romain; Bezzenine-Lafollée, Sophie; Gil, Richard; Prim, Damien; Schulz, Emmanuelle; Hannedouche, Jérôme

    2015-07-21

    This Perspective article provides an overview of the recent advancements in the field of intra- and inter-molecular C-N, C-O and C-P bond formation by hydroamination, hydroalkoxylation, hydrophosphination, hydrophosphonylation or hydrophosphinylation of unactivated alkenes, including allenes, 1,3-dienes and strained alkenes, promoted by (chiral) homogeneous catalysts based on earth abundant elements of the s and p blocks, the first row transition metals and the rare-earth metals. The relevant literature from 2009 until late 2014 has been covered.

  7. Using Formal Embedded Formative Assessments Aligned with a Short-Term Learning Progression to Promote Conceptual Change and Achievement in Science

    ERIC Educational Resources Information Center

    Yin, Yue; Tomita, Miki K.; Shavelson, Richard J.

    2014-01-01

    This study examined the effect of learning progression-aligned formal embedded formative assessment on conceptual change and achievement in middle-school science. Fifty-two sixth graders were randomly assigned to either an experimental group or a control group. Both groups were taught about sinking and floating by the same teacher with identical…

  8. Health promotion in Brazil.

    PubMed

    Buss, Paulo Marchiori; de Carvalho, Antonio Ivo

    2007-01-01

    The evolution of health promotion within the Brazilian health system is examined, including an assessment of the intersectoral and development policies that have influenced the process. Particular attention is paid to the legal characteristics of the Unified Health System. Human resources formation and research initiatives in health promotion are outlined, with a summary of the obstacles that need to be overcome in order to ensure the effective implementation of health promotion in the future. Up to the end of the 20th Century health promotion was not used as a term in the Brazilian public heath context. Health promoting activities were concentrated in the area of health education, although targeting the social determinants of health and the principle of intersectoral action were part of the rhetoric. The situation has changed during the last decade, with the publication of a national policy of health promotion, issued by the Ministry of Health and jointly implemented with the States and Municipals Health Secretaries. More recently there has been a re-emergence of the discourse on the social determinants of health and the formation of intersectoral public policies as the basis of a comprehensive health promotion. Health promotion infrastructure, particularly around human resources and financing, requires strengthening in order to ensure capacity and sustainability in health promotion practice.

  9. Sustained release poly (lactic-co-glycolic acid) microspheres of bone morphogenetic protein 2 plasmid/calcium phosphate to promote in vitro bone formation and in vivo ectopic osteogenesis

    PubMed Central

    Qiao, Chunyan; Zhang, Kai; Sun, Bin; Liu, Jinzhong; Song, Jiyu; Hu, Yue; Yang, Shihui; Sun, Hongchen; Yang, Bai

    2015-01-01

    Bone regeneration often requires continuous stimulation to promote local bone formation. In the present study, calcium phosphate (CaPi) was used to promote transfection of human bone morphogenetic protein 2 (BMP-2) cDNA plasmid, and poly (lactic-co-glycolic acid) (PLGA) was used to prepare microspheres of pBMP-2/CaPi (i.e., PLGA@pBMP-2/CaPi) using W/O/W double emulsion solvent evaporation method. We showed that PLGA@pBMP-2/CaPi microspheres were spherical with smooth surface, and the particle size ranged from 0.5 to 35 μm. Encapsulation efficiency was up to 30~50%. The release of BMP-2 cDNA from microspheres continued more than 30 days and constituted, less than 7.5% of total plasmid amount within the first 24 h. Real-time PCR results showed that co-culturing of PLGA@pBMP-2/CaPi with bone marrow-derived mesenchymal stem cells (BMSCs) increased calcium deposition and gene expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), SP7, and collagen type I (COLL I) in a time-dependent manner. Finally, X-ray analysis demonstrated that in vivo delivery of PLGA@pBMP-2/CaPi microspheres into the tibialis anterior muscles of rats promoted the generation of osteoblasts, bone tissue, and bone structure. The findings suggested that PLGA@pBMP-2/CaPi microspheres can promote ectopic osteogenesis in non-bone tissues, with strong prospects in promoting bone regeneration. PMID:26885257

  10. Role of Inn1 and its interactions with Hof1 and Cyk3 in promoting cleavage furrow and septum formation in S. cerevisiae.

    PubMed

    Nishihama, Ryuichi; Schreiter, Jennifer H; Onishi, Masayuki; Vallen, Elizabeth A; Hanna, Julia; Moravcevic, Katarina; Lippincott, Margaret F; Han, Haesun; Lemmon, Mark A; Pringle, John R; Bi, Erfei

    2009-06-15

    Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Delta cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow.

  11. Formation of Land Use Order in Hamamatsu City under the Original Criteria of the Farm Land Exclusion from the Agricultural Promotion Area

    NASA Astrophysics Data System (ADS)

    Arita, Hiroyuki; Miyazawa, Shingo

    While zoning has been practiced to prevent sprawling development and to preserve collective farmland under the Agriculture Promotion Act, The Agricultural Promotion Area (APA) has been reduced in area by the action of the Farm Land Exclusion from the APA (EAPA) aiming at urban-uses. Since the EAPA has a great impact on the regional land use, appropriate criteria application techniques ought to be formulated at the transaction level. However, most local governments seem to have no strategic measure so far. Hamamatsu city, meanwhile, has introduced a unique standard upon which approval of the EAPA aptitude is based in 2003. Since the number of EAPA registration was relatively large in Hamamatsu city owing to the zone bordering on the line of land which a building has erected the officials' willingness to establish an objective standard was high. In this research, we verified the effect of the criteria application over the land use ordering, and made proposals for improvement of the present state through the examination of the EAPA criterion application of Hamamatsu city.

  12. ADAM17/EGFR axis promotes transglutaminase-dependent skin barrier formation through phosholipase C γ1 and protein kinase C pathways

    PubMed Central

    Wolf , Cristina; Qian, Yawen; Brooke, Matthew A.; Kelsell, David P.; Franzke, Claus-Werner

    2016-01-01

    The vitally important skin barrier is formed by extensive cross-linking activity of transglutaminases (TGs) during terminal epidermal differentiation. We have previously shown that epidermal deficiency of a disintegrin and metalloproteinase 17 (ADAM17), the principal EGFR ligand sheddase, results in postnatal skin barrier defects in mice due to impeded TG activity. However, the mechanism by which ADAM17/EGFR signalling maintains TG activity during epidermal differentiation remains elusive. Here we demonstrate that ADAM17-dependent EGFR signalling promotes TG activity in keratinocytes committed to terminal differentiation by direct induction of TG1 expression. Restored TG1 expression of EGF-stimulated differentiated Adam17−/− keratinocytes was strongly repressed by inhibitors for PLCγ1 or protein kinase C (PKC) pathways, while treatment with the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate restored TG activity in the epidermis of keratinocyte-specific Adam17−/− (AD17ΔKC) mice. Further investigations emphasized the expression of PKCη, a mediator of TGM1 transcription, to be sensitive to EGFR activation. In agreement, topical skin application of cholesterol sulfate, an activator of PKCη, significantly improved TG activity in epidermis of AD17ΔKC mice. Our results suggest ADAM17/EGFR-driven PLCγ1 and PKC pathways as important promoters of TG1 expression during terminal keratinocyte differentiation. These findings may help to identify new therapeutic targets for inflammatory skin diseases related to epidermal barrier defects. PMID:28004780

  13. The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex

    PubMed Central

    Ge, Chunmin; Che, Lixiao; Du, Chunying

    2015-01-01

    BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response. PMID:26683461

  14. The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex.

    PubMed

    Ge, Chunmin; Che, Lixiao; Du, Chunying

    2015-01-01

    BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.

  15. The Class II Phosphatidylinositol 3-Phosphate Kinase PIK3C2A Promotes Shigella flexneri Dissemination through Formation of Vacuole-Like Protrusions

    PubMed Central

    Dragoi, Ana-Maria

    2015-01-01

    Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as a vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNA interference (RNAi)-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate [PtdIns(3)P] at the plasma membrane surrounding protrusions. PtdIns(3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns(3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system (T3SS). We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns(3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions. PMID:25667265

  16. Epithelial-mesenchymal transition delayed by E-cad to promote tissue formation in hepatic differentiation of mouse embryonic stem cells in vitro.

    PubMed

    Hu, Anbin; Shang, Changzhen; Li, Qiang; Sun, Nianfeng; Wu, Linwei; Ma, Yi; Jiao, Xingyuan; Min, Jun; Zeng, Gucheng; He, Xiaoshun

    2014-04-15

    Hepatic differentiation of embryonic stem cells (ESCs) usually results in a single cell lineage, and the formation of liver tissues remains difficult. Here, we examine the role of epithelial-mesenchymal transition (EMT) that is regulated by epithelial cadherin (E-cad) expression in hepatic tissue formation from ESCs. E-cad was transfected into mouse ESCs to enable a stable expression of E-cad. Hepatic differentiation of ESCs was then induced by hepatic growth factors. Wnt/β-catenin signaling and EMT speed were examined to determine the differentiation process. Hepatic and angiogenesis markers, as well as differentiated cell-adhesive force were also examined to identify the hepatic tissue differentiation. In our results, E-cad expression gradually decreased in normal ESC (N-ESC) differentiation, but remained stable in the E-cad transfected ESC (EC-ESC) group. In EC-ESC differentiation, expressions of cytoplastic β-catenin and EMT were much lower and significantly prolonged. Angiogenesis markers vascular endothelial growth factor receptor-1 (VEGFR-1) and CD31/PECAM-1 were expressed only on day 5-13 in N-ESC differentiation, whereas VEGFR-1 and CD31/PECAM-1 were expressed prolonged on day 5-17 in the EC-ESC group and were coincident with the expression of hepatic markers. Finally, EC-ESC differentiation maintained multilayer-growth patterns, and abundant vascular network structures appeared and migrated in albumin-positive cell areas. The cellular adhesion forces between embryonic body cells in EC-ESC differentiation during day 13-17 were similar to those of mouse liver tissue. In conclusion, accelerated EMT due to the decreased E-cad expression may partially contribute to the failure of hepatic tissue formation in N-ESC differentiation. E-cad can act in synergy with hepatic growth factors and facilitate the early-stage formation of hepatic tissues through down-regulating Wnt/β-catenin signaling and delaying EMT. This work provides a new insight into hepatic tissue

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

    PubMed

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

    2015-07-21

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

  18. Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype

    PubMed Central

    Cooper, James Ross; Abdullatif, Muhammad Bilal; Burnett, Edward C.; Kempsell, Karen E.; Conforti, Franco; Tolley, Howard; Collins, Jane E.; Davies, Donna E.

    2016-01-01

    Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line. PMID:27792742

  19. Cell confluency-induced Stat3 activation regulates NHE3 expression by recruiting Sp1 and Sp3 to the proximal NHE3 promoter region during epithelial dome formation.

    PubMed

    Su, Hsiao-Wen; Wang, Shainn-Wei; Ghishan, Fayez K; Kiela, Pawel R; Tang, Ming-Jer

    2009-01-01

    Activation of signal transducer and activator of transcription-3 (Stat3) during cell confluency is related to its regulatory roles in cell growth arrest- or survival-related physiological or developmental processes. We previously demonstrated that this signaling event triggers epithelial dome formation by transcriptional augmentation of sodium hydrogen exchanger-3 (NHE3) expression. However, the detailed molecular mechanism remained unclear. By using serial deletions, site-directed mutagenesis, and EMSA analysis, we now demonstrate Stat3 binding to an atypical Stat3-response element in the rat proximal NHE3 promoter, located adjacent to a cluster of Sp cis-elements (SpA/B/C), within -77/-36 nt of the gene. SpB (-58/-55 nt) site was more effective than SpA (-72/-69 nt) site for cooperative binding of Sp1/Sp3. Increasing cell density had no effect on Sp1/Sp3 expression but resulted in their increased binding to the SpA/B/C probe along with Stat3 and concurrently with enhanced nuclear pTyr705-Stat3 level. Immunoprecipitation performed with the nuclear extracts demonstrated physical interaction of Stat3 and Sp1/Sp3 triggered by cell confluency. Stat3 inhibition by overexpression of dominant-negative Stat3-D mutant in MDCK cells or by small interfering RNA-mediated knockdown in Caco-2 cells resulted in inhibition of the cell density-induced NHE3 expression, Sp1/Sp3 binding, and NHE3 promoter activity and in decreased dome formation. Thus, during confluency, ligand-independent Stat3 activation leads to its interaction with Sp1/Sp3, their recruitment to the SpA/B/C cluster in a Stat3 DNA-binding domain-dependent fashion, increased transcription, and expression of NHE3, to coordinate cell density-mediated epithelial dome formation.

  20. Interleukin-6 from Ovarian Mesenchymal Stem Cells Promotes Proliferation, Sphere and Colony Formation and Tumorigenesis of an Ovarian Cancer Cell Line SKOV3

    PubMed Central

    Ding, Dah-Ching; Liu, Hwan-Wun; Chu, Tang-Yuan

    2016-01-01

    The origin of the majority of epithelial ovarian cancers (EOC) is regarded as extraovarian, with the ovary being the secondary site. The aim of this study was to explore the possible role of ovarian mesenchymal stem cells (OvMSCs) and secreted IL-6 in the development of EOC. OvMSCs were derived from normal ovarian stroma. Cell surface markers and differentiation capability were determined. The effects of IL-6 and conditioned medium of OvMSCs on the malignant phenotype of SKOV3 ovarian cancer cells were tested, and the status of STAT3 and ERK phosphorylation was investigated. OvMSCs had similar surface marker profiles as bone marrow mesenchymal stem cells, i.e., CD44 (+), CD90 (+) and CD45 (-), and was readily inducible to osteogenic, adipogenic and chondrogenic differentiation. OvMSCs secreted an extremely high level (>2500 pg/ml) of IL-6. Treatment of SKOV3 cells with conditioned media from OvMSCs increased cell proliferation, tumor sphere formation and anchorage independent growth, and resulted in activation of STAT3 but not ERK. Coinjection of OvMSCs with SKOV3 cell enhanced tumorigenesis in NOD-SCID mice. All of these behaviors were blocked by IL-6 receptor blocking antibody administered in vitro or in vivo. The OvMSCs alone injected into mice had no tumor growth after 3 months. By secreting high levels of IL-6, OvMSCs enhance the proliferation, sphere and colony formation and tumorigenesis of SKOV3 cells. PMID:27698921

  1. Formation of a cobalt(III)-phenoxyl radical complex by acetic acid promoted aerobic oxidation of a Co(II)salen complex.

    PubMed

    Vinck, Evi; Murphy, Damien M; Fallis, Ian A; Strevens, Robert R; Van Doorslaer, Sabine

    2010-03-01

    The activation of N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexane-diamino Co(II), [Co(II)(1)], by the addition of acetic acid under aerobic conditions has been investigated by a range of spectroscopic techniques including continuous-wave EPR, HYSCORE, pulsed ENDOR, and resonance Raman. These measurements have revealed for the first time the formation of a coordinated cobalt(III)-bound phenoxyl radical labeled [Co(III)(1(*))(OAc)(n)](OAc)(m) (n = m = 1 or n = 2, m = 0). This cobalt(III)-bound phenoxyl radical is characterized by the following spin Hamiltonian parameters: g(x) = 2.0060, g(y) = 2.0031, g(z) = 1.9943, A(x) = 17 MHz, A(y) = 55 MHz, and A(z) = 14 MHz. Although the radical contains coordinated acetate(s), the experiments unambiguously proved that the phenoxyl radical is situated on ligand (1) as opposed to a phenoxyl radical ligated to cobalt in the axial position. Density functional theory computations on different models corroborate the stability of such a phenoxyl radical species and suggest the ligation of one or two acetate molecules to the complex. A mechanism is proposed, which accounts for the formation of this unusual and extremely robust phenoxyl radical, never previously observed for [Co(1)].

  2. Smooth Muscle Cell Foam Cell Formation, Apolipoproteins, and ABCA1 in Intracranial Aneurysms: Implications for Lipid Accumulation as a Promoter of Aneurysm Wall Rupture.

    PubMed

    Ollikainen, Eliisa; Tulamo, Riikka; Lehti, Satu; Lee-Rueckert, Miriam; Hernesniemi, Juha; Niemelä, Mika; Ylä-Herttuala, Seppo; Kovanen, Petri T; Frösen, Juhana

    2016-07-01

    Saccular intracranial aneurysm (sIA) aneurysm causes intracranial hemorrhages that are associated with high mortality. Lipid accumulation and chronic inflammation occur in the sIA wall. A major mechanism for lipid clearance from arteries is adenosine triphosphate-binding cassette A1 (ABCA1)-mediated lipid efflux from foam cells to apolipoprotein A-I (apoA-I). We investigated the association of wall degeneration, inflammation, and lipid-related parameters in tissue samples of 16 unruptured and 20 ruptured sIAs using histology and immunohistochemistry. Intracellular lipid accumulation was associated with wall remodeling (p = 0.005) and rupture (p = 0.020). Foam cell formation was observed in smooth muscle cells, in addition to CD68- and CD163-positive macrophages. Macrophage infiltration correlated with intracellular lipid accumulation and apolipoproteins, including apoA-I. ApoA-I correlated with markers of lipid accumulation and wall degeneration (p = 0.01). ApoA-I-positive staining colocalized with ABCA1-positive cells particularly in sIAs with high number of smooth muscle cells (p = 0.003); absence of such colocalization was associated with wall degeneration (p = 0.017). Known clinical risk factors for sIA rupture correlated inversely with apoA-I. We conclude that lipid accumulation associates with sIA wall degeneration and risk of rupture, possibly via formation of foam cells and subsequent loss of mural cells. Reduced removal of lipids from the sIA wall via ABCA1-apoA-I pathway may contribute to this process.

  3. Short-Term Spheroid Formation Enhances the Regenerative Capacity of Adipose-Derived Stem Cells by Promoting Stemness, Angiogenesis, and Chemotaxis

    PubMed Central

    Cheng, Nai-Chen; Chen, Szu-Yu; Li, Jia-Rong

    2013-01-01

    Adipose-derived stem cells (ASCs) represent an important source of mesenchymal stem cells for clinical application. During in vitro culture, ASCs quickly lose the expression of transcription factors associated with pluripotency and self-renewal (Sox-2, Oct-4, and Nanog) and CXCR4, the key receptor responsible for stem cell homing. To enhance their therapeutic potential despite in vitro passages, we examined whether ASCs exhibit superior regenerative capacity by expanding them in monolayers following short-term spheroid formation. Spheroid-derived ASCs retained the expression pattern of cell surface markers and adipogenic/osteogenic differentiation capabilities of ASCs constantly cultured in monolayers. However, spheroid-derived ASCs exhibited higher expansion efficiency with less senescence. Moreover, spheroid-derived ASCs expressed significantly higher levels of pluripotency markers, CXCR4, and angiogenic growth factors. Enhanced in vitro migration, associated with the increased expression of matrix metalloproteinases (MMP-9 and MMP-13), was also observed in spheroid-derived ASCs. The enhanced migration and MMP expression could be inhibited by a CXCR4-specific peptide antagonist, AMD3100. Using a murine model with healing-impaired cutaneous wounds, we observed faster healing and enhanced angiogenesis in the wounds treated with spheroid-derived ASCs. Significantly more cellular engraftment of spheroid-derived ASCs in the cutaneous wound tissue was also noted, with evidence of ASC differentiation toward endothelial and epidermal lineages. These findings suggest that short-term spheroid formation of ASCs before monolayer culture enhances their properties of stemness, angiogenesis, and chemotaxis and thereby increases their regenerative potential for therapeutic use. PMID:23847001

  4. Arp2/3 promotes junction formation and maintenance in the Caenorhabditis elegans intestine by regulating membrane association of apical proteins

    PubMed Central

    Bernadskaya, Yelena Y.; Patel, Falshruti B.; Hsu, Hsiao-Ting; Soto, Martha C.

    2011-01-01

    It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin–radixin–moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes. PMID:21697505

  5. A model for triple helix formation on human telomerase reverse transcriptase (hTERT) promoter and stabilization by specific interactions with the water soluble perylene derivative, DAPER.

    PubMed

    Rossetti, Luigi; D'Isa, Giuliana; Mauriello, Clementina; Varra, Michela; De Santis, Pasquale; Mayol, Luciano; Savino, Maria

    2007-08-01

    The promoter of human telomerase reverse transcriptase (hTERT) gene, in the region from -1000 to +1, contains two homopurine-homopyrimidine sequences (-835/-814 and -108/-90), that can be considered as potential targets to triple helix forming oligonucleotides (TFOs) for applying antigene strategy. We have chosen the sequence (-108/-90) on the basis of its unfavorable chromatin organization, evaluated by theoretical nucleosome positioning and nuclease hypersensitive sites mapping. On this sequence, anti-parallel triplex with satisfactory thermodynamic stability is formed by two TFOs, having different lengths. Triplex stability is significantly increased by specific interactions with the perylene derivative N,N'-bis[3,3'-(dimethylamino) propylamine]-3,4,9,10-perylenetetracarboxylic diimide (DAPER). Since DAPER is a symmetric molecule, the induced Circular Dichroism (CD) spectra in the range 400-600 nm allows us to obtain information on drug binding to triplex and duplex DNA. The drug-induced ellipticity is significantly higher in the case of triplex with respect to duplex and, surprisingly, it increases at decreasing of DNA. A model is proposed where self-stacked DAPER binds to triplex or to duplex narrow grooves.

  6. U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation.

    PubMed

    Park, Sung Mi; Ou, Jianhong; Chamberlain, Lynn; Simone, Tessa M; Yang, Huan; Virbasius, Ching-Man; Ali, Abdullah M; Zhu, Lihua Julie; Mukherjee, Siddhartha; Raza, Azra; Green, Michael R

    2016-05-19

    Recurrent mutations in the splicing factor U2AF35 are found in several cancers and myelodysplastic syndrome (MDS). How oncogenic U2AF35 mutants promote transformation remains to be determined. Here we derive cell lines transformed by the oncogenic U2AF35(S34F) mutant and identify aberrantly processed pre-mRNAs by deep sequencing. We find that in U2AF35(S34F)-transformed cells the autophagy-related factor 7 (Atg7) pre-mRNA is abnormally processed, which unexpectedly is not due to altered splicing but rather selection of a distal cleavage and polyadenylation (CP) site. This longer Atg7 mRNA is translated inefficiently, leading to decreased ATG7 levels and an autophagy defect that predisposes cells to secondary mutations, resulting in transformation. MDS and acute myeloid leukemia patient samples harboring U2AF35(S34F) have a similar increased use of the ATG7 distal CP site, and previous studies have shown that mice with hematopoietic cells lacking Atg7 develop an MDS-like syndrome. Collectively, our results reveal a basis for U2AF35(S34F) oncogenic activity.

  7. ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase hexamer formation at low micromolar concentrations.

    PubMed

    Kuriata, Agnieszka M; Chakraborty, Manas; Henderson, J Nathan; Hazra, Suratna; Serban, Andrew J; Pham, Tuong V T; Levitus, Marcia; Wachter, Rebekka M

    2014-11-25

    We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

  8. Promoting the bio-cathode formation of a constructed wetland-microbial fuel cell by using powder activated carbon modified alum sludge in anode chamber

    NASA Astrophysics Data System (ADS)

    Xu, Lei; Zhao, Yaqian; Doherty, Liam; Hu, Yuansheng; Hao, Xiaodi

    2016-05-01

    MFC centered hybrid technologies have attracted attention during the last few years due to their compatibility and dual advantages of energy recovery and wastewater treatment. In this study, a MFC was integrated into a dewatered alum sludge (DAS)- based vertical upflow constructed wetland (CW). Powder activate carbon (PAC) was used in the anode area in varied percentage with DAS to explore its influences on the performance of the CW-MFC system. The trial has demonstrated that the inclusion of PAC improved the removal efficiencies of COD, TN and RP. More significantly, increasing the proportion of PAC from 2% to 10% can significantly enhance the maximum power densities from 36.58 mW/m2 to 87.79 mW/m2. The induced favorable environment for bio-cathode formation might be the main reason for this improvement since the content of total extracellular polymeric substances (TEPS) of the substrate in the cathode area almost doubled (from 44.59 μg/g wet sludge to 87.70 μg/g wet sludge) as the percentage of PAC increased to 10%. This work provides another potential usage of PAC in CW-MFCs with a higher wastewater treatment efficiency and energy recovery.

  9. Promoting the bio-cathode formation of a constructed wetland-microbial fuel cell by using powder activated carbon modified alum sludge in anode chamber

    PubMed Central

    Xu, Lei; Zhao, Yaqian; Doherty, Liam; Hu, Yuansheng; Hao, Xiaodi

    2016-01-01

    MFC centered hybrid technologies have attracted attention during the last few years due to their compatibility and dual advantages of energy recovery and wastewater treatment. In this study, a MFC was integrated into a dewatered alum sludge (DAS)- based vertical upflow constructed wetland (CW). Powder activate carbon (PAC) was used in the anode area in varied percentage with DAS to explore its influences on the performance of the CW-MFC system. The trial has demonstrated that the inclusion of PAC improved the removal efficiencies of COD, TN and RP. More significantly, increasing the proportion of PAC from 2% to 10% can significantly enhance the maximum power densities from 36.58 mW/m2 to 87.79 mW/m2. The induced favorable environment for bio-cathode formation might be the main reason for this improvement since the content of total extracellular polymeric substances (TEPS) of the substrate in the cathode area almost doubled (from 44.59 μg/g wet sludge to 87.70 μg/g wet sludge) as the percentage of PAC increased to 10%. This work provides another potential usage of PAC in CW-MFCs with a higher wastewater treatment efficiency and energy recovery. PMID:27197845

  10. Novel apo E-derived ABCA1 agonist peptide (CS-6253) promotes reverse cholesterol transport and induces formation of preβ-1 HDL in vitro

    SciTech Connect

    Hafiane, Anouar; Bielicki, John K.; Johansson, Jan O.; Genest, Jacques; Zhu, Xuewei

    2015-07-24

    Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These

  11. Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium*

    PubMed Central

    Sheikh, M. Osman; Xu, Yuechi; van der Wel, Hanke; Walden, Paul; Hartson, Steven D.; West, Christopher M.

    2015-01-01

    O2 sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1–F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1–cullin-1–F-box protein–Rbx1 subcomplex of E3SCFUb ligases. E3SCFUb ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O2 availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA–Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modi