WorldWideScience

Sample records for monomeric rfp mrfp-based

  1. RFP tags for labeling secretory pathway proteins

    Energy Technology Data Exchange (ETDEWEB)

    Han, Liyang; Zhao, Yanhua [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Xi; Peng, Jianxin [College of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Xu, Pingyong, E-mail: pyxu@ibp.ac.cn [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Huan, Shuangyan, E-mail: shuangyanhuan@163.com [State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhang, Mingshu, E-mail: mingshu1984@gmail.com [Key Laboratory of Interdisciplinary Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

  2. A Structural Basis for Reversible Photoswitching of Absorbance Spectra in Red Fluorescent Protein rsTagRFP

    Energy Technology Data Exchange (ETDEWEB)

    Pletnev, Sergei; Subach, Fedor V.; Dauter, Zbigniew; Wlodawer, Alexander; Verkhusha, Vladislav V. (Einstein); (NCI)

    2012-09-05

    rsTagRFP is the first monomeric red fluorescent protein (FP) with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Foerster resonance energy transfer. Yellow FPs, YPet and mVenus, are demonstrated to be excellent photochromic Foerster resonance energy transfer donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans-isomers corresponding to its ON and OFF states, respectively. Unlike in other photoswitchable FPs, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.

  3. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    Science.gov (United States)

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

  4. RfpA, RfpB, and RfpC are the Master Control Elements of Far-Red Light Photoacclimation (FaRLiP).

    Science.gov (United States)

    Zhao, Chi; Gan, Fei; Shen, Gaozhong; Bryant, Donald A

    2015-01-01

    Terrestrial cyanobacteria often occur in niches that are strongly enriched in far-red light (FRL; λ > 700 nm). Some cyanobacteria exhibit a complex and extensive photoacclimation response, known as FRL photoacclimation (FaRLiP). During the FaRLiP response, specialized paralogous proteins replace 17 core subunits of the three major photosynthetic complexes: Photosystem (PS) I, PS II, and the phycobilisome. Additionally, the cells synthesize both chlorophyll (Chl) f and Chl d. Using biparental mating from Escherichia coli, we constructed null mutants of three genes, rfpA, rfpB, and rfpC, in the cyanobacteria Chlorogloeopsis fritschii PCC 9212 and Chroococcidiopsis thermalis PCC 7203. The resulting mutants were no longer able to modify their photosynthetic apparatus to absorb FRL, were no longer able to synthesize Chl f, inappropriately synthesized Chl d in white light, and were unable to transcribe genes of the FaRLiP gene cluster. We conclude that RfpA, RfpB, and RfpC constitute a FRL-activated signal transduction cascade that is the master control switch for the FaRLiP response. FRL is proposed to activate (or inactivate) the histidine kinase activity of RfpA, which leads to formation of the active state of RfpB, the key response regulator and transcription activator. RfpC may act as a phosphate shuttle between RfpA and RfpB. Our results show that reverse genetics via conjugation will be a powerful approach in detailed studies of the FaRLiP response.

  5. Gyrokinetic Studies of Microinstabilities in the RFP

    CERN Document Server

    Carmody, Daniel; Terry, P W

    2013-01-01

    An analytic equilibrium, the Toroidal Bessel Function Model, is used in conjunction with the gyrokinetic code GYRO to investigate the nature of microinstabilities in a reversed field pinch (RFP) plasma. The effect of the normalized electron plasma pressure ({\\beta}) on the characteristics of the microinstabilities is studied. A transition between an ion temperature gradient (ITG) driven mode and a microtearing mode as the dominant instability is found to occur at a {\\beta} value of approximately 4.5%. Suppression of the ITG mode occurs as in the tokamak, through coupling to shear Alfven waves, with a critical {\\beta} for stability higher than its tokamak equivalent due to a shorter parallel connection length. There is a steep dependence of the microtearing growth rate on temperature gradient suggesting high profile stiffness. There is evidence for a collisionless microtearing mode. The properties of this mode are investigated, and it is found that curvature drift plays an important role in the instability.

  6. Electron Bernstein Wave Emission from RFP Plasmas

    Science.gov (United States)

    Nornberg, M. D.; Thomas, M.; Anderson, J.; Forest, C. B.

    1998-11-01

    Electron cyclotron emission (ECE) has proven to be a powerfull diagnostic tool in tokamak plasmas for determining the time evolution of the electron temperature profile. The standard technique of observing O-mode or X-mode electromagnetic waves normal to the magnetic field is not applicable to reversed field pinch (RFP) plasmas since the plasma frequency is much larger than the electron cyclotron frequency. We are investigating the use of electron Bernstein waves (presumed to be in thermal equilibrium with the electrons) through the aip.org/journal_cgi/ getpdf?KEY=PRLTAO&cvips=PRLTAO000078000018003467000001>O-X-B mode conversion process. At oblique incidence, the evanescent layer separating the plamsa cutoff from the cyclotron cutoff vanishes, allowing conversion of the Bernstein mode waves to the extraordinary mode and finally to the ordinary mode. The O-mode radiation is received by a phased array antenna consisting of two waveguides on the edge of the plasma, and the spectrum of emitted radiation is measured using a radiometer spanning 4-8 GHz. In addition to providing information about the electron temperature profile, the spectrum can provide a novel method of measuring the central magnetic field strength for current profile reconstructions.

  7. RFP for the Auroral Multiscale Midex (AMM) Mission star tracker

    DEFF Research Database (Denmark)

    Riis, Troels; Betto, Maurizio; Jørgensen, John Leif;

    1999-01-01

    This document is in response to the John Hopkins University - Applied Physics Laboratory RFP for the Auroral Multiscale Midex Mission star tracker.It describes the functionality, the requirements and the performance of the ASC Star Tracker.......This document is in response to the John Hopkins University - Applied Physics Laboratory RFP for the Auroral Multiscale Midex Mission star tracker.It describes the functionality, the requirements and the performance of the ASC Star Tracker....

  8. Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein

    Directory of Open Access Journals (Sweden)

    Hadjantonakis Anna-Katerina

    2005-07-01

    permit deeper tissue imaging. Our work in generating CAG::mRFP1 transgenic ES cells and mice demonstrates the developmental neutrality of mRFP1 in an organismal context. It paves the way for the use of DsRed-based monomeric RFPs in transgenic and gene targeted approaches which often necessitate spatially and/or temporally restricted reporter expression. Moreover animals of the CAG::mRFP1 transgenic strain serve as a source of RFP tagged tissue for the derivation of cell lines and explant, transplant and embryo chimera experiments.

  9. RFX machine and power supply improvements for RFP advanced studies

    Energy Technology Data Exchange (ETDEWEB)

    Piovan, R. E-mail: piovan@igi.pd.cnr.it; Gnesotto, F.; Ortolani, S.; Baker, W.; Barana, O.; Bettini, P.; Cavazzana, R.; Chitarin, G.; Dal Bello, S.; De Lorenzi, A.; Fiorentin, P.; Gaio, E.; Grando, L.; Luchetta, A.; Manduchi, G.; Marchiori, G.; Marcuzzi, D.; Masiello, A.; Milani, F.; Peruzzo, S.; Pomaro, N.; Sonato, P.; Taliercio, C.; Toigo, V.; Zaccaria, P.; Zanotto, L.; Zollino, G

    2001-10-01

    Experimental results and theoretical studies call for Reversed Field Experiment (RFX) machine and power supply improvements to allow studies that go beyond those of a conventional Reversed Field Pinch (RFP) with passively stabilized turbulent MHD dynamo. The new paths opened by recent results in RFX and other RFP machines are introduced; then the goals and the design lines of the technical modifications of RFX, mainly addressed to improve the first wall, the plasma magnetic boundaries and to increase the operational flexibility of the toroidal field circuit power supply, are reported.

  10. Physics and optimization of plasma startup in the RFP

    Science.gov (United States)

    Mao, W.; Chapman, B. E.; Ding, W. X.; Lin, L.; Almagri, A. F.; Anderson, J. K.; Den Hartog, D. J.; Duff, J.; Ko, J.; Kumar, S. T. A.; Morton, L.; Munaretto, S.; Parke, E.; Reusch, J. A.; Sarff, J. S.; Waksman, J.; Brower, D. L.; Liu, W.

    2015-05-01

    In the tokamak and reversed-field pinch (RFP), inductively driven toroidal plasma current provides the confining poloidal magnetic field and ohmic heating power, but the magnitude and/or duration of this current is limited by the available flux swing in the poloidal field transformer. A portion of this flux is consumed during startup as the current is initiated and ramped to its final target value, and considerable effort has been devoted to understanding startup and minimizing the amount of flux consumed. Flux consumption can be reduced during startup in the RFP by increasing the toroidal magnetic field, Bti, applied to initiate the discharge, but the underlying physics is not yet entirely understood. Toward increasing this understanding, we have for the first time in the RFP employed advanced, non-invasive diagnostics on the Madison Symmetric Torus to measure the evolution of current, magnetic field, and kinetic profiles during startup. Flux consumption during startup is dominantly inductive, but we find that the inductive flux consumption drops as Bti increases. The resistive consumption of flux, while relatively small, apparently increases with Bti due to a smaller electron temperature. However, the ion temperature increases with Bti, exceeding the electron temperature and thus reflecting non-collisional heating. Magnetic fluctuations also increase with Bti, corresponding primarily to low-n modes that emerge sequentially as the safety factor profile evolves from tokamak-like to that of the RFP.

  11. 75 FR 40857 - Webinar About Advanced Defense Technologies RFP

    Science.gov (United States)

    2010-07-14

    ... ADMINISTRATION Webinar About Advanced Defense Technologies RFP AGENCY: U.S. Small Business Administration (SBA). ACTION: Notice of open webinar meeting to discuss Advanced Defense Technologies (ADT) Request for... webinar it is hosting to answer questions from potential Offerors about the Advanced Defense...

  12. 75 FR 40856 - Federal Register Meeting Notice; Webinar About Regional Innovation Clusters RFP

    Science.gov (United States)

    2010-07-14

    ... ADMINISTRATION Federal Register Meeting Notice; Webinar About Regional Innovation Clusters RFP AGENCY: U.S. Small Business Administration (SBA) ACTION: Notice of open webinar meeting to discuss Regional Innovation... Innovation Clusters RFP. For more information please go to http://www.sba.gov/clusters/index.html . The RFP...

  13. Effects of peripherally administered cholecystokinin-8 and secretin on feeding/drinking and oxytocin-mRFP1 fluorescence in transgenic rats.

    Science.gov (United States)

    Motojima, Yasuhito; Kawasaki, Makoto; Matsuura, Takanori; Saito, Reiko; Yoshimura, Mitsuhiro; Hashimoto, Hirofumi; Ueno, Hiromichi; Maruyama, Takashi; Suzuki, Hitoshi; Ohnishi, Hideo; Sakai, Akinori; Ueta, Yoichi

    2016-08-01

    Peripheral administration of cholecystokinin (CCK)-8 or secretin activates oxytocin (OXT)-secreting neurons in the hypothalamus. Although OXT is involved in the regulation of feeding behavior, detailed mechanism remains unclear. In the present study, we examined the central OXTergic pathways after intraperitoneally (i.p.) administration of CCK-8 and secretin using male OXT-monomeric red fluorescent protein 1 (mRFP1) transgenic rats and male Wistar rats. I.p. administration of CCK-8 (50μg/kg) and secretin (100μg/kg) decreased food intake in these rats. While i.p. administration of CCK-8 decreased water intake, i.p. administration of secretin increased water intake. Immunohistochemical study revealed that Fos-Like-Immunoreactive cells were observed abundantly in the brainstem and in the OXT neurons in the dorsal division of the parvocellular paraventricular nucleus (dpPVN). We could observe marked increase of mRFP1 fluorescence, as an indicator for OXT, in the dpPVN and mRFP1-positive granules in axon terminals of the dpPVN OXT neurons in the nucleus tractus solitarius (NTS) after i.p. administration of CCK-8 and secretin. These results provide us the evidence that, at least in part, i.p. administration of CCK-8 or secretin might be involved in the regulation of feeding/drinking via a OXTergic pathway from the dpPVN to the NTS.

  14. Incorporating Software Requirements into the System RFP: Survey of RFP Language for Software by Topic, v. 2.0

    Science.gov (United States)

    2009-05-01

    Bergey 2005]. EXAMPLE 2 The contractor will development and document scenarios required to conduct architecture evaluation using the method...requirements that are part of the RFP [ Bergey 2002]. The statement of work (SOW) describes what the supplier must accomplish. In terms of any evaluation...and the potential system supplier [ Bergey 2002]. 7.14.2 Section M - Evaluation EXAMPLE 1 To incorporate architecture evaluation, Section M must

  15. Effects of pulsed electric field on ULQ and RFP plasmas

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, M. [Iwate Univ., Morioka (Japan). Faculty of Engineering; Saito, K.; Suzuki, T. [and others

    1997-12-31

    Dynamo activity and self-organization processes are investigated using the application of pulsed poloidal and toroidal electric fields on ULQ and RFP plasmas. Synchronized to the application of the pulsed electric fields, the remarkable responses of the several plasma parameters are observed. The plasma has a preferential magnetic field structure, and the external perturbation activates fluctuation to maintain the structure through dynamo effect. This process changes the total dissipation with the variation of magnetic helicity in the system, showing that self organization accompanies an enhanced dissipation. (author)

  16. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  17. The plasma boundary in Single Helical Axis RFP plasmas

    CERN Document Server

    Martines, E; Momo, B; Munaretto, S; Innocente, P; Spolaore, M

    2009-01-01

    Single Helical Axis (SHAx) states obtained in high current reversed field pinch (RFP) plasmas display, aside from a dominant mode in the m=1 spectrum, also a dominant m=0 mode, with the same toroidal mode number as the m=1 one. The two modes have a fixed phase relationship. The island chain created by the m=0 mode across the reversal surface gives rise, at shallow reversal of the toroidal field, to an X-point structure which separates the last closed flux surface from the first wall, creating a divertor-like configuration. The plasma-wall interaction is found to be related to the connection length of the field lines intercepting the wall, which displays a pattern modulated by the dominant mode toroidal periodicity. This configuration, which occurs only for shallow toroidal field reversal, could be exploited to realize an island divertor in analogy to stellarators.

  18. Rfp-Y region polymorphism and Marek's disease resistance in multitrait immunocompetence-selected chicken lines.

    Science.gov (United States)

    Lakshmanan, N; Lamont, S J

    1998-04-01

    Although the influence of the chicken classical MHC in resistance to many diseases is well established, the role of the recently identified, genetically independent, MHC-like region known as Rfp-Y is unclear. The objectives of this study were to analyze the frequencies of DNA polymorphisms of the Rfp-Y region in White Leghorn lines, which were divergently selected in replicate for multitrait immunocompetence, and to determine the association of these polymorphisms with Marek's disease (MD) resistance. Chicks, either with or without herpes virus of turkey (HVT) vaccination, were challenged with 500 ffu of a very virulent Marek's disease virus (Md5) at 2 d of age. The MD-related data were collected for 10 wk. PvuII-digested genomic DNA was hybridized with an Rfp-Y region-specific probe, 18.1. Three Rfp-Y polymorphisms were observed. The frequency of one Rfp-Y polymorphism was significantly different between divergently selected multitrait immunocompetence lines in one replicate only; therefore, the impact of multitrait immunocompetence selection on Rfp-Y polymorphisms is inconclusive. The PvuII defined Rfp-Y region polymorphisms had no association with either innate or vaccine-induced MD resistance to Md5 virus challenge.

  19. Ion Heating Anisotropy during Dynamo Activity in the MST RFP

    Science.gov (United States)

    den Hartog, D. J.; Chapman, J. T.; Craig, D.; Fiksel, G.; Fontana, P. W.

    1999-11-01

    MHD dynamo activity is large in the MST Reversed-Field Pinch during sawtooth crashes, and small otherwise. During a sawtooth crash, ion temperature increases rapidly to a level several times as high as the temperature between sawteeth, which itself can be larger than the electron temperature. Several theories have been developed to explain this ion heating, some indicating a possible asymmetry in perpendicular to parallel heating [C. G. Gimblett, Europhys. Lett. 11, 541 (1990); Z. Yoshida, Nucl. Fusion 31, 386 (1991); N. Mattor, P. W. Terry, and S. C. Prager, Comments Plasma Phys. Controlled Fusion 15, 65 (1992)]. In standard MST discharges, impurity ion temperature measured perpendicular to the magnetic field (T_⊥) is higher than impurity ion temperature parallel to the magnetic field (T_allel) during a sawtooth crash. Throughout the rest of the sawtooth cycle, T_⊥ EXTRAP-T2 RFP which showed T_⊥ < T_allel throughout the discharge [K. Sasaki et al., Plasma Phys. Control. Fusion 39, 333 (1997)

  20. RFP for Smiles and Maxi projects to the Internationale Space Station

    DEFF Research Database (Denmark)

    Denver, Troelz; Thuesen, Gøsta; Jørgensen, Finn E

    1999-01-01

    The document for describes the functionality, the performance and the requirements of the ASC Star Tracker and it includes the RFP for the project's Smiles and Maxi on the International Space Station.......The document for describes the functionality, the performance and the requirements of the ASC Star Tracker and it includes the RFP for the project's Smiles and Maxi on the International Space Station....

  1. RFP for Smiles and Maxi projects to the Internationale Space Station

    DEFF Research Database (Denmark)

    Denver, Troelz; Thuesen, Gøsta; Jørgensen, Finn E

    1999-01-01

    The document for describes the functionality, the performance and the requirements of the ASC Star Tracker and it includes the RFP for the project's Smiles and Maxi on the International Space Station.......The document for describes the functionality, the performance and the requirements of the ASC Star Tracker and it includes the RFP for the project's Smiles and Maxi on the International Space Station....

  2. First molecular identification of the transgene red fluorescent protein (RFP) in transgenic ornamental zebrafish (Danio rerio) introduced in Peru

    OpenAIRE

    Carlos Scotto; Fernando Serna

    2013-01-01

    In this paper the transgenic fluorescent red, orange and pink zebra fish (Danio rerio), found in local aquariums in Peru, were identified using the PCR technique to amplify the transgene RFP sea anemone belonging to Discosoma spp. The gene expression of the red fluorescent protein (RFP) transgene was found to determine different gradients-of-bioluminescence (shades in color) in each GMO fish analyzed. We performed sequence analysis of the two variants of the RFP along with six variants of the...

  3. New chicken Rfp-Y haplotypes on the basis of MHC class II RFLP and MLC analyses

    DEFF Research Database (Denmark)

    Juul-Madsen, H R; Zoorob, R; Auffray, C;

    1997-01-01

    New chicken Rfp-Y haplotypes were determined by the use of restriction fragment length polymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B15, B19, B21, B201. The RFLP polymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps...... near a polymorphic lectin gene located in the Rfp-Y system and DNA from families with known segregation of the implicated RFLP polymorphism. For the first time it is shown that major histocompatibility complex class II genes in the Rfp-Y system have functional implications. Sequence information...

  4. Size separation of analytes using monomeric surfactants

    Science.gov (United States)

    Yeung, Edward S.; Wei, Wei

    2005-04-12

    A sieving medium for use in the separation of analytes in a sample containing at least one such analyte comprises a monomeric non-ionic surfactant of the of the general formula, B-A, wherein A is a hydrophilic moiety and B is a hydrophobic moiety, present in a solvent at a concentration forming a self-assembled micelle configuration under selected conditions and having an aggregation number providing an equivalent weight capable of effecting the size separation of the sample solution so as to resolve a target analyte(s) in a solution containing the same, the size separation taking place in a chromatography or electrophoresis separation system.

  5. Monomeric Friction Coefficient of Metalnanodispersible Polymeric Systems

    Directory of Open Access Journals (Sweden)

    B.B. Kolupayev

    2016-12-01

    Full Text Available Influence of a nanodispersible metal excipient in number of 0    5,0 vol.% Cu for the size of a monomeric friction coefficient of polyvinylchloride (PVC systems in temperature range 298  Т  (Tg + 10 K is investigated. It is shown that various types of coordination movements of building blocks are described by a friction coefficient which serves as a measure of influence of external fields and ingredients on viscoelastic behavior of a composite. The analysis of processes of a relaxation on the basis of the theory of flexible chains taking into account power and entropic factors is carried out.

  6. Critical parameters and TCLP performance of the RFP microwave solidification system

    Energy Technology Data Exchange (ETDEWEB)

    Sprenger, G.S.

    1993-01-01

    Two series of experiments were conducted at Rocky Flats Plant (RFP) to identify the critical operating parameters for microwave solidification and to evaluate the performance of the product against the EPA's Toxicity Characteristic Leach Procedure (TCLP). A surrogate hydroxide coprecipitation sludge spiked with heavy metals was used in the study. The RFP process uses microwave energy to heat and melt the waste into a vitreous final form that is suitable for land disposal. The results of the study indicate that waste loading and borax content in the glass forming frit are critical in the treatment of hydroxide sludge. Also, the product will easily satisfy EPA's limitations for land disposal. These results are very encouraging and support RFP's commitment to the use of microwave technology for treatment of various mixed waste streams at the facility.

  7. The RFP Process: Effective Management of the Acquisition of Library Materials.

    Science.gov (United States)

    Wilkinson, Frances C.; Thorson, Connie Capers

    Many librarians view procurement, with its myriad forms, procedures, and other organizational requirements, as a tedious or daunting challenge. This book simplifies the process, showing librarians how to successfully prepare a Request for Proposal (RFP) and make informed decisions when determining which vendors to use for purchasing library…

  8. Initial results from the rebuilt EXTRAP T2R RFP device

    Science.gov (United States)

    Brunsell, P. R.; Bergsåker, H.; Cecconello, M.; Drake, J. R.; Gravestijn, R. M.; Hedqvist\\ad{2 }, A.; Malmberg, J.-A.

    2001-11-01

    The EXTRAP T2R thin shell reversed-field pinch (RFP) device has recently resumed operation after a major rebuild including the replacement of the graphite armour with molybdenum limiters, a fourfold increase of the shell time constant, and the replacement of the helical coil used for the toroidal field with a conventional solenoid-type coil. Wall-conditioning using hydrogen glow discharge cleaning was instrumental for successful RFP operation. Carbon was permanently removed from the walls during the first week of operation. The initial results from RFP operation with relatively low plasma currents in the range Ip = 70-100 kA are reported. RFP discharges are sustained for more than three shell times. Significant improvements in plasma parameters are observed, compared to operation before the rebuild. There is a substantial reduction in the carbon impurity level. The electron density behaviour is more shot-to-shot reproducible. The typical density is ne = 0.5-1×1019 m-3. Monitors of Hα line radiation indicate that the plasma wall interaction is more toroidally symmetric and that there is less transient gas release from the wall. The minimum loop voltage is in the range Vt = 28-35 V, corresponding to a reduction by a factor of two to three compared to the value before the rebuild.

  9. Off-the-Shelf ILS: Library Issues RFP for Integrated Library System.

    Science.gov (United States)

    Library of Congress Information Bulletin, 1997

    1997-01-01

    Describes the Request for Proposal (RFP) issued by the Library of Congress for an integrated library system to help in the areas of inventory control, acquisitions, binding, cataloging, circulation, collection development, online catalogs, preservation, serials management, production of products, and the computer year 2000 problem. (LRW)

  10. Simultaneous measurement of electron temperature and density by a line pair method in the RFP plasma

    Science.gov (United States)

    Watanabe, Masayuki; Shimizu, S.; Ogawa, H.; Shinohara, T.

    2009-11-01

    A line-pair-method has been applied for a simultaneous measurement of the electron temperature and density in ATRAS RFP plasma. Three helium spectrum lines (668nm, 706nm, 728nm) were measured during the discharge at the same time and the electron temperature and density is estimated by using a Collision-Radiation model. To get the signal of the helium impunity line from the RFP discharge, the RFP plasma in the hydrogen gas with a few mixed helium gas was formed. In the typical ATRAS RFP discharge of the plasma current of 60kA, the electron temperature was approximately 50-150 eV and the electron density is the order of 10^18 m-3. During the discharge, the change of the temperature and density are mutually related and this correlation was the almost reverse phase. The periodically change of the temperature and density were also observed. This change synchronizes with a periodically increase of the averaged toroidal magnetic field, which is caused by the toroidal rotation of the increase of the toroidal magnetic field. This rotation, which is deeply related with dynamo effect, makes the plasma energy lose and particles also diffuse toward the plasma edge. As a result, the recycling of the particle and energy are occurred at the same time.

  11. Electrophysiological effects of kainic acid on vasopressin-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 neurones isolated from the supraoptic nucleus in transgenic rats.

    Science.gov (United States)

    Ohkubo, J; Ohbuchi, T; Yoshimura, M; Maruyama, T; Ishikura, T; Matsuura, T; Suzuki, H; Ueta, Y

    2014-01-01

    The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)-producing and oxytocin (OXT)-producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP-enhanced green fluorescent protein (eGFP) and the other expressing an OXT-monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP-eGFP and OXT-mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate-induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP-eGFP and OXT-mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA-induced responses were inhibited by the non-NMDA ionotrophic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3(1H4H)-dione in both AVP-eGFP and OXT-mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose-dependent manner. The KA-induced currents were significantly larger in OXT-mRFP1 neurones than in AVP-eGFP neurones. This significant difference in KA-induced currents was abolished by the GluK1-containing KAR antagonist UBP302. At high concentrations (250-500 μm), the specific GluK1-containing KAR agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) induced significantly larger currents in OXT-mRFP1 neurones than in AVP-eGFP neurones. Furthermore, the difference between the AVP-eGFP and OXT-mRFP1 neurones in the ATPA currents

  12. Assessing in vitro stem-cell function and tracking engraftment of stem cells in ischaemic hearts by using novel iRFP gene labelling.

    Science.gov (United States)

    Wang, Yingjie; Zhou, Mi; Wang, Xiaolong; Qin, Gangjian; Weintraub, Neal L; Tang, Yaoliang

    2014-09-01

    Near-infrared fluorescence (NIRF) imaging by using infrared fluorescent protein (iRFP) gene labelling is a novel technology with potential value for in vivo applications. In this study, we expressed iRFP in mouse cardiac progenitor cells (CPC) by lentiviral vector and demonstrated that the iRFP-labelled CPC (CPC(iRFP)) can be detected by flow cytometry and fluorescent microscopy. We observed a linear correlation in vitro between cell numbers and infrared signal intensity by using the multiSpectral imaging system. CPC(iRFP) injected into the non-ischaemic mouse hindlimb were also readily detected by whole-animal NIRF imaging. We then compared iRFP against green fluorescent protein (GFP) for tracking survival of engrafted CPC in mouse ischaemic heart tissue. GFP-labelled CPC (CPC(GFP)) or CPC labelled with both iRFP and GFP (CPC(iRFP) (GFP)) were injected intramyocardially into mouse hearts after infarction. Three days after cell transplantation, a strong NIRF signal was detected in hearts into which CPC(iRFP) (GFP), but not CPC(GFP), were transplanted. Furthermore, iRFP fluorescence from engrafted CPC(iRFP) (GFP) was detected in tissue sections by confocal microscopy. In conclusion, the iRFP-labelling system provides a valuable molecular imaging tool to track the fate of transplanted progenitor cells in vivo.

  13. Expression Levels of RFP in Normal and Cancer Human Tissues via Real-time RT-PCR Detection

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Ret finger protein(RFP) is a member of the tripartite motif family, which is characterized by a conserved RING finger of motif, a B-box, and a coiled-coil domain(they are called RBCC generally). Although RFP was known to be an oncogene when its RBCC moiety was connected with a tyrosine kinase domain by DNA rearrangement, its biological function was not well defined. In this study, by using real-time RT-PCR, the RFP expressions in human and mouse normal tissues, and in the cervical squamous cell carcinoma, endometrial adenocarcinoma, gastric adenocarcinoma, esophageal squamous cell carcinoma, and brain cancer tissues were analyzed. The result of the study proved that the highest level of mRNA reverse transcription appeared in the normal testical tissue, whereas that in other normal tissues of human and mice were low. The mRNA reverse transcription level of RFP was higher in the endometrial adenocarcinoma tissue than in the cervical squamous cell carcinoma tissue; the mRNA reverse transcription level of RFP in the gastric adenocarcinoma tissue was significantly higher than that in the esophageal squamous cell carcinoma tissue. It was also found that the mRNA reverse transcription level of RFP in the brain cancer tissue was higher than that in the normal brain tissue. These results suggested that RFP could possibly be a useful molecular target for the development of new therapeutics for malignant tumors.

  14. Fusion core start-up, ignition and burn simulations of reversed-field pinch (RFP) reactors

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Yuh-Yi

    1988-01-01

    A transient reactor simulation model is developed to investigate and simulate the start-up, ignition and burn of a reversed-field pinch reactor. The simulation is based upon a spatially averaged plasma balance model with field profiles obtained from MHD quasi-equilibrium analysis. Alpha particle heating is estimated from Fokker-Planck calculations. The instantaneous plasma current is derived from a self-consistent circuit analysis for plasma/coil/eddy current interactions. The simulation code is applied to the TITAN RFP reactor design which features a compact, high-power-density reversed-field pinch fusion system. A contour analysis is performed using the steady-state global plasma balance. The results are presented with contours of constant plasma current. A saddle point is identified in the contour plot which determines the minimum value of plasma current required to achieve ignition. An optimized start-up to ignition and burn path can be obtained by passing through the saddle point. The simulation code is used to study and optimize the start-up scenario. In the simulations of the TITAN RFP reactor, the OH-driven superconducting EF coils are found to deviate from the required equilibrium values as the induced plasma current increases. This results in the modification of superconducting EF coils and the addition of a set of EF trim coils. The design of the EF coil system is performed with the simulation code subject to the optimization of trim-coil power and current. In addition, the trim-coil design is subject to the constraints of vertical-field stability index and maintenance access. A power crowbar is also needed to prevent the superconducting EF coils from generating excessive vertical field. A set of basic results from the simulation of TITAN RFP reactor yield a picture of RFP plasma operation in a reactor. Investigations of eddy current are also presented. 145 refs., 37 figs., 2 tabs.

  15. Density-Gradient-Driven trapped-electron-modes in improved-confinement RFP plasmas

    Science.gov (United States)

    Duff, James

    2016-10-01

    Short wavelength density fluctuations in improved-confinement MST plasmas exhibit multiple features characteristic of the trapped-electron-mode (TEM), strong evidence that drift wave turbulence emerges in RFP plasmas when transport associated with MHD tearing is reduced. Core transport in the RFP is normally governed by magnetic stochasticity stemming from long wavelength tearing modes that arise from current profile peaking. Using inductive control, the tearing modes are reduced and global confinement is increased to values expected for a comparable tokamak plasma. The improved confinement is associated with a large increase in the pressure gradient that can destabilize drift waves. The measured density fluctuations have frequencies >50 kHz, wavenumbers k_phi*rho_sglobal tearing modes. Their amplitude increases with the local density gradient, and they exhibit a density-gradient threshold at R/L_n 15, higher than in tokamak plasmas by R/a. the GENE code, modified for RFP equilibria, predicts the onset of microinstability for these strong-gradient plasma conditions. The density-gradient-driven TEM is the dominant instability in the region where the measured density fluctuations are largest, and the experimental threshold-gradient is close to the predicted critical gradient for linear stability. While nonlinear analysis shows a large Dimits shift associated with predicted strong zonal flows, the inclusion of residual magnetic fluctuations causes a collapse of the zonal flows and an increase in the predicted transport to a level close to the experimentally measured heat flux. Similar circumstances could occur in the edge region of tokamak plasmas when resonant magnetic perturbations are applied for the control of ELMs. Work supported by US DOE.

  16. How acidic are monomeric structural units of heparin?

    Science.gov (United States)

    Remko, Milan; Broer, Ria; Van Duijnen, Piet Th.

    2013-12-01

    Density functional theory methods with the B3LYP functional have been used to letter the acidity of carboxyl, O-sulfo and N-sulfo groups in six basic monomeric structural units of heparin (1-OMe ΔUA-2S, 1-OMe GlcN-S6S, 1,4-DiOMe GlcA, 1,4-DiOMe GlcN-S3S6S, 1,4-DiOMe IdoA-2S, and 1,4-DiOMe GlcN-S6S). The predicted gas-phase acidity of the acidic functional groups in the monomeric structural units of heparin is: O-sulfo > N-sulfo > carboxyl. The computed pKa values provide the same order of acidity as was observed in water solution. This implies that hydration does not change ordering of acidity of major acidic groups of monomeric structural units of heparin.

  17. Plasma behaviour at high beta and high density in the Madison Symmetric Torus RFP

    Energy Technology Data Exchange (ETDEWEB)

    Wyman, M. [University of Wisconsin, Madison; Chapman, B. E. [University of Wisconsin, Madison; Ahn, J. W. [University of Wisconsin, Madison; Almagri, A. F. [University of Wisconsin, Madison; Anderson, J. [University of Wisconsin, Madison; Bonomo, F. [Consorzio RFX, Italy; Bower, D L [University of California, Los Angeles; Combs, Stephen Kirk [ORNL; Craig, D. [University of Wisconsin, Madison; Foust, Charles R [ORNL

    2009-01-01

    Pellet fuelling of improved confinement Madison Symmetric Torus (MST) plasmas has resulted in high density and high plasma beta. The density in improved confinement discharges has been increased fourfold, and a record plasma beta (beta(tot) = 26%) for the improved confinement reversed-field pinch (RFP) has been achieved. At higher beta, a new regime for instabilities is accessed in which local interchange and global tearing instabilities are calculated to be linearly unstable, but experimentally, no severe effect, e. g., a disruption, is observed. The tearing instability, normally driven by the current gradient, is driven by the pressure gradient in this case, and there are indications of increased energy transport ( as compared with low-density improved confinement). Pellet fuelling is also compared with enhanced edge fuelling of standard confinement RFP discharges for the purpose of searching for a density limit in MST. In standard-confinement discharges, pellet fuelling peaks the density profile where edge fuelling cannot, but transport appears unchanged. For a limited range of plasma current, MST discharges with edge fuelling are constrained to a maximum density corresponding to the Greenwald limit. This limit is surpassed in pellet-fuelled improved confinement discharges.

  18. First molecular identification of the transgene red fluorescent protein (RFP in transgenic ornamental zebrafish (Danio rerio introduced in Peru

    Directory of Open Access Journals (Sweden)

    Carlos Scotto

    2013-09-01

    Full Text Available In this paper the transgenic fluorescent red, orange and pink zebra fish (Danio rerio, found in local aquariums in Peru, were identified using the PCR technique to amplify the transgene RFP sea anemone belonging to Discosoma spp. The gene expression of the red fluorescent protein (RFP transgene was found to determine different gradients-of-bioluminescence (shades in color in each GMO fish analyzed. We performed sequence analysis of the two variants of the RFP along with six variants of the existing fluorescent protein GFP from the Genbank, this could help identify quickly if they are new genes or variants thereof as these novel fluorescent proteins may be introduced in aquatic GMO in the future. Thus, developing and improving biosecurity measures through its timely detection at the molecular genetic level.

  19. Homotropic cooperativity of monomeric cytochrome P450 3A4

    Energy Technology Data Exchange (ETDEWEB)

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G. (UIUC)

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  20. Monomeric tartrate resistant acid phosphatase induces insulin sensitive obesity.

    Directory of Open Access Journals (Sweden)

    Pernilla Lång

    Full Text Available BACKGROUND: Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer. PRINCIPAL FINDINGS: Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity. CONCLUSION: Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.

  1. 23 CFR 636.402 - What types of information exchange may take place after the release of the RFP document?

    Science.gov (United States)

    2010-04-01

    ... 23 Highways 1 2010-04-01 2010-04-01 false What types of information exchange may take place after... What types of information exchange may take place after the release of the RFP document? Certain types.... These communication methods are optional. Type of information exchange When Purpose Parties involved...

  2. 23 CFR 636.303 - May pre-qualification standards be used as proposal evaluation criteria in the RFP?

    Science.gov (United States)

    2010-04-01

    ... 23 Highways 1 2010-04-01 2010-04-01 false May pre-qualification standards be used as proposal evaluation criteria in the RFP? 636.303 Section 636.303 Highways FEDERAL HIGHWAY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION ENGINEERING AND TRAFFIC OPERATIONS DESIGN-BUILD CONTRACTING Proposal...

  3. Synthesis and structure of monomeric, trimeric, and mixed phenylcyanamides.

    Science.gov (United States)

    Brand, Harald; Mayer, Peter; Schulz, Axel; Soller, Thomas; Villinger, Alexander

    2008-06-02

    In a new synthetic approach phenylcyanamide (Hpca) was synthesized by methylation of phenylthiourea followed by a basic work-up. All products along the synthetic route have been fully characterized by means of NMR, IR, and X-ray studies. The first structural report of neutral mixed crystals of phenylcyanamide containing monomeric and trimeric Hpca is presented. Examination of these intriguing mixed crystals revealed the formation of distinct layers of monomeric and trimeric Hpca. These layers are interconnected by weak hydrogen bonds. The trimer represents triphenylisomelamine, which readily isomerizes to the triphenylmelamine in the melt, in accord with computations at the B3LYP level, indicating an exothermic process (DeltaH = -49.4 kcal mol(-1)). Pure trimeric Hpca (triphenylisomelamine) was obtained either by recrystallization of the mixed crystals from boiling water or by trimerization of monomeric Hpca in isopropanol for 12 h under reflux conditions. For comparison tritylcyanamide (Htca) and potassium phenylcyanamide as an [18]crown-6 complex [K([18]crown-6)pca] have been synthesized, and the solid-state structures were determined using X-ray diffraction techniques. The thermal behavior was studied by thermo-analytical experiments. In agreement with the experimental results, computations predict an exothermic cyclotrimerization process for Hpca (DeltaH = -41.3 kcal mol(-1)).

  4. Slow Unfolding of Monomeric Proteins from Hyperthermophiles with Reversible Unfolding

    Directory of Open Access Journals (Sweden)

    Atsushi Mukaiyama

    2009-03-01

    Full Text Available Based on the differences in their optimal growth temperatures microorganisms can be classified into psychrophiles, mesophiles, thermophiles, and hyperthermophiles. Proteins from hyperthermophiles generally exhibit greater stability than those from other organisms. In this review, we collect data about the stability and folding of monomeric proteins from hyperthermophilies with reversible unfolding, from the equilibrium and kinetic aspects. The results indicate that slow unfolding is a general strategy by which proteins from hyperthermophiles adapt to higher temperatures. Hydrophobic interaction is one of the factors in the molecular mechanism of the slow unfolding of proteins from hyperthermophiles.

  5. Addition-type polyimides from solutions of monomeric reactants

    Science.gov (United States)

    Delvigs, P.; Serafini, T. T.; Lightsey, G. R.

    1972-01-01

    The monomeric reactants approach was used to fabricate addition-type polyimide/graphite fiber composites with improved mechanical properties and thermal stability characteristics over those of composites derived from addition-type amide acid prepolymers. A screening study of 24 different monomer combinations was performed. The results of a more extensive investigation of a selected number of monomer combinations showed that the combination providing the best thermomechanical properties was 5-norbornene-2,3-dicarboxylic acid monomethyl ester/4,4'-methylenedianiline/3,3'4,4'-benzophenone tetracarboxylic acid dimethyl ester at a molar ratio of 2/3.09/2.09.

  6. Effects of Resistivity and Viscosity on m =0 Rise and Fall Time in the RFP

    Science.gov (United States)

    Futch, A. M.; Craig, D.; Hesse, R.; Jacobson, C. M.

    2016-10-01

    In the reversed field pinch (RFP), poloidal mode number m =0 fluctuations are driven in a sawtooth cycle via nonlinear coupling with unstable m =1 tearing modes. We explore how the rise and fall time of these m =0 fluctuations depends on resistivity and viscosity in visco-resistive MHD simulations using the DEBS code. Both the Lundquist number (S) and magnetic Prandtl number (Pr) affect the rise/fall time. Analysis of MST experimental data also shows that both the rise and fall times of the m =0 amplitude vary with S. The variation observed in experiment is consistent with simulation results for rise time, but shows some differences for fall time. Rise time is insensitive to the resistivity profile but depends slightly on the viscosity profile. Fall time is strongly correlated with the duration of the crash which depends on both resistivity and viscosity profiles. These results suggest that the rise and fall time of the m =0 modes at the sawtooth crash is not strongly influenced by the local resistivity near the resonant surface but instead is primarily determined by the overall dynamics of the entire sawtooth cycle. The role of viscosity is less clear though the edge viscosity affects the m =0 evolution more than the core. This work has been supported by the U.S.D.O.E.

  7. Measuring viscosity with a resonant magnetic perturbation in the MST RFP

    Science.gov (United States)

    Fridström, Richard; Munaretto, Stefano; Frassinetti, Lorenzo; Chapman, Brett; Brunsell, Per; Sarff, John; MST Team

    2016-10-01

    Application of an m = 1 resonant magnetic perturbation (RMP) causes braking and locking of naturally rotating m = 1 tearing modes (TMs) in the MST RFP. The experimental TM dynamics are replicated by a theoretical model including the interaction between the RMP and multiple TMs [Fridström PoP 23, 062504 (2016)]. The viscosity is the only free parameter in the model, and it is chosen such that model TM velocity evolution matches that of the experiment. The model does not depend on the means by which the natural rotation is generated. The chosen value of the viscosity, about 40 m2/s, is consistent with separate measurements in MST using a biased probe to temporarily spin up the plasma. This viscosity is about 100 times larger than the classical prediction, likely due to magnetic stochasticity in the core of these plasmas. Viscosity is a key parameter in visco-resistive MHD codes like NIMROD. The validation of these codes requires measurement of the viscosity over a broad parameter range, which will now be possible with the RMP technique that, unlike the biased probe, is not limited to low-energy-density plasmas. Estimation with the RMP technique of the viscosity in several MST discharges suggests that the viscosity decreases as the electron beta increases. Work supported by USDOE.

  8. Characterization of monomeric intermediates during VSV glycoprotein structural transition.

    Directory of Open Access Journals (Sweden)

    Aurélie A Albertini

    2012-02-01

    Full Text Available Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV glycoprotein G ectodomain (G(th, aa residues 1-422, the fragment that was previously crystallized. While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, G(th is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.

  9. Improved-confinement plasmas at high temperature and high beta in the MST RFP

    Energy Technology Data Exchange (ETDEWEB)

    Chapman, B. E. [University of Wisconsin, Madison; Ahn, J. W. [University of Wisconsin, Madison; Almagri, A. F. [University of Wisconsin, Madison; Anderson, J. [University of Wisconsin, Madison; Combs, Stephen Kirk [ORNL; Foust, Charles R [ORNL; Kaufman, M. [University of Wisconsin, Madison

    2009-01-01

    We have increased substantially the electron and ion temperatures, the electron density, and the total beta in plasmas with improved energy confinement in the Madison Symmetric Torus (MST). The improved confinement is achieved with a well-established current profile control technique for reduction of magnetic tearing and reconnection. A sustained ion temperature > 1 keV is achieved with intensified reconnection-based ion heating followed immediately by current profile control. In the same plasmas, the electron temperature reaches 2 keV, and the electron thermal diffusivity drops to about 2 m(2) s(-1). The global energy confinement time is 12 ms. This and the reported temperatures are the largest values yet achieved in the reversed-field pinch (RFP). These results were attained at a density similar to 10(19) m(-3). By combining pellet injection with current profile control, the density has been quadrupled, and total beta has nearly doubled to a record value of about 26%. The Mercier criterion is exceeded in the plasma core, and both pressure-driven interchange and pressure-driven tearing modes are calculated to be linearly unstable, yet energy confinement is still improved. Transient momentum injection with biased probes reveals that global momentum transport is reduced with current profile control. Magnetic reconnection events drive rapid momentum transport related to large Maxwell and Reynolds stresses. Ion heating during reconnection events occurs globally, locally, or not at all, depending on which tearing modes are involved in the reconnection. To potentially augment inductive current profile control, we are conducting initial tests of current drive with lower-hybrid and electron-Bernstein waves.

  10. Isolation and Characterization of Lewis Base Stabilized Monomeric Parent Stibanylboranes.

    Science.gov (United States)

    Marquardt, Christian; Hegen, Oliver; Hautmann, Matthias; Balázs, Gábor; Bodensteiner, Michael; Virovets, Alexander V; Timoshkin, Alexey Y; Scheer, Manfred

    2015-10-26

    The synthesis of the Lewis base stabilized monomeric parent compound of stibanylboranes, "H2 Sb-BH2 ", is reported. Through a salt metathesis route, the silyl-substituted compounds (Me3 Si)2 Sb-BH2 ⋅LB (LB=NMe3 , NHC(Me) ) were synthesized as representatives of derivatives with a Sb-B σ bond. Under very mild conditions, they could be transformed into the target compounds Me3 N⋅H2 B-HSb-BH2 ⋅NMe3 and H2 Sb-BH2 ⋅NHC(Me) , respectively. The products were characterized by X-ray structure analysis, NMR spectroscopy, IR spectroscopy, and mass spectrometry. DFT calculations give further insight into the stability and bonding of these unique compounds.

  11. Light-induced structural changes in a monomeric bacteriophytochrome

    Directory of Open Access Journals (Sweden)

    Heikki Takala

    2016-09-01

    Full Text Available Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of the phytochrome photosensory core is sufficient to perform the light-induced structural changes. This implies that allosteric cooperation with the other monomer is not needed for structural activation. The dimeric arrangement may instead be intrinsic to the biochemical output domains of bacterial phytochromes.

  12. Characterization of the Partially Folded Monomeric Intermediate of Creatine Kinase

    Institute of Scientific and Technical Information of China (English)

    朴龙斗; 周海梦

    2002-01-01

    The importance of understanding the protein folding pathway and intermediates is well recognized on the basis of extensive studies of protein folding in vitro and in vivo. Creatine kinase (CK) is a typical model for studying unfolding and refolding of proteins due to several interesting properties. Recent studies on the folding of CK show that its partially folded monomeric intermediate is present kinetically and is stable at equilibrium. The present paper contains 33 References as a mini review to characterize the properties of CK from studies on the CK folding pathway. Characterization of these intermediates is an essential step toward understanding the mechanism of protein folding. Some well-determined schemes are suggested as protein folding models.

  13. Single Molecule Spectroscopy of Monomeric LHCII: Experiment and Theory

    CERN Document Server

    Malý, Pavel; van Grondelle, Rienk; Mančal, Tomáš

    2015-01-01

    We derive approximate equations of motion for excited state dynamics of a multilevel open quantum system weakly interacting with light to describe fluorescence detected single molecule spectra. Based on the Frenkel exciton theory, we construct a model for the chlorophyll part of the LHCII complex of higher plants and its interaction with previously proposed excitation quencher in the form of the lutein molecule Lut 1. The resulting description is valid over a broad range of timescales relevant for single molecule spectroscopy, i.e. from ps to minutes. Validity of these equations is demonstrated by comparing simulations of ensemble and single-molecule spectra of monomeric LHCII with experiments. Using a conformational change of the LHCII protein as a switching mechanism, the intensity and spectral time traces of individual LHCII complexes are simulated, and the experimental statistical distributions are reproduced. Based on our model, it is shown that with reasonable assumptions about its interaction with chlo...

  14. mKikGR, a monomeric photoswitchable fluorescent protein.

    Directory of Open Access Journals (Sweden)

    Satoshi Habuchi

    Full Text Available The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.

  15. Computational design and characterization of a monomeric helical dinuclear metalloprotein.

    Science.gov (United States)

    Calhoun, Jennifer R; Kono, Hidetoshi; Lahr, Steven; Wang, Wei; DeGrado, William F; Saven, Jeffery G

    2003-12-12

    The de novo design of di-iron proteins is an important step towards understanding the diversity of function among this complex family of metalloenzymes. Previous designs of due ferro (DF) proteins have resulted in tetrameric and dimeric four-helix bundles having crystallographically well-defined structures and active-site geometries. Here, the design and characterization of DFsc, a 114 residue monomeric four-helix bundle, is presented. The backbone was modeled using previous oligomeric structures and appropriate inter-helical turns. The identities of 26 residues were predetermined, including the primary and secondary ligands in the active site, residues involved in active site accessibility, and the gamma beta gamma beta turn between helices 2 and 3. The remaining 88 amino acid residues were determined using statistical computer aided design, which is based upon a recent statistical theory of protein sequences. Rather than sampling sequences, the theory directly provides the site-specific amino acid probabilities, which are then used to guide sequence design. The resulting sequence (DFsc) expresses well in Escherichia coli and is highly soluble. Sedimentation studies confirm that the protein is monomeric in solution. Circular dichroism spectra are consistent with the helical content of the target structure. The protein is structured in both the apo and the holo forms, with the metal-bound form exhibiting increased stability. DFsc stoichiometrically binds a variety of divalent metal ions, including Zn(II), Co(II), Fe(II), and Mn(II), with micromolar affinities. 15N HSQC NMR spectra of both the apo and Zn(II) proteins reveal excellent dispersion with evidence of a significant structural change upon metal binding. DFsc is then a realization of complete de novo design, where backbone structure, activity, and sequence are specified in the design process.

  16. IL-6RFP can protect liver function in the model of mice infected with Schistosoma japonicum%IL-6RFP 在小鼠血吸虫感染中对肝细胞的保护作用

    Institute of Scientific and Technical Information of China (English)

    平春光; 程海燕; 高闻达; 计永胜; 刘淼; 任翠平; 邵延靖; 华梦晴; 沈际佳

    2015-01-01

    目的:探讨鼠白介素6受体融合蛋白(mIL-6RFP)在BALB/c 小鼠日本血吸虫感染模型中对肝细胞的保护作用。方法通过亲和层析方法获得可阻断白介素-6(IL-6)的mIL-6RFP,经人类肝癌细胞(HepG2)IL-6刺激及阻断实验检测纤维蛋白原(fibrinogen,FGG)和结合珠蛋白(haptoglobin, HP)的 mRNA 表达变化,体外验证重组蛋白 mIL-6RFP 的生物活性。建立 BALB/c 小鼠日本血吸虫感染模型,于感染第24天起经尾静脉注射 mIL-6RFP,隔天注射每次100μg/只,感染第42天剖杀小鼠。HE 染色法检测小鼠肝脏肉芽肿面积,荧光定量 PCR 法检测小鼠肝组织 IL-1β、IL-13、肿瘤坏死因子-α(TNF-α)和趋化因子1(CXCL1)的 mRNA 表达,连续监测法检测小鼠肝功能指标谷丙转氨酶(ALT)和谷草转氨酶(AST)。结果 HepG2细胞实验显示 mIL-6RFP 可降低IL-6对该细胞的刺激作用,显著下调 FGG 和 HP 的表达(P<0.05)。在小鼠感染模型中阻断 IL-6后,肝功能指标 ALT和 AST 与溶剂对照组相比均显著下降(P <0.05),肉芽肿病变无显著性差异。结论在 BALB/c 小鼠日本血吸虫感染模型中 mIL-6RFP 可明显改善肝细胞功能,但对肉芽肿形成未见明显作用。%Objective To explore the role of mouse interleukin-6 receptor fusion protein (mIL-6RFP)for the pro-tection of liver cells in the model of BALB /c mice infected with Schistosoma japonicum.Methods Nickel ion col-umn was used to obtain purified recombinant mIL-6RFP protein.In vitro,HepG2 cells were used to verify the bio-logical activity of mIL-6RFP.BALB /c mice were administrated by tail vein injection with 100 μg of mIL-6RFP or equal volume solvent,at 24,26,28,30,32,34,36,38,and 40 days after infection.Mice were sacrificed 48 h after the last injection.The granuloma size was measured in the mouse liver by HE staining.Interleukin-1β(IL-1β),interleukin-13 (IL-13 ),tumor

  17. Effect of Resonant Magnetic Perturbations on 3D equilibria in the MST RFP

    Science.gov (United States)

    Munaretto, Stefano

    2015-11-01

    The orientation of 3D, stellarator-like equilibria in the MST RFP can now be controlled with application of an m = 1 RMP. This has led to greatly improved diagnosis, revealing enhancements in both the central electron temperature and density. Coupled to a recent advance in the V3FIT code, reconstructions of the 3D equilibria have also been dramatically improved. The RMP also inhibits the generation of high-energy >20 keV electrons that is otherwise common with the 3D state. This state occurs when the normally broad spectrum of core-resonant m = 1 tearing modes condenses, with the innermost resonant mode growing to large amplitude, reaching ~ 8% of the axisymmetric field strength. This occurs in plasmas of sufficiently large Lundquist number ~ IpTe3/2, and the duration of the state is maximized with zero applied Bt (infinite toroidal beta). As the dominant mode grows, eddy current in MST's conducting shell slows the mode's rotation. This leads to locking of the 3D structure, but with an orientation that varies randomly shot to shot, making diagnosis difficult. An m = 1 RMP can now be applied with an array of saddle coils at the vertical insulated cut in the shell. With an amplitude br/B ~ 10% and a tailored temporal waveform, the RMP can force the 3D structure into any desired orientation relative to MST's diagnostics. A recent advance in V3FIT allows calculation of the substantial helical image current flowing in MST's shell, which has in turn allowed self-consistent utilization of both external and internal (Faraday rotation) measurements of the magnetic field. The ORBIT code predicts reduced stochasticity and improved confinement of high-energy electrons within the 3D structure. The suppression of these electrons by the m = 1 RMP may reflect a change to the central magnetic topology. The generation of these electrons is unaffected by non-resonant perturbations, such as m = 3. Supported by the US DOE.

  18. Fluorescent visualisation of the hypothalamic oxytocin neurones activated by cholecystokinin-8 in rats expressing c-fos-enhanced green fluorescent protein and oxytocin-monomeric red fluorescent protein 1 fusion transgenes.

    Science.gov (United States)

    Katoh, A; Shoguchi, K; Matsuoka, H; Yoshimura, M; Ohkubo, J-I; Matsuura, T; Maruyama, T; Ishikura, T; Aritomi, T; Fujihara, H; Hashimoto, H; Suzuki, H; Murphy, D; Ueta, Y

    2014-05-01

    The up-regulation of c-fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c-fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c-fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c-fos gene regulatory sequences that drive the expression of a c-fos-eGFP fusion protein. In c-fos-eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)-8, which selectively activates oxytocin (OXT)-secreting neurones in the hypothalamus. In double transgenic rats that express c-fos-eGFP and an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1-positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK-8. It is possible that not only a plane image, but also three-dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.

  19. Properties of monomeric paramyosin using a transient electric birefringence techniques.

    Science.gov (United States)

    DeLaney, D; Krause, S

    1976-01-01

    Paramyosin samples obtained from the chowder clam, Mercenaria mercenaria, by different extraction techniques were studied using transient electric birefringence techniques. The protein remain monomeric (unaggregated) in 1 mM buffer solution at pH 3.1 to 3.8 and near pH 10. At pH 3.2, the molecules obtained by different extraction techniques exhibit rotational diffusion constants that indicate a 5% difference in length between them, with the probable native form of paramyosin being the longer species. This difference in rotational diffusion constant disappears at higher pH, and, in addition, a large difference in dipole moment between the molecules observed at pH 3.2 also disappears at high pH. These results are used to hypothesize that the rodlike native paramyosin molecules have one or two partly flexible portions on their ends; at one end of each molecule this portion probably contains excess basic amino acids which are charged at low pH to account for the higher dipole moment of this form of paramyosin at these low pH values. At pH 3.2, these portions of the macromolecule are not flexible and act as stiff parts of the rodlike molecules, but they gradually become flexible at higher pH. Possible mechanisms for this change in flexibility are discussed.

  20. Labeling Monomeric Insulin with Renal-Clearable Luminescent Gold Nanoparticles.

    Science.gov (United States)

    Vinluan, Rodrigo D; Yu, Mengxiao; Gannaway, Melissa; Sullins, Justin; Xu, Jing; Zheng, Jie

    2015-12-16

    In the native physiological environment, inorganic nanoparticles (NPs) often induce nonspecific protein adsorption, which could significantly alter the function of the proteins they labeled. As a result, small fluorescent dyes are still widely used in the imaging of proteins in animals due to their minimal interference with protein function. Here, we used monomeric insulin as a model and compared its bioactivity before and after labeling with renal-clearable near-infrared-emitting gold NPs. These NPs were chosen because they have high resistance to serum protein adsorption and low nonspecific accumulation. We have found that a 1:1 insulin-NP ratio can be achieved, where the insulin-NPs show minimal serum protein binding with fully retained bioactivity comparable to that of unlabeled insulin. These results show a proof of concept that renal-clearable NPs can behave like small molecules in protein labeling without changing the individual protein's function, laying down a foundation for in vivo tracking of proteins with multimodality imaging techniques.

  1. Substrate-Induced Dimerization of Engineered Monomeric Variants of Triosephosphate Isomerase from Trichomonas vaginalis.

    Directory of Open Access Journals (Sweden)

    Samuel Lara-Gonzalez

    Full Text Available The dimeric nature of triosephosphate isomerases (TIMs is maintained by an extensive surface area interface of more than 1600 Å2. TIMs from Trichomonas vaginalis (TvTIM are held in their dimeric state by two mechanisms: a ball and socket interaction of residue 45 of one subunit that fits into the hydrophobic pocket of the complementary subunit and by swapping of loop 3 between subunits. TvTIMs differ from other TIMs in their unfolding energetics. In TvTIMs the energy necessary to unfold a monomer is greater than the energy necessary to dissociate the dimer. Herein we found that the character of residue I45 controls the dimer-monomer equilibrium in TvTIMs. Unfolding experiments employing monomeric and dimeric mutants led us to conclude that dimeric TvTIMs unfold following a four state model denaturation process whereas monomeric TvTIMs follow a three state model. In contrast to other monomeric TIMs, monomeric variants of TvTIM1 are stable and unexpectedly one of them (I45A is only 29-fold less active than wild-type TvTIM1. The high enzymatic activity of monomeric TvTIMs contrast with the marginal catalytic activity of diverse monomeric TIMs variants. The stability of the monomeric variants of TvTIM1 and the use of cross-linking and analytical ultracentrifugation experiments permit us to understand the differences between the catalytic activities of TvTIMs and other marginally active monomeric TIMs. As TvTIMs do not unfold upon dimer dissociation, herein we found that the high enzymatic activity of monomeric TvTIM variants is explained by the formation of catalytic dimeric competent species assisted by substrate binding.

  2. SGCC successfully developed large-capacity sodium-sulfur monomeric battery

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Through many years' cooperation,SGCC and Shanghai Silicate Research Institute of Chinese Academy of Science successfully developed 650 ampere-hours capacity sodium-sulfur monomeric storage battery with the independent intellectual property right

  3. Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane.

    Science.gov (United States)

    Ott, P; Brodbeck, U

    1984-08-08

    Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.

  4. 表达红荧光蛋白的重组痘苗病毒的包装与鉴定%Packing and identifcation of recombinant vaccinia virus expressing the RFP gene

    Institute of Scientific and Technical Information of China (English)

    沈百庆; 程乾; 罗砚曦; 阎辉倡; 郑骏年

    2015-01-01

    Objective To pack and identify the recombinant vaccinia virus ( VV-RFP) expressing red fluorescent protein ( RFP) gene.Methods The full-length sequence of RFP gene was PCR-amplified using plasmid pDsRed-express-C1 as the template.The RFP fragment was cloned into pCB vaccinia expression vector and the resulting recom-binant plasmid pCB-RFP was confirmed correct by restriction analysis and DNA sequencing.After 293 cells were trans-fected with the recombinant plasmid using liposome then infected with wild -type vaccinia virus, the VV-RFP was packed through homologous recombination and subsequently selected and screened by plaque-purified, taking advantage of RFP marker and gpt resistance.Results The recombinant vaccinia virus VV-RFP expressing RFP gene was con-structed successfully.Conclusion The packing of VV-RFP will provides a useful tool for the construction of recombi-nant vaccinia virus carrying therapeutic genes.%目的 包装并鉴定表达红荧光蛋白( RFP)基因的重组痘苗病毒( VV-RFP). 方法 以质粒pDsRed-express-C1为模板,PCR扩增出RFP基因,并克隆至痘苗病毒转移载体pCB中,经酶切分析和DNA测序鉴定正确,得到阳性重组质粒pCB-RFP. 将该重组质粒用脂质体转染293细胞后再用野生型痘苗病毒感染 ,通过同源重组获得重组痘苗病毒,利用RFP和gpt抗性标志筛选和噬斑纯化获得重组痘苗病毒VV-RFP. 结果 成功包装出表达RFP基因的重组痘苗病毒. 结论 包装的VV-RFP为进一步构建携带特定治疗基因的重组痘苗病毒奠定了基础.

  5. 23 CFR 636.401 - What types of information exchange may take place prior to the release of the RFP document?

    Science.gov (United States)

    2010-04-01

    ... 23 Highways 1 2010-04-01 2010-04-01 false What types of information exchange may take place prior... What types of information exchange may take place prior to the release of the RFP document? Verbal or written information exchanges (such as in the first-phase of a two-phase selection procedure) must...

  6. A monomeric variant of insulin degrading enzyme (IDE loses its regulatory properties.

    Directory of Open Access Journals (Sweden)

    Eun Suk Song

    Full Text Available BACKGROUND: Insulin degrading enzyme (IDE is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structure. METHODOLOGY/PRINCIPAL FINDINGS: IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained approximately 25% of the wild type activity. In contrast with the larger peptide substrates beta-endorphin and amyloid beta peptide 1-40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold. CONCLUSIONS/SIGNIFICANCE: These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.

  7. Anthocyanins and their variation in red wines I. Monomeric anthocyanins and their color expression.

    Science.gov (United States)

    He, Fei; Liang, Na-Na; Mu, Lin; Pan, Qiu-Hong; Wang, Jun; Reeves, Malcolm J; Duan, Chang-Qing

    2012-02-07

    Originating in the grapes, monomeric anthocyanins in young red wines contribute the majority of color and the supposed beneficial health effects related to their consumption, and as such they are recognized as one of the most important groups of phenolic metabolites in red wines. In recent years, our increasing knowledge of the chemical complexity of the monomeric anthocyanins, their stability, together with the phenomena such as self-association and copigmentation that can stabilize and enhance their color has helped to explain their color representation in red wine making and aging. A series of new enological practices were developed to improve the anthocyanin extraction, as well as their color expression and maintenance. This paper summarizes the most recent advances in the studies of the monomeric anthocyanins in red wines, emphasizing their origin, occurrence, color enhancing effects, their degradation and the effect of various enological practices on them.

  8. Anthocyanins and Their Variation in Red Wines I. Monomeric Anthocyanins and Their Color Expression

    Directory of Open Access Journals (Sweden)

    Chang-Qing Duan

    2012-02-01

    Full Text Available Originating in the grapes, monomeric anthocyanins in young red wines contribute the majority of color and the supposed beneficial health effects related to their consumption, and as such they are recognized as one of the most important groups of phenolic metabolites in red wines. In recent years, our increasing knowledge of the chemical complexity of the monomeric anthocyanins, their stability, together with the phenomena such as self-association and copigmentation that can stabilize and enhance their color has helped to explain their color representation in red wine making and aging. A series of new enological practices were developed to improve the anthocyanin extraction, as well as their color expression and maintenance. This paper summarizes the most recent advances in the studies of the monomeric anthocyanins in red wines, emphasizing their origin, occurrence, color enhancing effects, their degradation and the effect of various enological practices on them.

  9. B22 Glu Des-B30 Insulin: A Novel Monomeric Insulin

    Institute of Scientific and Technical Information of China (English)

    Hai-Juan DU; Jia-Hao SHI; Da-Fu CUI; You-Shang ZHANG

    2006-01-01

    Studies on monomeric insulin with reduced self-association are important in the development of insulin pharmaceutical preparations with rapid hypoglycemic action on patients with diabetes. Here we report a novel monomeric insulin, B22 Glu des-B30 insulin, prepared from a single chain insulin precursor with B22 Arg mutated to Glu, which was expressed in Pichia pastoris and converted to B22 Glu des-B30 insulin by tryptic digestion. It still retains 50% of the in vivo biological activity of porcine insulin and does not form a dimer even at a concentration of 10 mg/ml, showing that B22 Glu plays a key role in reducing the selfassociation of the insulin molecule without greatly reducing its biological activity. This novel monomeric insulin might have potential applications in the clinic.

  10. Pengaruh mimosa pada penyamakan kulit jaket domba samak nabati menggunakan sistem C-RFP, ditinjau dari sifat organoleptis, fisis, dan morfologi kulit

    Directory of Open Access Journals (Sweden)

    Sri Sutyasmi

    2016-06-01

    Full Text Available The purpose of this study was to determine the effect of the use of mimosa, to manufacture environmentally friendly leather for jacket by using the C-RFP system (C=Conditioning, R=Rapid, F=Fass (drum, P=Powder, on physical, organoleptic, and morphology properties of leather. It was also to obtain a formula for vegetable tanning with C-RFP system. Pickled skins are conditioned (pre-tanning using Sodotan TSN and Sodotan APR, and then Sodotan TSN was chosen due to it meets the requirements of SNI leather for jacket (SNI 4593:2011. Then, pickled skins were tanned with mimosa and applied C-RFP system or rapid tanning without water added. Mimosa, used in this research, were 15%, 20%, and 25% and fatliquor were 12.5%, 15%, and 17.5%. Furthermore, The leather were finished into an environmentally friendly leather for jackets, and then tested for physical and organoleptic properties based on the SNI 4593:2011 as well as leather morphology (SEM. The physical test result shows that for mimosa 15%, 20% and 25%, and for fatliquor 15% and 17.5% are fullfill the SNI.

  11. In vivo near-infrared fluorescence imaging of Leishmania amazonensis expressing infrared fluorescence protein (iRFP) for real-time monitoring of cutaneous leishmaniasis in mice.

    Science.gov (United States)

    Oliveira, Janaina Correia; da Silva, Aline Caroline; Oliveira, Renato Antonio Dos Santos; Pereira, Valéria Rêgo Alves; Gil, Laura Helena Vega Gonzales

    2016-11-01

    The use of Leishmania amazonensis-infected BALB/c mice is an important model for the study of experimental cutaneous leishmaniasis. Here we report the development of a non-invasive method to directly evaluate and measure parasite burden during the course of the infection, based on the near-infrared fluorescence detection of a recombinant L. amazonensis strain. So, we generated a L. amazonensis strain that stably expresses the near-infrared protein (iRFP) gene and compared the maintenance of its vitro and in vivo characteristics, such as fitness, pathogenicity and fluorescence emission. After that, we followed the disease development, as well as the parasite burden in BALB/c mice footpads infected with L. amazonensis-iRFP, by using an in vivo near-infrared fluorescence scanner. In vitro results showed a linear correlation between the fluorescence emission and the number of parasites. The in vivo study showed that the use of iRFP-transfected L. amazonensis enables the monitoring of parasite burden by measuring fluorescence signals. Therefore, this technique can be confidently used to directly monitor parasitic load and infection overtime and could be an excellent tool for in vitro and in vivo screening of anti-leishmanial drugs and vaccine efficiency. This is the first report of the use of the near-infrared fluorescence imaging technique for monitoring in vivo cutaneous leishmaniasis.

  12. A novel method to highly versatile monomeric PNA building blocks by multi component reactions

    NARCIS (Netherlands)

    Dömling, Alexander; Chi, Kai-Zu; Barrère, Mathieux

    1999-01-01

    A novel approach to monomeric PNA building blocks by a solution phase Ugi multi component reaction (MCR) is described. The reaction is easily performed in 96 well plates. The products precipitate from the reaction solution and are thus obtained in high yields and purity. Those products are not amena

  13. Monomeric G-proteins as signal transducers in airway physiology and pathophysiology

    NARCIS (Netherlands)

    Schaafsma, Dedmer; Roscioni, Sara S.; Meurs, Herman; Schmidt, Martina

    2008-01-01

    Monomeric G-proteins, also referred to as small GTPases, function as biological hubs being activated by extracellular stimuli and regulate downstream signalling events, which result in different cellular responses. The importance of these mechanisms is mirrored by the fact that several pathological

  14. Self-healing mechanism based on dispersed solid particles of various monomeric bismaleimides

    NARCIS (Netherlands)

    Turkenburg, D.H.; Fischer, H.R.

    2016-01-01

    In view of self-healing materials for high temperature applications we have studied the use of solid monomeric bismaleimide particles as embedded self-healing component dispersed in a host material. Below the self-healing activation temperature, bismaleimides remain inert while above it they may rap

  15. Monomeric red fluorescent protein variants used for imaging studies in different species

    NARCIS (Netherlands)

    Mueller-Taubenberger, Annette; Vos, Michel J.; Boettger, Angelika; Lasi, Margherita; Lai, Frank P. L.; Fischer, Markus; Rottner, Klemens

    2006-01-01

    Fluorescent proteins have proven to be excellent tools for live-cell imaging studies. In addition to green fluorescent protein (GFP) and its variants, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation

  16. Monomeric and dendritic second generation Grubbs- and Hoveyda-Grubbs-type catalysts for olefin metathesis Metallodendrimers Special Issue

    NARCIS (Netherlands)

    Pijnenburg, Niels J M; Tomás-Mendivil, Eder; Mayland, Kimberley E.; Kleijn, Henk; Lutz, Martin; Spek, Anthony L.; Van Koten, Gerard; Klein Gebbink, Bert

    2014-01-01

    The synthesis and characterization of monomeric and dendritic Grubbs II and Hoveyda-Grubbs II-based complexes are reported. These complexes were synthesized via a route based on the connection of monomeric or dendritic N-alkyl-N′-mesitylimidazol-2-ylidene pre-ligands to Grubbs I or Hoveyda-Grubbs I

  17. Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC.

    Science.gov (United States)

    Ionescu, Costin N; Shea, Kathleen A; Mehra, Rajendra; Prundeanu, Lucia; McAlear, Michael A

    2002-10-29

    Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.

  18. Monomeric carbohydrates production from olive tree pruning biomass: modeling of dilute acid hydrolysis.

    Science.gov (United States)

    Puentes, Juan G; Mateo, Soledad; Fonseca, Bruno G; Roberto, Inês C; Sánchez, Sebastián; Moya, Alberto J

    2013-12-01

    Statistical modeling and optimization of dilute sulfuric acid hydrolysis of olive tree pruning biomass has been performed using response surface methodology. Central composite rotatable design was applied to assess the effect of acid concentration, reaction time and temperature on efficiency and selectivity of hemicellulosic monomeric carbohydrates to d-xylose. Second-order polynomial model was fitted to experimental data to find the optimum reaction conditions by multiple regression analysis. The monomeric d-xylose recovery 85% (as predicted by the model) was achieved under optimized hydrolysis conditions (1.27% acid concentration, 96.5°C and 138 min), confirming the high validity of the developed model. The content of d-glucose (8.3%) and monosaccharide degradation products (0.1% furfural and 0.04% 5-hydroxymethylfurfural) provided a high quality subtract, ready for subsequent biochemical conversion to value-added products.

  19. The Roles of Monomeric GTP-Binding Proteins in Macroautophagy in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Shu Yang

    2014-10-01

    Full Text Available Autophagy is a cellular degradation process that sequesters components into a double-membrane structure called the autophagosome, which then fuses with the lysosome or vacuole for hydrolysis and recycling of building blocks. Bulk phase autophagy, also known as macroautophagy, controlled by specific Atg proteins, can be triggered by a variety of stresses, including starvation. Because autophagy relies extensively on membrane traffic to form the membranous structures, factors that control membrane traffic are essential for autophagy. Among these factors, the monomeric GTP-binding proteins that cycle between active and inactive conformations form an important group. In this review, we summarize the functions of the monomeric GTP-binding proteins in autophagy, especially with reference to experiments in Saccharomyces cerevisiae.

  20. Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

    Science.gov (United States)

    Vuong, H E; Pérez de Sevilla Müller, L; Hardi, C N; McMahon, D G; Brecha, N C

    2015-10-29

    Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.

  1. Multistage modeling of protein dynamics with monomeric Myc oncoprotein as an example

    Science.gov (United States)

    Liu, Jiaojiao; Dai, Jin; He, Jianfeng; Niemi, Antti J.; Ilieva, Nevena

    2017-03-01

    We propose to combine a mean-field approach with all-atom molecular dynamics (MD) into a multistage algorithm that can model protein folding and dynamics over very long time periods yet with atomic-level precision. As an example, we investigate an isolated monomeric Myc oncoprotein that has been implicated in carcinomas including those in colon, breast, and lungs. Under physiological conditions a monomeric Myc is presumed to be an example of intrinsically disordered proteins that pose a serious challenge to existing modeling techniques. We argue that a room-temperature monomeric Myc is in a dynamical state, it oscillates between different conformations that we identify. For this we adopt the C α backbone of Myc in a crystallographic heteromer as an initial ansatz for the monomeric structure. We construct a multisoliton of the pertinent Landau free energy to describe the C α profile with ultrahigh precision. We use Glauber dynamics to resolve how the multisoliton responds to repeated increases and decreases in ambient temperature. We confirm that the initial structure is unstable in isolation. We reveal a highly degenerate ground-state landscape, an attractive set towards which Glauber dynamics converges in the limit of vanishing ambient temperature. We analyze the thermal stability of this Glauber attractor using room-temperature molecular dynamics. We identify and scrutinize a particularly stable subset in which the two helical segments of the original multisoliton align in parallel next to each other. During the MD time evolution of a representative structure from this subset, we observe intermittent quasiparticle oscillations along the C-terminal α helix, some of which resemble a translating Davydov's Amide-I soliton. We propose that the presence of oscillatory motion is in line with the expected intrinsically disordered character of Myc.

  2. Fine blood vascular casting by monomeric methacrylate injection and microwave treatment

    OpenAIRE

    日根野谷, 仁

    1992-01-01

    A modified injection replica SEM method was introduced. Thorough injection of a resin mixture (monomeric metacrylate containing 1% benzoyl peroxide and 1% N, N-dimethylaniline) prior to the microwave treatment prepares good and fine blood vascular casts or replicas of brain, hypophysis, pineal body, thyroid gland and other organs. These casts sufficiently withstood ionbombardment and were useful for scanning electron microscopy. In this casting, preliminary perfusion fixation prior to the res...

  3. Heteroexpression and characterization of a monomeric isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680.

    Science.gov (United States)

    Wang, Ao; Cao, Zheng-Yu; Wang, Peng; Liu, Ai-Min; Pan, Wei; Wang, Jie; Zhu, Guo-Ping

    2011-08-01

    A monomeric NADP-dependent isocitrate dehydrogenase from the multicellular prokaryote Streptomyces avermitilis MA-4680 (SaIDH) was heteroexpressed in Escherichia coli, and the His-tagged enzyme was further purified to homogeneity. The molecular weight of SaIDH was about 80 kDa which is typical for monomeric isocitrate dehydrogenases. Structure-based sequence alignment reveals that the deduced amino acid sequence of SaIDH shows high sequence identity with known momomeric isocitrate dehydrogenase, and the coenzyme, substrate and metal ion binding sites are completely conserved. The optimal pH and temperature of SaIDH were found to be pH 9.4 and 45°C, respectively. Heat-inactivation studies showed that heating for 20 min at 50°C caused a 50% loss in enzymatic activity. In addition, SaIDH was absolutely specific for NADP+ as electron acceptor. Apparent Km values were 4.98 μM for NADP+ and 6,620 μM for NAD+, respectively, using Mn2+ as divalent cation. The enzyme performed a 33,000-fold greater specificity (kcat/Km) for NADP+ than NAD+. Moreover, SaIDH activity was entirely dependent on the presence of Mn2+ or Mg2+, but was strongly inhibited by Ca2+ and Zn2+. Taken together, our findings implicate the recombinant SaIDH is a divalent cation-dependent monomeric isocitrate dehydrogenase which presents a remarkably high cofactor preference for NADP+.

  4. Two mechanisms for dissipation of excess light in monomeric and trimeric light-harvesting complexes

    Energy Technology Data Exchange (ETDEWEB)

    Dall' Osto, Luca [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Cazzaniga, Stefano [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Bressan, Mauro [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Paleček, David [Lund Univ. (Sweden). Dept. of Chemical Physics; Židek, Karel [Lund Univ. (Sweden). Dept. of Chemical Physics; Niyogi, Krishna K. [Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst., Dept. of Plant and Microbial Biology; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Fleming, Graham R. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry, Graduate Group in Applied Science and Technology; Zigmantas, Donatas [Lund Univ. (Sweden). Dept. of Chemical Physics; Bassi, Roberto [Univ. di Verona, Verona (Italy). Dipartimento di Biotecnologie; Consiglio Nazionale delle Ricerche (CNR), Firenze (Italy). Istituto per la Protezione delle Piante (IPP)

    2017-04-10

    Oxygenic photoautotrophs require mechanisms for rapidly matching the level of chlorophyll excited states from light harvesting with the rate of electron transport from water to carbon dioxide. These photoprotective reactions prevent formation of reactive excited states and photoinhibition. The fastest response to excess illumination is the so-called non-photochemical quenching which, in higher plants, requires the luminal pH sensor PsbS and other yet unidentified components of the photosystem II antenna. Both trimeric light-harvesting complex II (LHCII) and monomeric LHC proteins have been indicated as site(s) of the heat-dissipative reactions. Different mechanisms have been proposed: Energy transfer to a lutein quencher in trimers, formation of a zeaxanthin radical cation in monomers. Here, we report on the construction of a mutant lacking all monomeric LHC proteins but retaining LHCII trimers. Its non-photochemical quenching induction rate was substantially slower with respect to the wild type. A carotenoid radical cation signal was detected in the wild type, although it was lost in the mutant. Here, we conclude that non-photochemical quenching is catalysed by two independent mechanisms, with the fastest activated response catalysed within monomeric LHC proteins depending on both zeaxanthin and lutein and on the formation of a radical cation. Trimeric LHCII was responsible for the slowly activated quenching component whereas inclusion in supercomplexes was not required. Finally, this latter activity does not depend on lutein nor on charge transfer events, whereas zeaxanthin was essential.

  5. Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds.

    Science.gov (United States)

    Ma, Ruoshui; Guo, Mond; Lin, Kuan-Ting; Hebert, Vincent R; Zhang, Jinwen; Wolcott, Michael P; Quintero, Melissa; Ramasamy, Karthikeyan K; Chen, Xiaowen; Zhang, Xiao

    2016-07-25

    Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer, it has been a challenge to effectively depolymerize lignin and produce high-value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) including 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPC yields obtained were 18 and 22 % based on the initial weight of the lignin in SESPL and DACSL, respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47 %. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated.

  6. Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Ruoshui [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Guo, Mond [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Lin, Kuan-ting [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Hebert, Vincent R. [Food and Environmental Laboratory, Washington State, University-TriCities, 2710 Crimson Way Richland WA 99354 USA; Zhang, Jinwen [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Wolcott, Michael P. [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Quintero, Melissa [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Ramasamy, Karthikeyan K. [Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland WA 99354 USA; Chen, Xiaowen [National Bioenergy Center, National Renewable Energy Lab, 1617 Cole Blvd Golden CO 80127 USA; Zhang, Xiao [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA

    2016-07-04

    Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer as well as its complex side chain structures, it has been a challenge to effectively depolymerize lignin and produce high value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) inclduing 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPCs yields obtained were 18% and 22% based on the initial weight of the lignin in SESPL and DACSL respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47%. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated.

  7. Peracetic Acid Depolymerization of Biorefinery Lignin for Production of Selective Monomeric Phenolic Compounds

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Ruoshui [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Guo, Mond [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Lin, Kuan-ting [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Hebert, Vincent R. [Food and Environmental Laboratory, Washington State, University-TriCities, 2710 Crimson Way Richland WA 99354 USA; Zhang, Jinwen [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Wolcott, Michael P. [Wood Materials and Engineering Laboratory, Washington State University, Pullman WA 99164 USA; Quintero, Melissa [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA; Ramasamy, Karthikeyan K. [Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland WA 99354 USA; Chen, Xiaowen [National Bioenergy Center, National Renewable Energy Lab, 1617 Cole Blvd Golden CO 80127 USA; Zhang, Xiao [Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science & Engineering Laboratory, Washington State University, 2710 Crimson Way Richland WA 99354 USA

    2016-07-04

    Lignin is the largest source of renewable material with an aromatic skeleton. However, due to the recalcitrant and heterogeneous nature of the lignin polymer, it has been a challenge to effectively depolymerize lignin and produce high-value chemicals with high selectivity. In this study, a highly efficient lignin-to-monomeric phenolic compounds (MPC) conversion method based on peracetic acid (PAA) treatment was reported. PAA treatment of two biorefinery lignin samples, diluted acid pretreated corn stover lignin (DACSL) and steam exploded spruce lignin (SESPL), led to complete solubilization and production of selective hydroxylated monomeric phenolic compounds (MPC-H) and monomeric phenolic acid compounds (MPC-A) including 4-hydroxy-2-methoxyphenol, p-hydroxybenzoic acid, vanillic acid, syringic acid, and 3,4-dihydroxybenzoic acid. The maximized MPC yields obtained were 18 and 22 % based on the initial weight of the lignin in SESPL and DACSL, respectively. However, we found that the addition of niobium pentoxide catalyst to PAA treatment of lignin can significantly improve the MPC yields up to 47 %. The key reaction steps and main mechanisms involved in this new lignin-to-MPC valorization pathway were investigated and elucidated.

  8. First synthesis and structural determination of a monomeric, unsolvated lithium amide, LiNH(2).

    Science.gov (United States)

    Grotjahn, D B; Sheridan, P M; Al Jihad, I; Ziurys, L M

    2001-06-13

    Alkali metal amides typically aggregate in solution and the solid phase, and even in the gas phase. In addition, even in the few known monomeric structures, the coordination number of the alkali metal is raised by binding of Lewis-basic solvent molecules, with concomitant changes in structure. In contrast, the simplest lithium amide LiNH(2) has never been made in a monomeric form, even though its structure has been theoretically predicted several times. Here, the first experimental structural data for a monomeric, unsolvated lithium amide are determined using a combination of gas-phase synthesis and millimeter/submillimeter-wave spectroscopy. All data point to a planar structure for LiNH(2). The r(o) structure of LiNH(2) has a Li-N distance of 1.736(3) A, an N-H distance of 1.022(3) A, and a H-N-H angle of 106.9(1) degrees. These results are compared with theoretical predictions for LiNH(2), and experimental data for oligomeric, solid-phase samples, which could not resolve the question of whether LiNH(2) is planar or not. In addition, comparisons are made with revised gas-phase and solid-phase data and calculated structures of NaNH(2).

  9. The peroxisomal protein import machinery displays a preference for monomeric substrates.

    Science.gov (United States)

    Freitas, Marta O; Francisco, Tânia; Rodrigues, Tony A; Lismont, Celien; Domingues, Pedro; Pinto, Manuel P; Grou, Cláudia P; Fransen, Marc; Azevedo, Jorge E

    2015-04-01

    Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.

  10. Treatment of iron deficiency anemia: are monomeric iron compounds suitable for parenteral administration?

    Science.gov (United States)

    Gupta, A; Crumbliss, A L

    2000-11-01

    Iron deficiency is the most common nutritional problem worldwide, especially in the developing countries. Oral iron supplementation programs have failed because of noncompliance and gastrointestinal toxicity, thereby necessitating parenteral administration of iron. For parenteral administration, only iron-carbohydrate complexes are currently used, because monomeric iron salts release free iron, thereby causing oxidant injury. However, iron-carbohydrate complexes also have significant toxicity, and they are expensive. We have proposed the hypothesis that monomeric iron salts can be safely administered by the parenteral route if iron is tightly complexed to the ligand, thereby causing clinically insignificant release of free iron, and the kinetic properties of the compound allow rapid transfer of iron to plasma transferrin. A detailed analysis of the physicochemical and kinetic properties reveals that ferric iron complexed to pyrophosphate or acetohydroxamate anions may be suitable for parenteral administration. We have demonstrated that infusion of ferric pyrophosphate into the circulation via the dialysate is safe and effective in maintaining iron balance in patients undergoing maintenance hemodialysis. Parenteral administration of monomeric iron compounds is a promising approach to the treatment of iron deficiency in the general population and merits further investigation.

  11. Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

    DEFF Research Database (Denmark)

    Schønning, Kristian; Bolmstedt, A; Novotny, J;

    1998-01-01

    An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to...... immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.......An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected...

  12. The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP.

    Science.gov (United States)

    Buhrke, David; Velazquez Escobar, Francisco; Sauthof, Luisa; Wilkening, Svea; Herder, Nico; Tavraz, Neslihan N; Willoweit, Mario; Keidel, Anke; Utesch, Tillmann; Mroginski, Maria-Andrea; Schmitt, Franz-Josef; Hildebrandt, Peter; Friedrich, Thomas

    2016-06-22

    Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.

  13. The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

    Science.gov (United States)

    Buhrke, David; Velazquez Escobar, Francisco; Sauthof, Luisa; Wilkening, Svea; Herder, Nico; Tavraz, Neslihan N.; Willoweit, Mario; Keidel, Anke; Utesch, Tillmann; Mroginski, Maria-Andrea; Schmitt, Franz-Josef; Hildebrandt, Peter; Friedrich, Thomas

    2016-01-01

    Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores. PMID:27329837

  14. Monomeric banana lectin at acidic pH overrules conformational stability of its native dimeric form.

    Directory of Open Access Journals (Sweden)

    Javed M Khan

    Full Text Available Banana lectin (BL is a homodimeric protein categorized among jacalin-related family of lectins. The effect of acidic pH was examined on conformational stability of BL by using circular dichroism, intrinsic fluorescence, 1-anilino-8-napthalene sulfonate (ANS binding, size exclusion chromatography (SEC and dynamic light scattering (DLS. During acid denaturation of BL, the monomerization of native dimeric protein was found at pH 2.0. The elution profile from SEC showed two different peaks (59.65 ml & 87.98 ml at pH 2.0 while single peak (61.45 ml at pH 7.4. The hydrodynamic radii (R h of native BL was 2.9 nm while at pH 2.0 two species were found with R h of 1.7 and 3.7 nm. Furthermore at, pH 2.0 the secondary structures of BL remained unaltered while tertiary structure was significantly disrupted with the exposure of hydrophobic clusters confirming the existence of molten globule like state. The unfolding of BL with different subunit status was further evaluated by urea and temperature mediated denaturation to check their stability. As inferred from high Cm and ΔG values, the monomeric form of BL offers more resistance towards chemical denaturation than the native dimeric form. Besides, dimeric BL exhibited a Tm of 77°C while no loss in secondary structures was observed in monomers even up to 95°C. To the best of our knowledge, this is the first report on monomeric subunit of lectins showing more stability against denaturants than its native dimeric state.

  15. Conformational equilibria in monomeric alpha-synuclein at the single-molecule level.

    Directory of Open Access Journals (Sweden)

    Massimo Sandal

    2008-01-01

    Full Text Available Human alpha-Synuclein (alphaSyn is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of alphaSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between alphaSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy-based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant alphaSyn. The conformational heterogeneity of monomeric alphaSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and "beta-like" structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the "beta-like" structures significantly increased in different conditions promoting the aggregation of alphaSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric alphaSyn conformers, positively correlated with conditions known to promote the formation of

  16. Isolation of monomeric photosystem II that retains the subunit PsbS.

    Science.gov (United States)

    Haniewicz, Patrycja; De Sanctis, Daniele; Büchel, Claudia; Schröder, Wolfgang P; Loi, Maria Cecilia; Kieselbach, Thomas; Bochtler, Matthias; Piano, Dario

    2013-12-01

    Photosystem II has been purified from a transplastomic strain of Nicotiana tabacum according to two different protocols. Using the procedure described in Piano et al. (Photosynth Res 106:221-226, 2010) it was possible to isolate highly active PSII composed of monomers and dimers but depleted in their PsbS protein content. A "milder" procedure than the protocol reported by Fey et al. (Biochim Biophys Acta 1777:1501-1509, 2008) led to almost exclusively monomeric PSII complexes which in part still bind the PsbS protein. This finding might support a role for PSII monomers in higher plants.

  17. Monomeric CH3: A Small, Stable Antibody Domain with Therapeutic Promise | Poster

    Science.gov (United States)

    By Ashley DeVine, Staff Writer Antibody domains are emerging as promising biopharmaceuticals because of their relatively small size compared to full-sized antibodies, which are too large to effectively penetrate tumors and bind to sterically restricted therapeutic targets. In an article published in The Journal of Biological Chemistry, Tianlei Ying, Ph.D., Dimiter Dimitrov, Ph.D., and their colleagues in the Protein Interactions Group, Cancer and Inflammation Program, Center for Cancer Research, reported their design of a novel antibody domain, monomeric CH3 (mCH3).

  18. The Beckman DxI 800 prolactin assay demonstrates superior specificity for monomeric prolactin.

    LENUS (Irish Health Repository)

    Byrne, Brendan

    2010-02-01

    Commercially available prolactin immunoassays detect macroprolactin to variable degrees. Best practice requires laboratories to assess the cross-reactivity of their prolactin assay with macroprolactin, and where appropriate, introduce a screen for the presence of macroprolactin. Our policy has been to reanalyse hyperprolactinaemic samples following polyethylene glycol (PEG) precipitation and to report the resultant value as the monomeric prolactin content of the sample. The goal of this study was to determine the need to continue PEG precipitation when prolactin measurements with the Wallac AutoDELFIA were replaced by the Beckman DxI 800.

  19. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

    Science.gov (United States)

    Verespy III, Stephen; Mehta, Akul Y.; Afosah, Daniel; Al-Horani, Rami A.; Desai, Umesh R.

    2016-01-01

    Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. PMID:27053426

  20. Nortriptyline inhibits aggregation and neurotoxicity of alpha-synuclein by enhancing reconfiguration of the monomeric form.

    Science.gov (United States)

    Collier, Timothy J; Srivastava, Kinshuk R; Justman, Craig; Grammatopoulous, Tom; Hutter-Paier, Birgit; Prokesch, Manuela; Havas, Daniel; Rochet, Jean-Christophe; Liu, Fang; Jock, Kevin; de Oliveira, Patrícia; Stirtz, Georgia L; Dettmer, Ulf; Sortwell, Caryl E; Feany, Mel B; Lansbury, Peter; Lapidus, Lisa; Paumier, Katrina L

    2017-10-01

    The pathology of Parkinson's disease and other synucleinopathies is characterized by the formation of intracellular inclusions comprised primarily of misfolded, fibrillar α-synuclein (α-syn). One strategy to slow disease progression is to prevent the misfolding and aggregation of its native monomeric form. Here we present findings that support the contention that the tricyclic antidepressant compound nortriptyline (NOR) has disease-modifying potential for synucleinopathies. Findings from in vitro aggregation and kinetics assays support the view that NOR inhibits aggregation of α-syn by directly binding to the soluble, monomeric form, and by enhancing reconfiguration of the monomer, inhibits formation of toxic conformations of the protein. We go on to demonstrate that NOR inhibits the accumulation, aggregation and neurotoxicity of α-syn in multiple cell and animal models. These findings suggest that NOR, a compound with established safety and efficacy for treatment of depression, may slow progression of α-syn pathology by directly binding to soluble, native, α-syn, thereby inhibiting pathological aggregation and preserving its normal functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Monomeric, Oligomeric and Polymeric Proteins in Huntington Disease and Other Diseases of Polyglutamine Expansion

    Directory of Open Access Journals (Sweden)

    Guylaine Hoffner

    2014-03-01

    Full Text Available Huntington disease and other diseases of polyglutamine expansion are each caused by a different protein bearing an excessively long polyglutamine sequence and are associated with neuronal death. Although these diseases affect largely different brain regions, they all share a number of characteristics, and, therefore, are likely to possess a common mechanism. In all of the diseases, the causative protein is proteolyzed, becomes abnormally folded and accumulates in oligomers and larger aggregates. The aggregated and possibly the monomeric expanded polyglutamine are likely to play a critical role in the pathogenesis and there is increasing evidence that the secondary structure of the protein influences its toxicity. We describe here, with special attention to huntingtin, the mechanisms of polyglutamine aggregation and the modulation of aggregation by the sequences flanking the polyglutamine. We give a comprehensive picture of the characteristics of monomeric and aggregated polyglutamine, including morphology, composition, seeding ability, secondary structure, and toxicity. The structural heterogeneity of aggregated polyglutamine may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective.

  2. Synthesis and characterization of a monomeric mutant Cu/Zn superoxide dismutase with partially reconstituted enzymic activity.

    Science.gov (United States)

    Banci, L; Bertini, I; Chiu, C Y; Mullenbach, G T; Viezzoli, M S

    1995-12-15

    A monomeric analog of human Cu/Zn superoxide dismutase (F50E/G51E SOD), previously characterized and found to have reduced enzymic activity, was here further modified by replacing Glu133 with Gln. This substitution does not dramatically affect the coordination geometry at the active site, but enhances enzymic activity, and also increases the affinity for anions at the active site. This behavior parallels earlier published results in which this point mutation was made in the dimeric wild-type enzyme. The analog described here has afforded for the first time a monomeric superoxide dismutase with substantial activity. This point mutation does not significantly influence the protein structure but interactions with anions, including superoxide, are altered with respect to the monomeric form. The present monomeric Glu133Gln mutant has partially restored enzymic activity. The diminished activity of the monomeric analogs is discussed in the light of possible minor structural changes and some of their characteristics are compared with those of naturally occurring mutants associated with various neurological diseases.

  3. Two-step cultivation for production of astaxanthin in Chlorella zofingiensis using a patented energy-free rotating floating photobioreactor (RFP).

    Science.gov (United States)

    Zhang, Zhao; Huang, Jim Junhui; Sun, Dongzhe; Lee, Yuankun; Chen, Feng

    2017-01-01

    In the present study, high light and nitrogen starvation with glucose-fed to the culture was found efficient to induce astaxanthin accumulation in Chlorella zofingiensis. Therefore, a two-step cultivation strategy including high biomass yield fermentation and outdoor induction with an energy-free RFP was conducted. During the fermentation, the highest cell density of 98.4gL(-1) and astaxanthin yield of 73.3mgL(-1) were achieved, which were higher than those so far reported in C. zofingiensis. During the outdoor induction, astaxanthin content was further increased by 1.5-fold leading to the highest astaxanthin productivity of 5.26mgL(-1)day(-1) under an optimal dilution of 5-fold. Our work thus provided an effective two-step cultivation strategy for production of astaxanthin by C. zofingiensis.

  4. Properties and metathesis activity of monomeric and dimeric Mo centres variously located on γ-alumina A DFT study

    Science.gov (United States)

    Handzlik, Jarosław

    2007-05-01

    Ethene metathesis proceeding on monomeric and dimeric Mo species on the (1 0 0) and (1 1 0) γ-alumina is investigated by density functional theory, applying the cluster approach. The calculated vibrational frequencies of the surface OH groups are assigned to the experimental IR bands. It is shown that both monomeric and dimeric Mo forms can be the active sites of olefin metathesis. Metathesis activity and stability of the Mo-methylidene centres depend on their location on alumina. The differences in the sites reactivity are explained on the basis of their geometrical and electronic structure parameters. For the monomeric centres, isomerisation of the trigonal bipyramidal intermediate to the stable square pyramidal molybdacyclobutane is kinetically favoured over the cycloreversal step. The situation is opposite in the case of the dimeric species.

  5. Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase.

    Science.gov (United States)

    Pikal-Cleland, K A; Carpenter, J F

    2001-09-01

    During freezing in phosphate buffers, selective precipitation of a less soluble buffer component and subsequent pH shifts may induce protein denaturation. Previous reports indicate significantly more inactivation and secondary structural perturbation of monomeric and tetrameric beta-galactosidase (beta-gal) during freeze-thawing in sodium phosphate (NaP) buffer as compared with potassium phosphate (KP) buffer. This observation was attributed to the significant pH shifts (from 7.0 to as low as 3.8) observed during freezing in the NaP buffer (1). In the current study, we investigated the impact of the additional stress of dehydration after freezing on the recovery of active protein on reconstitution and the retention of the native structure in the dried state. Freeze-drying monomeric and tetrameric beta-gal in either NaP or KP buffer resulted in significant secondary structural perturbations, which were greatest for the NaP samples. However, similar recoveries of active monomeric protein were observed after freeze-thawing and freeze-drying, indicating that most dehydration-induced unfolding was reversible on reconstitution of the freeze-dried protein. In contrast, the tetrameric protein was more susceptible to dehydration-induced denaturation as seen by the greater loss in activity after reconstitution of the freeze-dried samples relative to that measured after freeze-thawing. To ensure optimal protein stability during freeze-drying, the protein must be protected from both freezing and dehydration stresses. Although poly(ethylene glycol) and dextran are preferentially excluded solutes and should confer protection during freezing, they were unable to prevent lyophilization-induced denaturation. In addition, Tween did not foster maintenance of native protein during freeze-drying. However, sucrose, which hydrogen bonds to dried protein in the place of lost water, greatly reduced freezing- and drying-induced denaturation, as observed by the high retention of native

  6. Extracellular monomeric tau protein is sufficient to initiate the spread of tau protein pathology.

    Science.gov (United States)

    Michel, Claire H; Kumar, Satish; Pinotsi, Dorothea; Tunnacliffe, Alan; St George-Hyslop, Peter; Mandelkow, Eckhard; Mandelkow, Eva-Maria; Kaminski, Clemens F; Kaminski Schierle, Gabriele S

    2014-01-10

    Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.

  7. Monomeric GLP-1/GIP/glucagon triagonism corrects obesity, hepatosteatosis, and dyslipidemia in female mice

    DEFF Research Database (Denmark)

    Jall, Sigrid; Sachs, Stephan; Clemmensen, Christoffer

    2017-01-01

    . RESULTS: Our results show that GLP-1/GIP/glucagon triple agonism inhibits food intake and decreases body weight and body fat mass with comparable potency in male and female mice that have been matched for body fat mass. Treatment improved dyslipidemia in both sexes and reversed diet......OBJECTIVE: Obesity is a major health threat that affects men and women equally. Despite this fact, weight-loss potential of pharmacotherapies is typically first evaluated in male mouse models of diet-induced obesity (DIO). To address this disparity we herein determined whether a monomeric peptide...... mice and a cohort of fatmass-matched C57BL/6J male mice were treated for 27 days via subcutaneous injections with either the GLP-1/GIP/glucagon triagonist or PBS. A second cohort of C57BL/6J male mice was included to match the females in the duration of the high-fat, high-sugar diet (HFD) exposure...

  8. Novel Monomeric Phenanthroline—Thallium(Ⅲ) Complexes Multinuclear NMR Characterization in Organic Solvents

    Institute of Scientific and Technical Information of China (English)

    GuiBinMA; JuliusGLASER

    2002-01-01

    A novel complex of monomeric thallium (Ⅲ) with the nitrogen donor ligand phenanthroline (phen) has been prepared and characterized by multimuclear NMR(1H,13C,205Tl). The three complexes exist in equilibria in DMSO and acetonitrile solution, which was proved by the 205Tl NMR spectra. The 1H and 13C NMR spectra of tris-phen T1(Ⅲ) complex have been measured, where the spin-spin coupling between T1(I=1/2) and 13C or 1H signals were observed with the 1H and 13C NMR spectroscopy in acetonitrile. The coupling constants are presented and the chemical shifts of complexes are discussed in detail.

  9. Novel Monomeric Phenanthroline - Thallium(Ⅲ) Complexes Multinuclear NMR Characterization in Organic Solvents

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A novel complex of monomeric thallium(III) with the nitrogen donor ligand phenanthrolinc (phen) has been prepared and characterized by multinuclear NMR (1H, 13C, 205T1).The three complexes exist in equilibria in DMSO and acetonitrile solution, which was proved by the 205Tl NMR spectra. The 1H and 13C NMR spectra of tris-phen Tl(III) complex have been measured, where the spin-spin coupling between TI (1 = 1/2) and 13C or 1H signals were observed with the 1H and 13C NMR spectroscopy in acetonitrile. The coupling constants are presented and the chemical shifts of complexes are discussed in detail.

  10. Kinetics of carbon monoxide binding to monomeric hemoproteins. Role of the proximal histidine.

    Science.gov (United States)

    Coletta, M; Ascenzi, P; Traylor, T G; Brunori, M

    1985-04-10

    The effect of pH on (i) the second-order rate constant for CO binding and (ii) the spectral properties of the deoxygenated derivative of several monomeric hemoproteins has been investigated in the pH range between 2.3 and 9.0. As in the case of 3-[1-imidazolyl]-propylamide monomethyl ester mesoheme, the rate constant for CO binding to sperm whale, horse, Dermochelys coriacea, Coryphaena hippurus, and Aplysia limacina myoglobins (the latter only in the presence of acetate/acetic acid mixture) increases, as the pH is lowered, to a value at least 1 order of magnitude higher than at pH 7.0. Such an effect is not observed in A. limacina myoglobin (in the absence of the acetate/acetic acid mixture) and Chironomus thummi thummi erythrocruorin. Moreover, the absorption spectrum, in the visible region, of the deoxy derivative of all these monomeric hemoproteins (with the exception of A. limacina myoglobin in the absence of the acetate/acetic acid mixture) undergoes a transition as the pH is lowered, an effect observed previously with 3-[1-imidazolyl]-propylamide monomethyl ester protoheme. On the basis of analogous spectroscopic and kinetic properties of chelated heme model compounds we attribute this behavior to the protonation of the N epsilon of the proximal imidazole involved in the bond with the iron atom. On the basis of this model the movement of the iron atom to the heme plane appears as a crucial step for CO binding, the activation free energy of the process amounting to approximately 2 kcal/mol.

  11. LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State

    Directory of Open Access Journals (Sweden)

    Claus U. Pietrzik

    2017-04-01

    Full Text Available The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aβ secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin β and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin β, with the latter exhibiting a preference for APP in its dimeric state.

  12. LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State.

    Science.gov (United States)

    Herr, Uta-Mareike; Strecker, Paul; Storck, Steffen E; Thomas, Carolin; Rabiej, Verena; Junker, Anne; Schilling, Sandra; Schmidt, Nadine; Dowds, C Marie; Eggert, Simone; Pietrzik, Claus U; Kins, Stefan

    2017-01-01

    The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aβ secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third) but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin β and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin β, with the latter exhibiting a preference for APP in its dimeric state.

  13. Quantification of the predominant monomeric catechins in baking chocolate standard reference material by LC/APCI-MS.

    Science.gov (United States)

    Nelson, Bryant C; Sharpless, Katherine E

    2003-01-29

    Catechins are polyphenolic plant compounds (flavonoids) that may offer significant health benefits to humans. These benefits stem largely from their anticarcinogenic, antioxidant, and antimutagenic properties. Recent epidemiological studies suggest that the consumption of flavonoid-containing foods is associated with reduced risk of cardiovascular disease. Chocolate is a natural cocoa bean-based product that reportedly contains high levels of monomeric, oligomeric, and polymeric catechins. We have applied solid-liquid extraction and liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry to the identification and determination of the predominant monomeric catechins, (+)-catechin and (-)-epicatechin, in a baking chocolate Standard Reference Material (NIST Standard Reference Material 2384). (+)-Catechin and (-)-epicatechin are detected and quantified in chocolate extracts on the basis of selected-ion monitoring of their protonated [M + H](+) molecular ions. Tryptophan methyl ester is used as an internal standard. The developed method has the capacity to accurately quantify as little as 0.1 microg/mL (0.01 mg of catechin/g of chocolate) of either catechin in chocolate extracts, and the method has additionally been used to certify (+)-catechin and (-)-epicatechin levels in the baking chocolate Standard Reference Material. This is the first reported use of liquid chromatography/mass spectrometry for the quantitative determination of monomeric catechins in chocolate and the only report certifying monomeric catechin levels in a food-based Standard Reference Material.

  14. Principal component regression analysis of the relation between CIELAB color and monomeric anthocyanins in young Cabernet Sauvignon wines.

    Science.gov (United States)

    Han, Fu-Liang; Zhang, Wen-Na; Pan, Qiu-Hong; Zheng, Cheng-Rong; Chen, Hai-Yan; Duan, Chang-Qing

    2008-11-17

    Color is one of the key characteristics used to evaluate the sensory quality of red wine, and anthocyanins are the main contributors to color. Monomeric anthocyanins and CIELAB color values were investigated by HPLC-MS and spectrophotometry during fermentation of Cabernet Sauvignon red wine, and principal component regression (PCR), a statistical tool, was used to establish a linkage between the detected anthocyanins and wine coloring. The results showed that 14 monomeric anthocyanins could be identified in wine samples, and all of these anthocyanins were negatively correlated with the L*, b* and H*ab values, but positively correlated with a* and C*ab values. On an equal concentration basis for each detected anthocyanin, cyanidin-3-O-glucoside (Cy3-glu) had the most influence on CIELAB color value, while malvidin 3-O-glucoside (Mv3-glu) had the least. The color values of various monomeric anthocyanins were influenced by their structures, substituents on the B-ring, acyl groups on the glucoside and the molecular steric structure. This work develops a statistical method for evaluating correlation between wine color and monomeric anthocyanins, and also provides a basis for elucidating the effect of intramolecular copigmentation on wine coloring.

  15. Principal Component Regression Analysis of the Relation Between CIELAB Color and Monomeric Anthocyanins in Young Cabernet Sauvignon Wines

    Directory of Open Access Journals (Sweden)

    Chang-Qing Duan

    2008-11-01

    Full Text Available Color is one of the key characteristics used to evaluate the sensory quality of red wine, and anthocyanins are the main contributors to color. Monomeric anthocyanins and CIELAB color values were investigated by HPLC-MS and spectrophotometry during fermentation of Cabernet Sauvignon red wine, and principal component regression (PCR, a statistical tool, was used to establish a linkage between the detected anthocyanins and wine coloring. The results showed that 14 monomeric anthocyanins could be identified in wine samples, and all of these anthocyanins were negatively correlated with the L*, b* and H*ab values, but positively correlated with a* and C*ab values. On an equal concentration basis for each detected anthocyanin, cyanidin-3-O-glucoside (Cy3-glu had the most influence on CIELAB color value, while malvidin 3-O-glucoside (Mv3-glu had the least. The color values of various monomeric anthocyanins were influenced by their structures, substituents on the B-ring, acyl groups on the glucoside and the molecular steric structure. This work develops a statistical method for evaluating correlation between wine color and monomeric anthocyanins, and also provides a basis for elucidating the effect of intramolecular copigmentation on wine coloring.

  16. Reactivity studies on [Cp'FeI]2: monomeric amido, phenoxo, and alkyl complexes.

    Science.gov (United States)

    Walter, Marc D; White, Peter S

    2012-11-05

    A series of monomeric mono(cyclopentadienyl) iron amido, phenoxo, and alkyl complexes were synthesized, and their structure and reactivity are presented. The iron(II) centers in these 14VE one-legged piano stool complexes are high spin (S = 2) in solid state and solution independent of solvent. The silylamide compound [Cp'FeN(SiMe(3))(2)] (2a, Cp' = 1,2,4-(Me(3)C)(3)C(5)H(2)) is an excellent starting material for the reaction with more acidic substrates such as phenols. Sterically encumbered phenols 2,6-(Me(3)C)(2)(4-R)C(6)H(2)OH (R = H, Me, and tBu) were investigated. In all cases monomeric iron phenoxo half-sandwich complexes [Cp'FeOR'] (4-R) are initially formed. Rearrangement of 4-R to the diamagnetic oxocyclohexadienyl complex [Cp'Fe(η(5)-O═C(6)H(2)R'(2)R")] (5-R) is observed for 2,6-(Me(3)C)(2)(4-R)C(6)H(2)OH (R = H and Me) and the Gibbs free enthalpy of activation (ΔG(‡)) was determined. In contrast this rearrangement is inhibited when the 4-position is blocked by a tBu group. Removing the steric bulk from the 2,6-positions leads to the formation of a μ-phenoxo dimer, [Cp'Fe(μ-OC(6)H(3)tBu(2)-3,5)](2) (5). Density functional theory (DFT) was used to further elucidate the structure-reactivity relationship in these molecules. The one-legged piano stool anilido complex [Cp'Fe(NHC(6)H(2)tBu(3)-2,4,6)] (7) is not accessible via acid-base reaction between 2a and H(2)NC(6)H(2)tBu(3)-2,4,6, but can be prepared by conventional salt metathesis reaction from [Cp'FeI](2) and [Li(NHC(6)H(2)tBu(3)-2,4,6)(OEt(2))](2). In contrast, reaction of 2a with Ph(2)NH yields the bimetallic [Cp'Fe(N,C-κ(1),η(5)-C(6)H(5)NPh)Fe(N-κ(1)-NPh(2))Cp'] (8) which combines two iron centers in the same oxidation state (+2), but different spin-states (S = 0 and S = 2) which is reflected in very different Cp(cent)-Fe distances of 1.68 and 2.04 Å, respectively. A monomeric iron alkyl half-sandwich complex [Cp'FeCH(SiMe(3))(2)] (9) was prepared that exhibits no reactivity toward H(2), C

  17. Synthesis and structural characterization of monomeric mercury(II) selenolate complexes derived from 2-phenylbenzamide ligands.

    Science.gov (United States)

    Patel, Saket; Meenakshi; Hodage, Ananda S; Verma, Ajay; Agrawal, Shailendra; Yadav, Abhimanyu; Kumar, Sangit

    2016-03-07

    Monomeric Hg(II) selenolate complexes derived from 2-phenylbenzamide ligands were prepared by oxidative addition of diselenides [{C6H4(CONR2)Se}2, R = Me, Et, iPr] to elemental Hg and reductive cleavage of the Se–N bond of isoselenazolone derivatives [(NO2)C6H3(CONSe)R, (R = allyl, nbutyl)] followed by the treatment with HgCl2. The complexes have been characterized by multinuclear NMR (1H, 13C and 77Se) spectroscopy and mass spectrometry which suggest the monomeric form of these in solution. The molecular structures of diselenides [C6H4(CONR2)Se]2 and mercury selenolates [Hg{(NO2)C6H3(CONH-C3H5) Se}2], [Hg{C6H4(CONiPr2)Se}2] and [Hg{C6H4(CONMe2)Se}2] were established by a single crystal X-ray diffraction study. Diselenides show strong intramolecular non-bonded Se⋯O interactions, which are influenced by the nature of C(O)NR̲2 and decrease with the sterically bulky alkyl substituent (Se⋯O =2.823 Å for R = di-Me, 2.760 Å for R = allyl, and 3.157 Å for R = di-iPr). Mercury complexes derived from less bulky 2-phenyl-N,N-dialkylbenzamide ligands associated with poor or no intramolecular nonbonded Hg⋯O interactions (4.91 Å for R = di-Me, 4.199 Å for R = allyl) and instead strong intermolecular Hg⋯O [2.792(3) and 2.820(4) Å] for di-Me and allyl and Hg⋯Se [3.3212(5) and 3.4076(8) Å] interactions were observed which lead to a dimeric form in the crystals. On the other hand, the mercury complex derived from the sterically bulky diisopropyl amide ligand shows a strong intramolecular non-bonded Hg⋯O (2.860 Å) interaction, adopts linear geometry and exists as a monomer. Thermogravimetric analysis (TGA) of the mercury selenolate complexes revealed two-step decomposition which leads to the formation of HgSe. The mercury selenolate complex 3c derived from the sterically bulky 2-phenyl-N,Ndiisopropylbenzamide ligand decomposed to give HgSe in the range of 220-300 °C.

  18. 11 Efficacy and Tolerability of HDM Injective Immunotherapy With Monomeric Allergoid

    Science.gov (United States)

    Compalati, Enrico; Atzeni, Isabella; Cabras, Sergio; Fancello, Paolo; Gaspardini, Giulio; Longo, Rocco; Patella, Vincenzo; Tore, Giorgio

    2012-01-01

    Background Subcutaneous immunotherapy (SCIT) is an effective treatment of respiratory allergy and carbamylated monomeric allergoids (monoids), by virtue of their reduced IgE-binding activity, resulted clinically safe by sublingual administration. Purpose of this study was to investigate the efficacy and tolerability of immunotherapy with house dust mites (HDM) monoid administered by injective route in patients with allergic rhinoconjunctivitis (AR). Methods A preparation of 0.70 mL of 10 BU/mL containing modified extract with 50% Dermatophagoides pteronyssinus and 50% Dermatophagoides farinae (amount of major allergen: 4 μg of group 1 per milliliter) was delivered monthly for 12 months, following a 5-week build-up induction phase (0.10–0.20–0.30–0.50–0.70 mL), to 58 patients (60% males, mean age 25.1 ± 12.7) suffering from AR due to mites for at least 2 years, whereas 60 patients with similar baseline characteristics were observed as controls. All patients were allowed to assume traditional drug therapy for their condition. At the end of the study changes from baseline in symptoms scores, in number of days with drug assumption, in severity of AR (according to ARIA classification) were compared between the 2 groups; moreover an overall assessment of clinical efficacy and tolerability was based on patients' and physicians' judgements (unsatisfactory, mild, good, optimal). Results In respect to baseline both groups showed, after 1 year, an improvement in symptoms score (P < 0.001) with a significant difference in favour of SCIT group (P < 0.05). Days of drug intake were significantly lower in patients receiving SCIT (P < 0.05). The number of patients with severe AR decreased in the first group while no variation was observed in controls. The subjective clinical overall assessment was optimal in 31 cases and good in 24 according to physicians' and patients' judgements; similarly 38 patients judged tolerability as optimal and 18 as good, whereas according to

  19. Holistic versus monomeric strategies for hydrological modelling of human-modified hydrosystems

    Science.gov (United States)

    Nalbantis, I.; Efstratiadis, A.; Rozos, E.; Kopsiafti, M.; Koutsoyiannis, D.

    2011-03-01

    The modelling of human-modified basins that are inadequately measured constitutes a challenge for hydrological science. Often, models for such systems are detailed and hydraulics-based for only one part of the system while for other parts oversimplified models or rough assumptions are used. This is typically a bottom-up approach, which seeks to exploit knowledge of hydrological processes at the micro-scale at some components of the system. Also, it is a monomeric approach in two ways: first, essential interactions among system components may be poorly represented or even omitted; second, differences in the level of detail of process representation can lead to uncontrolled errors. Additionally, the calibration procedure merely accounts for the reproduction of the observed responses using typical fitting criteria. The paper aims to raise some critical issues, regarding the entire modelling approach for such hydrosystems. For this, two alternative modelling strategies are examined that reflect two modelling approaches or philosophies: a dominant bottom-up approach, which is also monomeric and, very often, based on output information, and a top-down and holistic approach based on generalized information. Critical options are examined, which codify the differences between the two strategies: the representation of surface, groundwater and water management processes, the schematization and parameterization concepts and the parameter estimation methodology. The first strategy is based on stand-alone models for surface and groundwater processes and for water management, which are employed sequentially. For each model, a different (detailed or coarse) parameterization is used, which is dictated by the hydrosystem schematization. The second strategy involves model integration for all processes, parsimonious parameterization and hybrid manual-automatic parameter optimization based on multiple objectives. A test case is examined in a hydrosystem in Greece with high complexities

  20. Holistic versus monomeric strategies for hydrological modelling of human-modified hydrosystems

    Directory of Open Access Journals (Sweden)

    I. Nalbantis

    2011-03-01

    Full Text Available The modelling of human-modified basins that are inadequately measured constitutes a challenge for hydrological science. Often, models for such systems are detailed and hydraulics-based for only one part of the system while for other parts oversimplified models or rough assumptions are used. This is typically a bottom-up approach, which seeks to exploit knowledge of hydrological processes at the micro-scale at some components of the system. Also, it is a monomeric approach in two ways: first, essential interactions among system components may be poorly represented or even omitted; second, differences in the level of detail of process representation can lead to uncontrolled errors. Additionally, the calibration procedure merely accounts for the reproduction of the observed responses using typical fitting criteria. The paper aims to raise some critical issues, regarding the entire modelling approach for such hydrosystems. For this, two alternative modelling strategies are examined that reflect two modelling approaches or philosophies: a dominant bottom-up approach, which is also monomeric and, very often, based on output information, and a top-down and holistic approach based on generalized information. Critical options are examined, which codify the differences between the two strategies: the representation of surface, groundwater and water management processes, the schematization and parameterization concepts and the parameter estimation methodology. The first strategy is based on stand-alone models for surface and groundwater processes and for water management, which are employed sequentially. For each model, a different (detailed or coarse parameterization is used, which is dictated by the hydrosystem schematization. The second strategy involves model integration for all processes, parsimonious parameterization and hybrid manual-automatic parameter optimization based on multiple objectives. A test case is examined in a hydrosystem in Greece

  1. Prospective separation and transcriptome analyses of cortical projection neurons and interneurons based on lineage tracing by Tbr2 (Eomes)-GFP/Dcx-mRFP reporters.

    Science.gov (United States)

    Liu, Jiancheng; Wu, Xiwei; Zhang, Heying; Qiu, Runxiang; Yoshikawa, Kazuaki; Lu, Qiang

    2016-06-01

    In the cerebral cortex, projection neurons and interneurons work coordinately to establish neural networks for normal cortical functions. While the specific mechanisms that control productions of projection neurons and interneurons are beginning to be revealed, a global characterization of the molecular differences between these two neuron types is crucial for a more comprehensive understanding of their developmental specifications and functions. In this study, using lineage tracing power of combining Tbr2(Eomes)-GFP and Dcx-mRFP reporter mice, we prospectively separated intermediate progenitor cell (IPC)-derived neurons (IPNs) from non-IPC-derived neurons (non-IPNs) of the embryonic cerebral cortex. Molecular characterizations revealed that IPNs and non-IPNs were enriched with projection neurons and interneurons, respectively. Expression profiling documented cell-specific genes including differentially expressed transcriptional regulators that might be involved in cellular specifications, for instance, our data found that SOX1 and SOX2, which were known for important functions in neural stem/progenitor cells, continued to be expressed by interneurons but not by projection neurons. Transcriptome analyses of cortical neurons isolated at different stages of neurogenesis revealed distinct temporal patterns of expression of genes involved in early-born or late-born neuron specification. These data present a resource useful for further investigation of the molecular regulations and functions of projection neurons and interneurons.

  2. Primera identificación molecular del transgen de la proteína fluorescente roja (RFP en peces Cebra (Danio rerio transgénicos ornamentales introducidos en el Perú

    Directory of Open Access Journals (Sweden)

    Carlos Scotto

    2013-01-01

    Full Text Available En el presente trabajo se identificó por primera vez peces Cebra transgénicos (Danio rerio fluorescentes de color rojo, naranja y rosado introducidos al territorio peruano de acuarios locales utilizando la técnica de PCR para amplificar el transgen RFP perteneciente a la anémona marina Discosoma spp. Se encontró una expresión génica diferencial del transgen de la proteína fluorescente roja (RFP que determinaría una gradiente de bioluminiscencia para cada color entre los peces OVM analizados. Se realizó un análisis de secuencias de las dos variantes de la RFP junto con las seis variantes de la GFP de proteínas fluorescentes existentes en el Genbank que podrían ayudar a identificar rápidamente si son nuevos genes o si son nuevas variantes de éstas proteínas fluorescentes y que podrían ser utilizadas en otros OVMs hidrobiológicos a futuro. De este modo, desarrollar y optimizar las medidas de bioseguridad mediante su oportuna detección a nivel genético molecular.

  3. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

    Science.gov (United States)

    Chamma, Ingrid; Letellier, Mathieu; Butler, Corey; Tessier, Béatrice; Lim, Kok-Hong; Gauthereau, Isabel; Choquet, Daniel; Sibarita, Jean-Baptiste; Park, Sheldon; Sainlos, Matthieu; Thoumine, Olivier

    2016-01-01

    The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures. PMID:26979420

  4. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation.

    Science.gov (United States)

    Takemoto, Kiwamu; Matsuda, Tomoki; Sakai, Naoki; Fu, Donald; Noda, Masanori; Uchiyama, Susumu; Kotera, Ippei; Arai, Yoshiyuki; Horiuchi, Masataka; Fukui, Kiichi; Ayabe, Tokiyoshi; Inagaki, Fuyuhiko; Suzuki, Hiroshi; Nagai, Takeharu

    2013-01-01

    Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

  5. Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Yunli; Wang, Jianji; Liu, Zhen; Ren, Yunlai; Li, Guozhi [School of Chemical Engineering and Pharmaceutics, Henan University of Science and Technology, Luoyang 471039, Henan (China)

    2009-12-15

    Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose. (author)

  6. Retention mechanism for polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography with monomeric stationary phases.

    Science.gov (United States)

    Rafferty, Jake L; Siepmann, J Ilja; Schure, Mark R

    2011-12-23

    Reversed-phase liquid chromatography (RPLC) is the foremost technique for the separation of analytes that have very similar chemical functionalities, but differ only in their molecular shape. This ability is crucial in the analysis of various mixtures with environmental and biological importance including polycyclic aromatic hydrocarbons (PAHs) and steroids. A large amount of effort has been devoted to studying this phenomenon experimentally, but a detailed molecular-level description remains lacking. To provide some insight on the mechanism of shape selectivity in RPLC, particle-based simulations were carried out for stationary phases and chromatographic parameters that closely mimic those in an experimental study by Sentell and Dorsey [J. Chromatogr. 461 (1989) 193]. The retention of aromatic hydrocarbons ranging in size from benzene to the isomeric PAHs of the formula C(18)H(12) was examined for model RPLC systems consisting of monomeric dimethyl octadecylsilane (ODS) stationary phases with surface coverages ranging from 1.6 to 4.2 μmol/m(2) (i.e., stationary phases yielding low to intermediate shape selectivity) in contact with a 67/33 mol% acetonitrile/water mobile phase. The simulations show that the stationary phase acts as a very heterogeneous environment where analytes with different shapes prefer different spatial regions with specific local bonding environments of the ODS chains. However, these favorable retentive regions cannot be described as pre-existing cavities because the chain conformation in these local stationary phase regions adapts to accommodate the analytes.

  7. Contribution of Monomeric Anthocyanins to the Color of Young Red Wine: Statistical and Experimental Approaches.

    Science.gov (United States)

    Han, Fu Liang; Li, Zheng; Xu, Yan

    2015-12-01

    Monomeric anthocyanin contributions to young red wine color were investigated using partial least square regression (PLSR) and aqueous alcohol solutions in this study. Results showed that the correlation between the anthocyanin concentration and the solution color fitted in a quadratic regression rather than linear or cubic regression. Malvidin-3-O-glucoside was estimated to show the highest contribution to young red wine color according to its concentration in wine, whereas peonidin-3-O-glucoside in its concentration contributed the least. The PLSR suggested that delphinidin-3-O-glucoside and peonidin-3-O-glucoside under the same concentration resulted in a stronger color of young red wine compared with malvidin-3-O-glucoside. These estimates were further confirmed by their color in aqueous alcohol solutions. These results suggested that delphinidin-3-O-glucoside and peonidin-3-O-glucoside were primary anthocyanins to enhance young red wine color by increasing their concentrations. This study could provide an alternative approach to improve young red wine color by adjusting anthocyanin composition and concentration.

  8. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    Directory of Open Access Journals (Sweden)

    Françoise Paquet

    Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

  9. Crystal structure of monomeric photosystem II from Thermosynechococcus elongatus at 3.6-a resolution.

    Science.gov (United States)

    Broser, Matthias; Gabdulkhakov, Azat; Kern, Jan; Guskov, Albert; Müh, Frank; Saenger, Wolfram; Zouni, Athina

    2010-08-20

    The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C222(1), contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 A. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone Q(A), two heme groups, 11 beta-carotenes, 22 lipids, seven detergent molecules, and the Mn(4)Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular ( approximately 87 degrees ) to the crystallographic b-axis, further information about the structure of the Mn(4)Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.

  10. An Intrinsically Disordered Motif Mediates Diverse Actions of Monomeric C-reactive Protein.

    Science.gov (United States)

    Li, Hai-Yun; Wang, Jing; Meng, Fan; Jia, Zhe-Kun; Su, Yang; Bai, Qi-Feng; Lv, Ling-Ling; Ma, Fu-Rong; Potempa, Lawrence A; Yan, Yong-Bin; Ji, Shang-Rong; Wu, Yi

    2016-04-15

    Most proinflammatory actions of C-reactive protein (CRP) are only expressed following dissociation of its native pentameric assembly into monomeric form (mCRP). However, little is known about what underlies the greatly enhanced activities of mCRP. Here we show that a single sequence motif, i.e. cholesterol binding sequence (CBS; a.a. 35-47), is responsible for mediating the interactions of mCRP with diverse ligands. The binding of mCRP to lipoprotein component ApoB, to complement component C1q, to extracellular matrix components fibronectin and collagen, to blood coagulation component fibrinogen, and to membrane lipid component cholesterol, are all found to be markedly inhibited by the synthetic CBS peptide but not by other CRP sequences tested. Likewise, mutating CBS in mCRP also greatly impairs these interactions. Functional experiments further reveal that CBS peptide significantly reduces the effects of mCRP on activation of endothelial cells in vitro and on acute induction of IL-6 in mice. The potency and specificity of CBS are critically determined by the N-terminal residues Cys-36, Leu-37, and His-38; while the versatility of CBS appears to originate from its intrinsically disordered conformation polymorphism. Together, these data unexpectedly identify CBS as the major recognition site of mCRP and suggest that this motif may be exploited to tune the proinflammatory actions of mCRP.

  11. Design of monomeric water-soluble β-hairpin and β-sheet peptides.

    Science.gov (United States)

    Jiménez, M Angeles

    2014-01-01

    Since the first report in 1993 (JACS 115, 5887-5888) of a peptide able to form a monomeric β-hairpin structure in aqueous solution, the design of peptides forming either β-hairpins (two-stranded antiparallel β-sheets) or three-stranded antiparallel β-sheets has become a field of growing interest and activity. These studies have yielded great insights into the principles governing the stability and folding of β-hairpins and antiparallel β-sheets. This chapter provides an overview of the reported β-hairpin/β-sheet peptides focussed on the applied design criteria, reviews briefly the factors contributing to β-hairpin/β-sheet stability, and describes a protocol for the de novo design of β-sheet-forming peptides based on them. Guidelines to select appropriate turn and strand residues and to avoid self-association are provided. The methods employed to check the success of new designed peptides are also summarized. Since NMR is the best technique to that end, NOEs and chemical shifts characteristic of β-hairpins and three-stranded antiparallel β-sheets are given.

  12. Lipoamino acid-based micelles as promising delivery vehicles for monomeric amphotericin B.

    Science.gov (United States)

    Serafim, Cláudia; Ferreira, Inês; Rijo, Patrícia; Pinheiro, Lídia; Faustino, Célia; Calado, António; Garcia-Rio, Luis

    2016-01-30

    Lipoamino acid-based micelles have been developed as delivery vehicles for the hydrophobic drug amphotericin B (AmB). The micellar solubilisation of AmB by a gemini lipoamino acid (LAA) derived from cysteine and its equimolar mixtures with the bile salts sodium cholate (NaC) and sodium deoxycholate (NaDC), as well as the aggregation sate of the drug in the micellar systems, was studied under biomimetic conditions (phosphate buffered-saline, pH 7.4) using UV-vis spectroscopy. Pure surfactant systems and equimolar mixtures were characterized by tensiometry and important parameters were determined, such as critical micelle concentration (CMC), surface tension at the CMC (γCMC), maximum surface excess concentration (Γmax), and minimum area occupied per molecule at the water/air interface (Amin). Rheological behaviour from viscosity measurements at different shear rates was also addressed. Solubilisation capacity was quantified in terms of molar solubilisation ratio (χ), micelle-water partition coefficient (KM) and Gibbs energy of solubilisation (ΔGs°). Formulations of AmB in micellar media were compared in terms of drug loading, encapsulation efficiency, aggregation state of AmB and in vitro antifungal activity against Candida albicans. The LAA-containing micellar systems solubilise AmB in its monomeric and less toxic form and exhibit in vitro antifungal activity comparable to that of the commercial formulation Fungizone.

  13. Monomeric malonate precursors for the MOCVD of HfO2 and ZrO2 thin films.

    Science.gov (United States)

    Pothiraja, Ramasamy; Milanov, Andrian; Parala, Harish; Winter, Manuela; Fischer, Roland A; Devi, Anjana

    2009-01-28

    New Hf and Zr malonate complexes have been synthesized by the reaction of metal amides with different malonate ligands (L = dimethyl malonate (Hdmml), diethyl malonate (Hdeml), di-tert-butyl malonate (Hdbml) and bis(trimethylsilyl) malonate (Hbsml)). Homoleptic eight-coordinated monomeric compounds of the type ML4 were obtained for Hf with all the malonate ligands employed. In contrast, for Zr only Hdmml and Hdeml yielded the eight-coordinated monomeric compounds of the type ML4, while using the bulky Hdbml and Hbsml ligands resulted into mixed alkoxo-malonato six-coordinated compounds of the type [ML2(OR)2]. Single crystal X-ray diffraction studies of all the compounds are presented and discussed, and they are found to be monomeric. The complexes are solids and in solution, they retain their monomeric nature as evidenced by NMR measurements. Compared to the classical beta-diketonate complexes, [M(acac)4] and [M(thd)4] (M = Hf, Zr; acac: acetylacetonate; thd: tetramethylheptadione), the new malonate compounds are more volatile, decompose at lower temperatures and have lower melting points. In particular, the homoleptic diethyl malonate complexes of Hf and Zr melt at temperatures as low as 62 degrees C. In addition, the compounds are very stable in air and can be sublimed quantitatively. The promising thermal properties makes these compounds interesting for metal-organic chemical vapor deposition (MOCVD). This was demonstrated by depositing HfO2 and ZrO2 thin films successfully with two representative Hf and Zr complexes.

  14. Neurodevelopmental Expression Profile of Dimeric and Monomeric Group 1 mGluRs: Relevance to Schizophrenia Pathogenesis and Treatment

    Science.gov (United States)

    Lum, Jeremy S.; Fernandez, Francesca; Matosin, Natalie; Andrews, Jessica L.; Huang, Xu-Feng; Ooi, Lezanne; Newell, Kelly A.

    2016-01-01

    Group 1 metabotropic glutamate receptors (mGluR1/mGluR5) play an integral role in neurodevelopment and are implicated in psychiatric disorders, such as schizophrenia. mGluR1 and mGluR5 are expressed as homodimers, which is important for their functionality and pharmacology. We examined the protein expression of dimeric and monomeric mGluR1α and mGluR5 in the prefrontal cortex (PFC) and hippocampus throughout development (juvenile/adolescence/adulthood) and in the perinatal phencyclidine (PCP) model of schizophrenia. Under control conditions, mGluR1α dimer expression increased between juvenile and adolescence (209–328%), while monomeric levels remained consistent. Dimeric mGluR5 was steadily expressed across all time points; monomeric mGluR5 was present in juveniles, dramatically declining at adolescence and adulthood (−97–99%). The mGluR regulators, Homer 1b/c and Norbin, significantly increased with age in the PFC and hippocampus. Perinatal PCP treatment significantly increased juvenile dimeric mGluR5 levels in the PFC and hippocampus (37–50%) but decreased hippocampal mGluR1α (−50–56%). Perinatal PCP treatment also reduced mGluR1α dimer levels in the PFC at adulthood (−31%). These results suggest that Group 1 mGluRs have distinct dimeric and monomeric neurodevelopmental patterns, which may impact their pharmacological profiles at specific ages. Perinatal PCP treatment disrupted the early expression of Group 1 mGluRs which may underlie neurodevelopmental alterations observed in this model. PMID:27721389

  15. Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity.

    Science.gov (United States)

    Moulaei, Tinoush; Shenoy, Shilpa R; Giomarelli, Barbara; Thomas, Cheryl; McMahon, James B; Dauter, Zbigniew; O'Keefe, Barry R; Wlodawer, Alexander

    2010-09-08

    Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviral activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.

  16. Identification of the chromophores involved in aggregation-dependent energy quenching of the monomeric photosystem II antenna protein Lhcb5.

    Science.gov (United States)

    Ballottari, Matteo; Girardon, Julien; Betterle, Nico; Morosinotto, Tomas; Bassi, Roberto

    2010-09-03

    Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light-harvesting complex II in ADQ and exclusively monomeric Lhcb proteins in charge transfer quenching. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin, a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than light-harvesting complex II during aggregation and that this is enhanced by binding to zeaxanthin, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll 612 and 613, in close contact with lutein bound at site L1, are important facilitators of ADQ.

  17. Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

    DEFF Research Database (Denmark)

    Schønning, Kristian; Bolmstedt, A; Novotny, J

    1998-01-01

    An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected...... to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321....... HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase...

  18. Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

    DEFF Research Database (Denmark)

    Schønning, Kristian; Bolmstedt, A; Novotny, J

    1998-01-01

    An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected...... to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321....... HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase...

  19. Monomeric and gemini surfactants as antimicrobial agents - influence on environmental and reference strains.

    Science.gov (United States)

    Koziróg, Anna; Brycki, Bogumił

    2015-01-01

    Quaternary ammonium salts (QAS) belong to surfactant commonly used both, in the household and in different branches of industry, primarily in the process of cleaning and disinfection. They have several positive features inter alia effectively limiting the development of microorganisms on many surfaces. In the present work, two compounds were used as biocides: hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) that belongs to the gemini surfactant (GS), and its single analogue - dodecyl(trimethyl)ammonium bromide (DTAB). Two fold dilution method was used to determine the minimum concentration of compounds (MIC) which inhibit the growth of bacteria: Staphylococcus aureus (ATCC 6538 and an environmental strain), Pseudomonas aeruginosa (ATCC 85327 and an environmental strain), and yeast Candida albicans (ATCC 11509 and an environmental strain). The viability of cells in liquid cultures with addition of these substances at ¼ MIC, ½ MIC and MIC concentrations were also determined. The obtained results show that DTAB inhibits the growth of bacteria at the concentration of 0.126-1.010 µM/ml, and gemini surfactant is active at 0.036-0.029 µM/ml. Therefore, GS is active at more than 17-70-fold lower concentrations than its monomeric analogue. Strains isolated from natural environment are less sensitive upon testing biocides than the references strains. Both compounds at the MIC value reduced the number of cells of all strains. The use of too low concentration of biocides can limit the growth of microorganisms, but often only for a short period of time in case of special environmental strains. Later on, they can adapt to adverse environmental conditions and begin to evolve defence mechanisms.

  20. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

    Science.gov (United States)

    Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

  1. Crystal structure of a monomeric thiolase-like protein type 1 (TLP1 from Mycobacterium smegmatis.

    Directory of Open Access Journals (Sweden)

    Neelanjana Janardan

    Full Text Available An analysis of the Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1, has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 Å resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six β-strands forming an anti-parallel β-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.

  2. Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein.

    Science.gov (United States)

    Lecours, Katia; Tremblay, Marie-Hélène; Gagné, Marie-Eve Laliberté; Gagné, Stéphane M; Leclerc, Denis

    2006-05-01

    Papaya mosaic virus (PapMV) is a flexuous rod shape virus made of 1400 subunits that assemble around a plus sense genomic RNA. The structure determination of PapMV and of flexuous viruses in general is a major challenge for both NMR and X-ray crystallography. In this report, we present the characterization of a truncated version of the PapMV coat protein (CP) that is suitable for NMR study. The deletion of the N-terminal 26 amino acids of the PapMV CP (CP27-215) generates a monomer that can be expressed to high level and easily purified for production of an adequate NMR sample. The RNA gel shift assay showed that CP27-215 lost its ability to bind RNA in vitro, suggesting that the multimerization of the subunit is important for this function. The fusion of a 6x His tag at the C-terminus improved the solubility of the monomer and allowed its concentration to 0.2 mM. The CD spectra of the truncated and the wild-type proteins were similar, suggesting that both proteins are well ordered and have a similar secondary structure. CP27-215 was 15N labeled for NMR studies and a 2D 1H-15N-HSQC spectrum confirmed the presence of a well-ordered structure and the monomeric form of the protein. These results show that CP27-215 is amenable to a complete and exhaustive NMR study that should lead to the first three-dimensional structure determination of a flexuous rod shape virus.

  3. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    Science.gov (United States)

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  4. Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

    Directory of Open Access Journals (Sweden)

    Rodrigo Jácome

    Full Text Available The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

  5. Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation

    Directory of Open Access Journals (Sweden)

    Elena Grossini

    2016-09-01

    Full Text Available We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article “Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions” (Grossini et al., in press [1].

  6. A monomeric methyl and hydroxypropyl methacrylate injection medium and its utility in casting blood capillaries and liver bile canaliculi for scanning electron microscopy.

    Science.gov (United States)

    Murakami, T; Itoshima, T; Hitomi, K; Ohtsuka, A; Jones, A L

    1984-06-01

    A mixture of 50-60% monomeric methyl methacrylate and 40-50% monomeric 2-hydroxypropyl methacrylate was supplemented with 1.5% benzoyl peroxide (catalyst) and 1.5% N,N-dimethylaniline (accelerator) and injected into glutaraldehyde-perfusion fixed rat hypophyseal and other endocrine organ blood vessels and biliary tracts. This injection medium rapidly polymerized at room temperature and did not require partial polymerization prior to injection. Good casts of blood vessels, including the hypophyseal capillaries, were obtained for scanning electron microscopy. The monomeric methacrylate medium possesses a great advantage over previous ones, as its fluidity enables the casting of very fine vessels such as bile canaliculi. In the case of non-fixed tissues, the monomeric methacrylate medium should be injected carefully, as it is toxic and destructive to the vessels.

  7. Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation

    DEFF Research Database (Denmark)

    Hatzakis, Nikos S.; Wei, Li; Jørgensen, Sune Klamer

    2012-01-01

    allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus Lipase) while titrating...... its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate...

  8. Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water.

    Science.gov (United States)

    Willhite, Calvin C; Ball, Gwendolyn L; McLellan, Clifton J

    2012-05-01

    Maximum contaminant levels are used to control potential health hazards posed by chemicals in drinking water, but no primary national or international limits for aluminum (Al) have been adopted. Given the differences in toxicological profiles, the present evaluation derives total allowable concentrations for certain water-soluble inorganic Al compounds (including chloride, hydroxide, oxide, phosphate and sulfate) and for the hydrated Al silicates (including attapulgite, bentonite/montmorillonite, illite, kaolinite) in drinking water. The chemistry, toxicology and clinical experience with Al materials are extensive and depend upon the particular physical and chemical form. In general, the water solubility of the monomeric Al materials depends on pH and their water solubility and gastrointestinal bioavailability are much greater than that of the hydrated Al silicates. Other than Al-containing antacids and buffered aspirin, food is the primary source of Al exposure for most healthy people. Systemic uptake of Al after ingestion of the monomeric salts is somewhat greater from drinking water (0.28%) than from food (0.1%). Once absorbed, Al accumulates in bone, brain, liver and kidney, with bone as the major site for Al deposition in humans. Oral Al hydroxide is used routinely to bind phosphate salts in the gut to control hyperphosphatemia in people with compromised renal function. Signs of chronic Al toxicity in the musculoskeletal system include a vitamin D-resistant osteomalacia (deranged membranous bone formation characterized by accumulation of the osteoid matrix and reduced mineralization, reduced numbers of osteoblasts and osteoclasts, decreased lamellar and osteoid bands with elevated Al concentrations) presenting as bone pain and proximal myopathy. Aluminum-induced bone disease can progress to stress fractures of the ribs, femur, vertebrae, humerus and metatarsals. Serum Al ≥100 µg/L has a 75-88% positive predictive value for Al bone disease. Chronic Al

  9. Use of the quartz crystal microbalance to determine the monomeric friction coefficient of polyimides

    Science.gov (United States)

    Bechtold, Mary M.

    1995-01-01

    When a thin film of polymer is coated on to a quartz crystal microbalance (QCM), the QCM can be used to detect the rate of increase in weight of the polymer film as the volatile penetrant diffuses into the polymer. From this rate information the diffusion coefficient of the penetrant into the polymer can be computed. Calculations requiring this diffusion coefficient lead to values which approximate the monomeric friction coefficient of the polymer. This project has been concerned with the trial of crystal oscillating circuits suitable for driving polymer coated crystals in an atmosphere of penetrant. For these studies done at room temperature, natural rubber was used as an easily applied polymer that is readily penetrated by toluene vapors, qualities anticipated with polyimides when they are tested at T(g) in the presence of toluene. Three quartz crystal oscillator circuits were tested. The simplest circuit used +/- 5 volt dc and had a transistor to transistor logic (TTL) inverter chip that provides a 180 deg phase shift via a feed back loop. This oscillator circuit was stable but would not drive the crystal when the crystal was coated with polymer and subjected to toluene vapors. Removal of a variable resistor from this circuit increased stability but did not otherwise increase performance. Another driver circuit tested contained a two stage differential input, differential output, wide band video amplifier and also contain a feed back loop. The circuit voltage could not be varied and operated at +/- 5 volts dc; this circuit was also stable but failed to oscillate the polymer coated crystal in an atmosphere saturated with toluene vapors. The third oscillator circuit was of similar construction and relied on the same video amplifier but allowed operation with variable voltage. This circuit would drive the crystal when the crystal was submerged in liquid toluene and when the crystal was coated with polymer and immersed in toluene vapors. The frequency readings

  10. MANPRINT Handbook for RFP Development

    Science.gov (United States)

    1990-06-01

    requirement documents such as the O&O Plan, Mission Need Statement (MNS), Joint Service Operational Requirement ( JSOR ) and ROC are circulated to potential...B Abbreviations/Acronyms J JMSNS Justification for Major System New Start (Obsolete term, see MNS) JSOR Joint Service Operational Requirement L LAV

  11. Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

    Directory of Open Access Journals (Sweden)

    Ben Buelow

    Full Text Available Poly adenosine diphosphate-ribose polymerase-1 (PARP-1 is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR, and formation of the soluble 2(nd messenger monomeric adenosine diphosphate-ribose (mADPR. Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

  12. Monomeric Cu(Ⅱ) Complex Containing Chiral Phase-transfer Catalyst as Ligand and Its Asymmetrically Catalytic Reaction

    Institute of Scientific and Technical Information of China (English)

    QU Zhi-Rong; XIONG Ren-Gen

    2008-01-01

    The thermal treatment of CuCl2 with N-(4'-vinylbenzyl)cinchonidinitim chloride(L1)afforded a monomeric discrete homochiral copper(Ⅱ)complex N-4'-(vinylbenzyl)cinchonidinium trichlorocoprate(Ⅱ)(1).Their applications to the enantioselectively catalytic alkylation reaction of N-(diphenylmethylidene)glycine tert-butyl ester(3)show that the higher ee value observed in catalyst 1 than that in the corresponding free ligand L1 is probably due to the rigidity enhancement after the coordination of N atom of quinoline ring to the copper ion.

  13. Two-dimensional crystallization of monomeric bovine cytochrome c oxidase with bound cytochrome c in reconstituted lipid membranes.

    Science.gov (United States)

    Osuda, Yukiho; Shinzawa-Itoh, Kyoko; Tani, Kazutoshi; Maeda, Shintaro; Yoshikawa, Shinya; Tsukihara, Tomitake; Gerle, Christoph

    2016-06-01

    Mitochondrial cytochrome c oxidase utilizes electrons provided by cytochrome c for the active vectorial transport of protons across the inner mitochondrial membrane through the reduction of molecular oxygen to water. Direct structural evidence on the transient cytochrome c oxidase-cytochrome c complex thus far, however, remains elusive and its physiological relevant oligomeric form is unclear. Here, we report on the 2D crystallization of monomeric bovine cytochrome c oxidase with tightly bound cytochrome c at a molar ratio of 1:1 in reconstituted lipid membranes at the basic pH of 8.5 and low ionic strength.

  14. Conditions for homogeneous preparation of stable monomeric and oligomeric forms of activated Vip3A toxin from Bacillus thuringiensis.

    Science.gov (United States)

    Kunthic, Thittaya; Surya, Wahyu; Promdonkoy, Boonhiang; Torres, Jaume; Boonserm, Panadda

    2016-07-29

    Bacillus thuringiensis vegetative insecticidal proteins like Vip3A have been used for crop protection and to delay resistance to existing insecticidal Cry toxins. However, little is known about Vip3A's behavior or its mechanism of action, and a structural model is required. Herein, in an effort to facilitate future crystallization and functional studies, we have used the orthogonal biophysical techniques of light scattering and sedimentation to analyze the aggregation behavior and stability of trypsin-activated Vip3A toxin in solution. Both scattering and sedimentation data suggest that at pH 10 the toxin is monomeric and adopts an elongated shape, but after overnight incubation aggregation was observed at all pH values tested (5-12). The narrowest size distribution was observed at pH 7, but it was consistent with large oligomers of ~50 nm on average. The addition of β-D-glucopyranoside (OG) helped in achieving preparations that were stable and with a narrower particle size distribution. In this case, scattering was consistent with a 4-nm monomeric globular Vip3A form. After OG dialysis, 40-nm particles were detected, with a molecular weight consistent with homotetramers. Therefore, OG is proposed as the detergent of choice to obtain a Vip3A crystal for structural studies, either before (monomers) or after dialysis (tetramers).

  15. Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution.

    Science.gov (United States)

    Clavel, Damien; Gotthard, Guillaume; von Stetten, David; De Sanctis, Daniele; Pasquier, Hélène; Lambert, Gerard G; Shaner, Nathan C; Royant, Antoine

    2016-12-01

    Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent protein lanYFP from Branchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures of lanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV-visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.

  16. Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blueberry products.

    Science.gov (United States)

    Brownmiller, C; Howard, L R; Prior, R L

    2008-06-01

    This study evaluated the effects of processing and 6 mo of storage on total monomeric anthocyanins, percent polymeric color, and antioxidant capacity of blueberries that were canned in syrup (CS), canned in water (CW), pureed, and juiced (clarified and nonclarified). Total monomeric anthocyanins, percent polymeric color, and oxygen radical absorbing capacity (ORAC) assay using fluorescein (ORAC(FL)) were determined postprocessing after 1 d, and 1, 3, and 6 mo of storage. Thermal processing resulted in marked losses in total anthocyanins (28% to 59%) and ORAC(FL) values (43% to 71%) in all products, with the greatest losses occurring in clarified juices and the least in nonclarified juices. Storage at 25 degrees C for 6 mo resulted in dramatic losses in total anthocyanins, ranging from 62% in berries CW to 85% in clarified juices. This coincided with marked increases in percent polymeric color values of these products over the 6-mo storage. The ORAC(FL) values showed little change during storage, indicating that the formation of polymers compensated for the loss of antioxidant capacity due to anthocyanin degradation. Methods are needed to retain anthocyanins in thermally processed blueberries.

  17. Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed black raspberry products.

    Science.gov (United States)

    Hager, A; Howard, L R; Prior, R L; Brownmiller, C

    2008-08-01

    This study evaluated the effects of processing and 6 mo of storage on total monomeric anthocyanins, percent polymeric color, and antioxidant capacity of black raspberries that were individually quick-frozen (IQF), canned-in-syrup, canned-in-water, pureed, and juiced (clarified and nonclarified). Total monomeric anthocyanins, percent polymeric color, and ORAC(FL) were determined 1 d postprocessing and after 1, 3, and 6 mo of storage. Thermal processing resulted in marked losses in total anthocyanins ranging from 37% in puree to 69% to 73% in nonclarified and clarified juices, respectively, but only the juices showed substantial losses (38% to 41%) in ORAC(FL). Storage at 25 degrees C of all thermally processed products resulted in dramatic losses in total anthocyanins ranging from 49% in canned-in-syrup to 75% in clarified juices. This coincided with marked increases in percent polymeric color values of these products over the 6-mo storage. ORAC(FL) values showed little change during storage, indicating that the formation of polymers compensated for the loss of antioxidant capacity due to anthocyanin degradation. Total anthocyanins and ORACFL of IQF berries were well retained during long-term storage at -20 degrees C.

  18. Interaction of Classical Platinum Agents with the Monomeric and Dimeric Atox1 Proteins: A Molecular Dynamics Simulation Study

    Directory of Open Access Journals (Sweden)

    Xiaolei Wang

    2013-12-01

    Full Text Available We carried out molecular dynamics simulations and free energy calculations for a series of binary and ternary models of the cisplatin, transplatin and oxaliplatin agents binding to a monomeric Atox1 protein and a dimeric Atox1 protein to investigate their interaction mechanisms. All three platinum agents could respectively combine with the monomeric Atox1 protein and the dimeric Atox1 protein to form a stable binary and ternary complex due to the covalent interaction of the platinum center with the Atox1 protein. The results suggested that the extra interaction from the oxaliplatin ligand–Atox1 protein interface increases its affinity only for the OxaliPt + Atox1 model. The binding of the oxaliplatin agent to the Atox1 protein might cause larger deformation of the protein than those of the cisplatin and transplatin agents due to the larger size of the oxaliplatin ligand. However, the extra interactions to facilitate the stabilities of the ternary CisPt + 2Atox1 and OxaliPt + 2Atox1 models come from the α1 helices and α2-β4 loops of the Atox1 protein–Atox1 protein interface due to the cis conformation of the platinum agents. The combinations of two Atox1 proteins in an asymmetric way in the three ternary models were analyzed. These investigations might provide detailed information for understanding the interaction mechanism of the platinum agents binding to the Atox1 protein in the cytoplasm.

  19. Diagnóstico bioquímico de la hiperprolactinemia monomérica Biochemical diagnosis of monomeric hyperprolactinemia

    Directory of Open Access Journals (Sweden)

    A. Rivero

    2011-08-01

    Full Text Available Fundamento. La prolactina se puede presentar bajo varias formas moleculares siendo la forma monomérica (PRLm la biológicamente activa. La presencia de macroprolactina (MPRL puede originar un falso diagnóstico de hiperprolactinemia debido a la interferencia en el procedimiento de medida. El objetivo ha sido desarrollar un protocolo que permita diagnosticar la hiperprolactinemia monomérica, que además sea complementario al procedimiento que detecta MPRL. Material y métodos. La población de referencia para PRLm estaba formada por 122 mujeres y 140 hombres aparentemente sanos a los que se les extrajo sangre para la cuantificación de PRL. Además, se recogieron 49 sueros (33 mujeres y 16 hombres hiperprolactinémicos. Se cuantificó PRL en todas las muestras en un Immulite 2000. La detección de MPRL y de PRLm se realizó tras precipitación con polietilenglicol. Se confirmó el resultado por cromatografía de filtración en gel. Para la obtención de los valores de referencia se siguieron las indicaciones del Panel de Expertos de la IFCC. Resultados. Los valores de referencia de PRLm fueron 3,4-26,6 μg/L y 4,6-16,4 μg/L en mujeres y varones, respectivamente. De los 49 pacientes hiperprolactinémicos, en el 57 % la concentración de PRLm tras PEG se encontraba fuera del intervalo de referencia previamente obtenido, confirmándose la presencia de hiperprolactinemia monomérica. Conclusiones. Se ha desarrollado e implantado un protocolo para la cuantificación de PRLm. La obtención de los valores de referencia de PRLm permite el diagnóstico de la hiperprolactinemia monomérica o activa de forma complementaria a la identificación de MPRL.Background. Prolactin can take several molecular forms of which the most biologically active is the monomeric form (PRLm. The presence of macroprolactin (MPRL can give rise to a false diagnosis of hyperprolactinemia due to interference in the measuring procedure. The aim was to develop a protocol that

  20. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

    DEFF Research Database (Denmark)

    Whorton, Matthew R; Bokoch, Michael P; Rasmussen, Søren Gøgsig Faarup;

    2007-01-01

    G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet...... the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However......, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein...

  1. Synthesis of monomeric and polymeric alkali and alkaline earth metal complexes using a phosphinoselenoic amide ligand in metal coordination sphere

    Indian Academy of Sciences (India)

    Jayeeta Bhattacharjee; Ravi K Kottalanka; Harinath Adimulam; Tarun K Panda

    2014-09-01

    We report the monomeric complexes of magnesium and calcium of composition [M(THF){2-Ph2P(Se)N(CMe3)}2] [M= Mg (3), n = 1 andM = Ca (4), n = 2)] and polymeric complexes of potassium and barium of composition [K(THF)2{Ph2P(Se)N(CMe3)}] (2) and [K(THF)Ba{Ph2P(Se)N(CMe3)}3](5) respectively. The potassium complex 2 was readily prepared by the reaction of potassium bis(trimethylsilyl)amide with phosphinoselenoic amide ligand (1) at ambient temperature. The calcium complex 4 was prepared by two synthetic routes: in the first method, commonly known as salt metathesis reaction, the potassium complex 2 was made to react with alkaline earth metal diiodide at room temperature to afford the corresponding calcium complex. The metal bis(trimethylsilyl)amides were made to react with protic ligand 1 in the second method to eliminate the volatile bis(trimethyl)silyl amine. The magnesium complex 3 and barium complex 5 were prepared only through the first method. Solid-state structures of all the new complexes were established by single crystal X-ray diffraction analysis. The smaller ionic radii of Mg2+ (0.72Å) and Ca2+ (0.99Å) ions form the monomeric complex, whereas the larger ions K+ (1.38Å) and Ba2+ (1.35Å) were found to form onedimensional polymeric complexes with monoanionic ligand 1. Compound 2 serves an example of magnesium complex with a Mg-Se direct bond.

  2. Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

    Directory of Open Access Journals (Sweden)

    Baisakhi Banerjee

    2015-01-01

    Full Text Available E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700 forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS.

  3. Crystallization and preliminary crystallographic analysis of decameric and monomeric forms of C49S mutant thioredoxin-dependent AhpC from Helicobacter pylori

    Energy Technology Data Exchange (ETDEWEB)

    Supangat [Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Seo, Kyung Hye; Furqoni, Ahmad [Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Kwon, Young-Chul; Cho, Myung-Je; Rhee, Kwang-Ho [Department of Microbiology, School of Medicine, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Lee, Sang Yeol; Lee, Kon Ho, E-mail: lkh@gsnu.ac.kr [Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of)

    2008-05-01

    Decameric and monomeric forms of recombinant C49S mutant AhpC from H. pylori have been crystallized. Diffraction data were collected to 2.8 and 2.25 Å, respectively. Cys49Ser mutant Helicobacter pylori alkyl hydroperoxide reductase (C49S HpAhpC) was purified under reducing conditions in monomeric and decameric forms. The monomeric form was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.25 Å resolution and belonged to space group C2, with unit-cell parameters a = 245.8, b = 140.7, c = 189.5 Å, β = 127°, and contained 20 molecules in the asymmetric unit. A crystal of the decameric form was obtained by the microbatch crystallization method and diffracted to 2.8 Å resolution. It belonged to space group C222, with unit-cell parameters a = 257.5, b = 417.5, c = 95.6 Å. The structure of the monomeric form of C49S HpAhpC has been solved by the molecular-replacement method.

  4. Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

    Science.gov (United States)

    Banerjee, Baisakhi; Banerjee, Rajat

    2015-01-01

    E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700) forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D) also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS. PMID:26617997

  5. Calcium, acylation, and molecular confinement favor folding of Bordetella pertussis adenylate cyclase CyaA toxin into a monomeric and cytotoxic form.

    Science.gov (United States)

    Karst, Johanna C; Ntsogo Enguéné, V Yvette; Cannella, Sara E; Subrini, Orso; Hessel, Audrey; Debard, Sylvain; Ladant, Daniel; Chenal, Alexandre

    2014-10-31

    The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

  6. Analysis of by-product formation and sugar monomerization in sugarcane bagasse pretreated at pilot plant scale: Differences between autohydrolysis, alkaline and acid pretreatment

    NARCIS (Netherlands)

    Pol, van der E.C.; Bakker, R.; Zeeland, van A.N.T.; Sanchez Garcia, D.; Punt, A.M.; Eggink, G.

    2015-01-01

    Sugarcane bagasse is an interesting feedstock for the biobased economy since a large fraction is polymerized sugars. Autohydrolysis, alkaline and acid pretreatment conditions combined with enzyme hydrolysis were used on lignocellulose rich bagasse to acquire monomeric. By-products found after pretre

  7. Tgf2转座系统在转RFP基因斑马鱼上的应用%Application of the Tgf2 transposon system to RFP transgenic Danio rerio construction

    Institute of Scientific and Technical Information of China (English)

    吴芳; 叶星; 邹曙明; 张莉莉; 孙成飞; 田园园; 白俊杰

    2014-01-01

    Improving the efficiency of gene transfer and integration is a key objective in transgenic fish research. To evaluate its use for transgenic fish applications, we used the Tgf2 transposon system to construct a transgenic zebrafish line. The Tgf2 transposon donor plasmid pTgf2-EF1α-eGFP was re-constructed by replacement of the transgenic ele-ments, EF1α-eGFP with the myosin light chain 2 promoter (mylz2) and the red fluorescent protein (RFP) gene, mylz2-RFP. The recombinant Tgf2 transposon donor plasmid, pTgf2-Mylz2-RFP was intended to serve as a transposon donor plasmid that specifically expresses red fluorescent protein in muscle tissue. The donor plasmid and the Tgf2 transposase mRNA were co-injected into zebra fish fertilized eggs. A total of 972 eggs were microinjected and 803 lar-vae survived, with a positive rate of 76.6%. The red fluorescence (RFP) gene transgenic zebrafish (F0) were subse-quently cultivated and 10 sexually mature transgenic individuals were mated with wild-type zebrafish, respectively. F1 individuals that exhibited red fluorescence on their body surface were obtained from one of the mating pairs yielding an integration efficiency of 10%. The positive rate of F2 individuals, which were generated by mating the mature trans-genic F1 with the wild-type fish, was 69%. The mature RFP positive F1 individual was then mated with wild-type fish, yielding a 69%positive rate in the F2 generation. Our results demonstrate that the Tgf2 transposon improves the effi-ciency of gene transfer and integration during transgene treatments. Thus, the Tgf2 transposon can be used to construct transgenic fish and in other transgenic fish research.%提高目的基因的转植效率与可遗传效率是转基因鱼研制的关键点之一。本研究利用近年开发的金鱼转座子系统进行转基因斑马鱼的研制,探讨其在转基因鱼上应用的可行性。通过 PCR 方法,改造 Tgf2转座子供体质粒pTgf2-EF1α-eGFP,将肌球蛋白轻链2

  8. Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

    Directory of Open Access Journals (Sweden)

    Nilanjana Banerjee

    Full Text Available BACKGROUND: Allium sativum leaf agglutinin (ASAL is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL. METHODOLOGY/PRINCIPAL FINDINGS: By introduction of 5 site-specific mutations (-DNSNN-, a β turn was incorporated between the 11(th and 12(th β strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL. mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and Alternaria brassicicola in a disc diffusion assay. A propidium iodide uptake assay suggested that the inhibitory activity of mASAL might be associated with the alteration of the membrane permeability of the fungus. Furthermore, a ligand blot assay of the membrane subproteome of R. solani with mASAL detected a glycoprotein receptor having interaction with mASAL. CONCLUSIONS/SIGNIFICANCE: Conversion of ASAL into a stable monomer resulted in antifungal activity. From an evolutionary aspect, these data implied that variable quaternary organization of lectins might be the outcome of defense-related adaptations to diverse situations in plants. Incorporation of m

  9. Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

    Science.gov (United States)

    Banerjee, Nilanjana; Sengupta, Subhadipa; Roy, Amit; Ghosh, Prithwi; Das, Kalipada; Das, Sampa

    2011-04-07

    Allium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL. By introduction of 5 site-specific mutations (-DNSNN-), a β turn was incorporated between the 11(th) and 12(th) β strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL). mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and Alternaria brassicicola in a disc diffusion assay. A propidium iodide uptake assay suggested that the inhibitory activity of mASAL might be associated with the alteration of the membrane permeability of the fungus. Furthermore, a ligand blot assay of the membrane subproteome of R. solani with mASAL detected a glycoprotein receptor having interaction with mASAL. Conversion of ASAL into a stable monomer resulted in antifungal activity. From an evolutionary aspect, these data implied that variable quaternary organization of lectins might be the outcome of defense-related adaptations to diverse situations in plants. Incorporation of mASAL into agronomically-important crops could be an alternative method to protect them

  10. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

    Science.gov (United States)

    Tamaddon, M.; Burrows, M.; Ferreira, S. A.; Dazzi, F.; Apperley, J. F.; Bradshaw, A.; Brand, D. D.; Czernuszka, J.; Gentleman, E.

    2017-03-01

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS – which was released from scaffolds quickly – significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  11. Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro.

    Science.gov (United States)

    Tamaddon, M; Burrows, M; Ferreira, S A; Dazzi, F; Apperley, J F; Bradshaw, A; Brand, D D; Czernuszka, J; Gentleman, E

    2017-03-03

    Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

  12. Age-dependent preferential dense-core vesicle exocytosis in neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein.

    Science.gov (United States)

    Tsuboi, Takashi; Kitaguchi, Tetsuya; Karasawa, Satoshi; Fukuda, Mitsunori; Miyawaki, Atsushi

    2010-01-01

    Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K(+)] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.

  13. Differing modes of interaction between monomeric Aβ(1-40) peptides and model lipid membranes: an AFM study.

    Science.gov (United States)

    Sheikh, Khizar; Giordani, Cristiano; McManus, Jennifer J; Hovgaard, Mads Bruun; Jarvis, Suzanne P

    2012-02-01

    Membrane interactions with β-amyloid peptides are implicated in the pathology of Alzheimer's disease and cholesterol has been shown to be key modulator of this interaction, yet little is known about the mechanism of this interaction. Using atomic force microscopy, we investigated the interaction of monomeric Aβ(1-40) peptides with planar mica-supported bilayers composed of DOPC and DPPC containing varying concentrations of cholesterol. We show that below the bilayer melting temperature, Aβ monomers adsorb to, and assemble on, the surface of DPPC bilayers to form layers that grow laterally and normal to the bilayer plane. Above the bilayer melting temperature, we observe protofibril formation. In contrast, in DOPC bilayers, Aβ monomers exhibit a detergent-like action, forming defects in the bilayer structure. The kinetics of both modes of interaction significantly increases with increasing membrane cholesterol content. We conclude that the mode and rate of the interaction of Aβ monomers with lipid bilayers are strongly dependent on lipid composition, phase state and cholesterol content.

  14. The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane

    Science.gov (United States)

    Zeev-Ben-Mordehai, Tzviya; Vasishtan, Daven; Siebert, C. Alistair; Grünewald, Kay

    2014-05-01

    Fusogens are membrane proteins that remodel lipid bilayers to facilitate membrane merging. Although several fusogen ectodomain structures have been solved, structural information on full-length, natively membrane-anchored fusogens is scarce. Here we present the electron cryo microscopy three-dimensional reconstruction of the Caenorhabditis elegans epithelial fusion failure 1 (EFF-1) protein natively anchored in cell-derived membrane vesicles. This reveals a membrane protruding, asymmetric, elongated monomer. Flexible fitting of a protomer of the EFF-1 crystal structure, which is homologous to viral class-II fusion proteins, shows that EFF-1 has a hairpin monomeric conformation before fusion. These structural insights, when combined with our observations of membrane-merging intermediates between vesicles, enable us to propose a model for EFF-1 mediated fusion. This process, involving identical proteins on both membranes to be fused, follows a mechanism that shares features of SNARE-mediated fusion while using the structural building blocks of the unilaterally acting class-II viral fusion proteins.

  15. Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

    Science.gov (United States)

    Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

    2016-02-01

    Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.

  16. New strategy for high-level expression and purification of biologically active monomeric TGF-β1/C77S in Escherichia coli.

    Science.gov (United States)

    Kim, Yana V; Gasparian, Marine E; Bocharov, Eduard V; Chertkova, Rita V; Tkach, Elena N; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2015-02-01

    Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

  17. Recognition profile of Morus nigra agglutinin (Morniga G) expressed by monomeric ligands, simple clusters and mammalian polyvalent glycotopes.

    Science.gov (United States)

    Singh, Tanuja; Wu, June H; Peumans, Willy J; Rougé, Pierre; Van Damme, Els J M; Wu, Albert M

    2007-01-01

    The carbohydrate binding properties of a novel member of the subfamily of galactose-specific jacalin-related lectin isolated from the bark of black mulberry (Morus nigra) (Morniga G) was studied in detail by enzyme-linked lectinosorbent and inhibition assays using panels of monomeric saccharides, mammalian polyvalent glycotopes and polysaccharides. Among the natural glycans tested for lectin binding, Morniga G reacted best with glycoproteins (gps) presenting a high density of tumor-associated carbohydrate antigens Tn (GalNAcalpha1-Ser/Thr) and Talpha (Galbeta1-3GalNAcalpha1-). Their reactivities, on a nanogram basis, were up to 72.5, 3.9x10(3), 6.0x10(3), 8.8x10(3) and 2.9x10(4) times higher than that of Tn-containing glycopeptides (M.W.Man/Glc, GlcNAc and lFuc; (ii) the mammalian glycotope specificity is Talpha1-benzyl>T>Tn>GalNAcbeta1-3Gal (P), while B/E (Galalpha1-3/4Gal), I/II (Galbeta1-3/4GlcNAc), S (GalNAcbeta1-4Gal), F/A (GalNAcalpha1-3GalNAc/Gal) and L (Galbeta1-4Glc) are inactive; (iii) the most active ligand is T/Tn; (iv) simple clustered Tn or triantennary N-glycans with II termini (Tri-II) have limited impact; (v) high-density polyvalent glycotopes play a prominent role for enhancing Morniga G reactivity. These results provide evidence for the binding of this lectin to dense cell surface T/Tn glycoconjugates and facilitate future usage of this lectin in biotechnological and medical applications.

  18. Pleiotropic benefit of monomeric and oligomeric flavanols on vascular health--a randomized controlled clinical pilot study.

    Directory of Open Access Journals (Sweden)

    Antje R Weseler

    Full Text Available BACKGROUND: Cardiovascular diseases are expanding to a major social-economic burden in the Western World and undermine man's deep desire for healthy ageing. Epidemiological studies suggest that flavanol-rich foods (e.g. grapes, wine, chocolate sustain cardiovascular health. For an evidenced-based application, however, sound clinical data on their efficacy are strongly demanded. METHODS: In a double-blind, randomized, placebo-controlled intervention study we supplemented 28 male smokers with 200 mg per day of monomeric and oligomeric flavanols (MOF from grape seeds. At baseline, after 4 and 8 weeks we measured macro- and microvascular function and a cluster of systemic biomarkers for major pathological processes occurring in the vasculature: disturbances in lipid metabolism and cellular redox balance, and activation of inflammatory cells and platelets. RESULTS: In the MOF group serum total cholesterol and LDL decreased significantly (P ≤ 0.05 by 5% (n = 11 and 7% (n = 9, respectively in volunteers with elevated baseline levels. Additionally, after 8 weeks the ratio of glutathione to glutathione disulphide in erythrocytes rose from baseline by 22% (n = 15, P<0.05 in MOF supplemented subjects. We also observed that MOF supplementation exerts anti-inflammatory effects in blood towards ex vivo added bacterial endotoxin and significantly reduces expression of inflammatory genes in leukocytes. Conversely, alterations in macro- and microvascular function, platelet aggregation, plasma levels of nitric oxide surrogates, endothelin-1, C-reactive protein, fibrinogen, prostaglandin F2alpha, plasma antioxidant capacity and gene expression levels of antioxidant defense enzymes did not reach statistical significance after 8 weeks MOF supplementation. However, integrating all measured effects into a global, so-called vascular health index revealed a significant improvement of overall vascular health by MOF compared to placebo (P ≤ 0.05. CONCLUSION: Our

  19. Aggregation of a slow-folding mutant of a beta-clam protein proceeds through a monomeric nucleus.

    Science.gov (United States)

    Ignatova, Zoya; Gierasch, Lila M

    2005-05-17

    Mechanistic understanding of protein aggregation, leading either to structured amyloid fibrils or to amorphous inclusion body-like deposits, should facilitate the identification of potential therapeutic intervention strategies for the devastating amyloid-based diseases. Here we focus on the in vitro aggregation of a slow-folding mutant of the beta-clam protein, cellular retinoic acid-binding protein I (P39A CRABP I), which forms inclusion bodies when expressed in Escherichia coli. Aggregation was monitored by observing the fluorescence of a fluorescein-based biarsenical dye (FlAsH) that ligates to a tetra-Cys motif, here incorporated into a flexible Omega-loop. The fluorescence signal of FlAsH on the tetra-Cys-containing P39A CRABP I is sensitive to whether this protein is native or unfolded, and was used in combination with other techniques to follow aggregate formation. The aggregation time course is compatible with a nucleation-dependent polymerization model, and detailed kinetic analysis showed that the energetically unfavorable nucleus is monomeric. A similar conclusion was reached previously for poly(Gln) species [Chen, S., Ferrone, F. A., and Wetzel, R. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11884-11889] and points to an unfavorable equilibrium between the misfolded intermediate and the bulk pool of monomers as causative in aggregation. The P39A mutation, which removes a helix-stop signal, may slow closure of the beta-barrel in P39A CRABP I relative to the wild type, leaving it vulnerable to aggregation. Wide-angle X-ray scattering showed that the amorphous aggregates formed by the aggregation-prone intermediates of P39A CRABP I contain predominantly beta-strands structured in a lamellar fashion with 10.03 A spacing between adjacent beta-sheets.

  20. Occurrence and Speciation of Polymeric Chromium(III), Monomeric Chromium(III) and Chromium(VI) in Environmental Samples

    Science.gov (United States)

    HU, LIGANG; CAI, YONG; JIANG, GUIBIN

    2016-01-01

    Laboratory experiments suggest that polymeric Cr(III) could exist in aqueous solution for a relative long period of time. However, the occurrence of polymeric Cr(III) has not been reported in environmental media due partially to the lack of method for speciating polymeric Cr. We observed an unknown Cr species during the course of study on speciation of Cr in the leachates of chromated-copper-arsenate (CCA)-treated wood. Efforts were made to identify structure of the unknown Cr species. Considering the forms of Cr existed in the CCA-treated woods, we mainly focused our efforts to determine if the unknown species were polymeric Cr(III), complex of Cr/As or complex of Cr with dissolved organic matter (DOM). In order to evaluate whether polymeric Cr(III) largely exist in wood leachates, high performance liquid chromatography coupled with inductively coupled mass spectrometry (HPLC-ICPMS was used) for simultaneous speciation of monomeric Cr(III), polymeric Cr(III), and Cr(VI). In addition to wood leachates where polymeric Cr (III) ranged from 39.1 to 67.4 %, occurrence of the unknown Cr species in other environmental matrices, including surface waters, tap and waste waters, was also investigated. It was found that polymeric Cr(III) could exist in environmental samples containing μg/L level of Cr, at a level up to 60% of total Cr, suggesting that polymeric Cr(III) could significantly exist in natural environments. Failure in quantifying polymeric Cr(III) would lead to the underestimation of total Cr and bias in Cr speciation. The environmental implication of the presence of polymeric Cr(III) species in the environment deserves further study. PMID:27156211

  1. Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

    Science.gov (United States)

    Cannella, Sara E.; Ntsogo Enguéné, Véronique Yvette; Davi, Marilyne; Malosse, Christian; Sotomayor Pérez, Ana Cristina; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Ladant, Daniel; Chenal, Alexandre

    2017-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins. PMID:28186111

  2. Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods.

    Science.gov (United States)

    Tang, Pei-Ling; Hassan, Osman; Maskat, Mohamad Yusof; Badri, Khairiah

    2015-01-01

    In this study, oil palm empty fruit bunch (OPEFBF) was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen) and enzymatic [cutinase versus manganese peroxidase (MnP)] approaches was investigated. The effects of sodium hydroxide concentration (2, 5, and 10% wt.) and reaction time (30, 90, and 180 minutes) on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield (333.17 ± 49.44 ppm hydroxybenzoic acid, 5.67 ± 0.25 ppm p-hydroxybenzaldehyde, 25.57 ± 1.64 ppm vanillic acid, 168.68 ± 23.23 ppm vanillin, 75.44 ± 6.71 ppm syringic acid, 815.26 ± 41.77 ppm syringaldehyde, 15.21 ± 2.19 ppm p-coumaric acid, and 44.75 ± 3.40 ppm ferulic acid), among the tested methods. High sodium hydroxide concentration (10% wt.) was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid). Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g(-1) lignin at pH 8 and 55°C for 24 hours, about 642.83 ± 14.45 ppm hydroxybenzoic acid, 70.19 ± 3.31 ppm syringaldehyde, 22.80 ± 1.04 ppm vanillin, 27.06 ± 1.20 ppm p-coumaric acid, and 50.19 ± 2.23 ppm ferulic acid were produced.

  3. Stable divalent germanium, tin and lead amino(ether)-phenolate monomeric complexes: structural features, inclusion heterobimetallic complexes, and ROP catalysis.

    Science.gov (United States)

    Wang, Lingfang; Roşca, Sorin-Claudiu; Poirier, Valentin; Sinbandhit, Sourisak; Dorcet, Vincent; Roisnel, Thierry; Carpentier, Jean-François; Sarazin, Yann

    2014-03-21

    Stable germanium(II) and lead(II) amido complexes {LO(i)}M(N(SiMe3)2) (M = Ge(II), Pb(II)) bearing amino(ether)phenolate ligands are readily available using the proteo-ligands {LO(i)}H of general formula 2-CH2NR2-4,6-tBu2-C6H2OH (i = 1, NR2 = N((CH2)2OCH3)2; i = 2, NR2 = NEt2; i = 3, NR2 = aza-15-crown-5) and M(N(SiMe3)2)2 precursors. The molecular structures of these germylenes and plumbylenes, as well as those of {LO(3)}GeCl, {LO(3)}SnCl and of the congeneric {LO(4)}Sn(II)(N(SiMe3)2) where NR2 = aza-12-crown-4, have been determined crystallographically. All complexes are monomeric, with 3-coordinate metal centres. The phenolate systematically acts as a N^O(phenolate) bidentate ligand, with no interactions between the metal and the O(side-arm) atoms in these cases (for {LO(1)}(-), {LO(3)}(-) and {LO(4)}(-)) where they could potentially arise. For each family, the lone pair of electrons essentially features ns(2) character, and there is little, if any, hybridization of the valence orbitals. Heterobimetallic complexes {LO(3)}M(N(SiMe3)2)·LiOTf, where the Li(+) cation sits inside the tethered crown-ether, were prepared by reaction of {LO(3)}M(N(SiMe3)2) and LiOTf (M = Ge(II), Sn(II)). The inclusion of Li(+) (featuring a close contact with the triflate anion) in the macrocycle bears no influence on the coordination sphere of the divalent tetrel element. In association with iPrOH, the amido germylenes, stannylenes and plumbylenes catalyse the controlled polymerisation of L- and racemic lactide. The activity increases linearly according to Ge(II) ≪ Sn(II) ≪ Pb(II). The simple germylenes generate very sluggish catalysts, but the activity is significantly boosted if the heterobimetallic complex {LO(3)}Ge(N(SiMe3)2)·LiOTf is used instead. On the other hand, with 10-25 equiv. of iPrOH, the plumbylenes afford highly active binary catalysts, converting 1000 or 5000 equiv. of monomer at 60 °C within 3 or 45 min, respectively, in a controlled fashion.

  4. Production of Monomeric Aromatic Compounds from Oil Palm Empty Fruit Bunch Fiber Lignin by Chemical and Enzymatic Methods

    Directory of Open Access Journals (Sweden)

    Pei-Ling Tang

    2015-01-01

    Full Text Available In this study, oil palm empty fruit bunch (OPEFBF was pretreated with alkali, and lignin was extracted for further degradation into lower molecular weight phenolic compounds using enzymes and chemical means. Efficiency of monomeric aromatic compounds production from OPEFBF lignin via chemical (nitrobenzene versus oxygen and enzymatic [cutinase versus manganese peroxidase (MnP] approaches was investigated. The effects of sodium hydroxide concentration (2, 5, and 10% wt. and reaction time (30, 90, and 180 minutes on the yield of aromatic compounds were studied. The results obtained indicated that nitrobenzene oxidation produced the highest yield (333.17±49.44 ppm hydroxybenzoic acid, 5.67±0.25 ppm p-hydroxybenzaldehyde, 25.57±1.64 ppm vanillic acid, 168.68±23.23 ppm vanillin, 75.44±6.71 ppm syringic acid, 815.26±41.77 ppm syringaldehyde, 15.21±2.19 ppm p-coumaric acid, and 44.75±3.40 ppm ferulic acid, among the tested methods. High sodium hydroxide concentration (10% wt. was needed to promote efficient nitrobenzene oxidation. However, less severe oxidation condition was preferred to preserve the hydroxycinnamic acids (p-coumaric acid and ferulic acid. Cutinase-catalyzed hydrolysis was found to be more efficient than MnP-catalyzed oxidation in the production of aromatic compounds. By hydrolyzed 8% wt. of lignin with 0.625 mL cutinase g−1 lignin at pH 8 and 55°C for 24 hours, about 642.83±14.45 ppm hydroxybenzoic acid, 70.19±3.31 ppm syringaldehyde, 22.80±1.04 ppm vanillin, 27.06±1.20 ppm p-coumaric acid, and 50.19±2.23 ppm ferulic acid were produced.

  5. Development and characterization of mouse monoclonal antibodies against monomeric dengue virus non-structural glycoprotein 1 (NS1).

    Science.gov (United States)

    Gelanew, Tesfaye; Poole-Smith, B Katherine; Hunsperger, Elizabeth

    2015-09-15

    Dengue virus (DENV) nonstructural-1 (NS1) glycoprotein is useful for diagnosis of DENV infections in the first 8 days of illness with any of the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). However, NS1 diagnostics are less sensitive for secondary DENV infections so the utility of NS1 diagnostics in dengue endemic countries where there is predominantly secondary infections is being questioned. Heat-mediated immunecomplex dissociation (ICD) prior to testing serum samples can significantly improve NS1 test sensitivity in secondary infections but requires monoclonal antibodies (MAbs) reactive to heat-denatured NS1. In order to incorporate a simple heat-mediated ICD step, a crucial step was to develop new MAbs with high affinity and specificity to heat-denatured DENV NS1 protein. In the present study, six new MAbs were isolated from BALB/c mice immunized with recombinant monomeric NS1 of DENV-1 and DENV-2. Characterization using three different methods: indirect ELISA, fixed cell ELISA and western blot revealed that all six MAbs are serotype-cross-reactive and capable of recognizing dimeric and hexameric isoforms as well as heat-denatured NS1 from all four DENV serotypes. No cross-reactivity to NS1 of West Nile virus and Yellow fever virus was observed on western blot and indirect ELISA. Five of the six MAbs mapped to the DENV NS1 region of 105-119 amino acids. The remaining MAb mapped to DENV NS1 region of 25-39 amino acids. These two NS1 regions were found to be highly conserved among all four DENV serotypes by sequences analysis and database comparison. These MAbs were used to develop an NS1 capture ELISA and tested using a small panel of clinical specimens. The results from the NS1 capture ELISA indicated at least a three-fold increase in NS1 antigen detection in heat-denatured samples compared to untreated specimens. Furthermore, artificial immunecomplexed results also demonstrated the binding efficiency of these MAbs to heat denatured NS1. Taken together

  6. Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate.

    Science.gov (United States)

    Stump, Matthew R; Gloss, Lisa M

    2008-02-01

    Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.

  7. Trapping of Vibrio cholerae cytolysin in the membrane-bound monomeric state blocks membrane insertion and functional pore formation by the toxin.

    Science.gov (United States)

    Rai, Anand Kumar; Chattopadhyay, Kausik

    2014-06-13

    Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytolytic toxin that belongs to the family of β barrel pore-forming protein toxins. VCC induces lysis of its target eukaryotic cells by forming transmembrane oligomeric β barrel pores. The mechanism of membrane pore formation by VCC follows the overall scheme of the archetypical β barrel pore-forming protein toxin mode of action, in which the water-soluble monomeric form of the toxin first binds to the target cell membrane, then assembles into a prepore oligomeric intermediate, and finally converts into the functional transmembrane oligomeric β barrel pore. However, there exists a vast knowledge gap in our understanding regarding the intricate details of the membrane pore formation process employed by VCC. In particular, the membrane oligomerization and membrane insertion steps of the process have only been described to a limited extent. In this study, we determined the key residues in VCC that are critical to trigger membrane oligomerization of the toxin. Alteration of such key residues traps the toxin in its membrane-bound monomeric state and abrogates subsequent oligomerization, membrane insertion, and functional transmembrane pore-formation events. The results obtained from our study also suggest that the membrane insertion of VCC depends critically on the oligomerization process and that it cannot be initiated in the membrane-bound monomeric form of the toxin. In sum, our study, for the first time, dissects membrane binding from the subsequent oligomerization and membrane insertion steps and, thus, defines the exact sequence of events in the membrane pore formation process by VCC.

  8. The impact of single nucleotide polymorphism in monomeric alpha-amylase inhibitor genes from wild emmer wheat, primarily from Israel and Golan

    Directory of Open Access Journals (Sweden)

    Yan Ze-Hong

    2010-06-01

    Full Text Available Abstract Background Various enzyme inhibitors act on key insect gut digestive hydrolases, including alpha-amylases and proteinases. Alpha-amylase inhibitors have been widely investigated for their possible use in strengthening a plant's defense against insects that are highly dependent on starch as an energy source. We attempted to unravel the diversity of monomeric alpha-amylase inhibitor genes of Israeli and Golan Heights' wild emmer wheat with different ecological factors (e.g., geography, water, and temperature. Population methods that analyze the nature and frequency of allele diversity within a species and the codon analysis method (comparing patterns of synonymous and non-synonymous changes in protein coding sequences were used to detect natural selection. Results Three hundred and forty-eight sequences encoding monomeric alpha-amylase inhibitors (WMAI were obtained from 14 populations of wild emmer wheat. The frequency of SNPs in WMAI genes was 1 out of 16.3 bases, where 28 SNPs were detected in the coding sequence. The results of purifying and the positive selection hypothesis (p Conclusions Great diversity at the WMAI locus, both between and within populations, was detected in the populations of wild emmer wheat. It was revealed that WMAI were naturally selected for across populations by a ratio of dN/dS as expected. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs. A sharp genetic divergence over very short geographic distances compared to a small genetic divergence between large geographic distances also suggested that the SNPs were subjected to natural selection, and ecological factors had an important evolutionary role in polymorphisms at this locus. According to population and codon analysis, these results suggested that monomeric alpha-amylase inhibitors are adaptively selected under different environmental conditions.

  9. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

    Energy Technology Data Exchange (ETDEWEB)

    Al, Hui-wang; Henderson, J. Nathan; Remington, S. James; Campbell, Robert E. (Alberta); (Oregon)

    2008-05-07

    The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 {angstrom} crystal structure (1 {angstrom}=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.

  10. Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins

    DEFF Research Database (Denmark)

    Andreasen, Mette Findal; Landbo, A K; Christensen, L P

    2001-01-01

    Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were...... investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4-diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid...

  11. Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins

    DEFF Research Database (Denmark)

    Andreasen, M.F.; Landbo, Anne-Katrine Regel; Christensen, L.P.

    2001-01-01

    Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were...... investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4- diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid...

  12. Absorption spectrum of monomeric pseudoisocyanine: A new perspective and its implications for formation and spectral response of J-aggregates in solution and in thin films

    Energy Technology Data Exchange (ETDEWEB)

    Guelen, Demet [Physics Department, Middle East Technical University (METU), 06531 Ankara (Turkey)], E-mail: dgul@metu.edu.tr; Ozcelik, Serdar [Chemistry Department, Izmir Institute of Technology, Urla 35430, Izmir (Turkey)

    2008-05-15

    We argued against the current spectral assignment for absorption spectrum of monomeric PIC which is widely accepted since the pioneering works of Scheibe and Jelley [G. Scheibe, Angew. Chem. 49 (1936) 563; E.E. Jelly, Nature 138 (1936) 1009]. A new spectrum is presented along with its conceptual basis. The hypothesized spectrum attributes the previous 0-0 ({approx}525 nm) and 0-1 ({approx}490 nm) assignments, respectively, to intermediates acting as the precursor of J-aggregates and to the 0-0 transition of monomeric PIC and brings the spectrum in accord with the seemingly universal spectral fingerprint of cyanines. The hypothesis is used to analyze and interpret the temperature dependence of the UV-vis absorption of PIC aggregates in saline aqueous solution by incorporating the J-band simulations within frenkel exciton formalism. Its implications for aggregate formation kinetics are given on the basis of current spectroscopic evidence. The hypothesis readily answers several long-standing questions: Why compared to many other cyanines at least an order of magnitude higher dye concentration is needed to form J-aggregates of PIC? Why are there no precursors, since aggregation is expected to be a consecutive process? A large number of observations on steady-state and time-resolved spectral properties, and aggregation kinetics in solution/thin films are likely to find reasonable explanations within this hypothesis.

  13. Binding of the monomeric form of C-reactive protein to enzymatically-modified low-density lipoprotein: effects of phosphoethanolamine

    Science.gov (United States)

    Singh, Sanjay K.; Suresh, Madathilparambil V.; Hammond, David J.; Rusiñol, Antonio E.; Potempa, Lawrence A.; Agrawal, Alok

    2009-01-01

    Background The 5 subunits of native pentameric C-reactive protein (CRP) are dissociated to generate monomeric form of CRP (mCRP) in some in vitro conditions, both physiological and non-physiological, and also in vivo. Many bioactivities of mCRP generated by urea-treatment of CRP and of mCRP generated by mutating the primary structure of CRP have been reported. The bioactivities of mCRP generated by spontaneous dissociation of CRP are largely unexplored. Methods We purified mCRP generated by spontaneous dissociation of CRP and investigated the binding of mCRP to enzymatically-modified low-density lipoprotein (E-LDL). Results mCRP was approximately 60 times more potent than CRP in binding to E-LDL. In the presence of the small-molecule compound phosphoethanolamine (PEt), at 37°C, the binding of mCRP to E-LDL was enhanced 10-fold. In contrast, PEt inhibited the binding of both CRP and mCRP to pneumococcal C-polysaccharide, another phosphocholine-containing ligand to which CRP and mCRP were found to bind. We have not investigated yet whether PEt alters the structure of CRP at 37°C. Conclusions Combined data suggest that the targeting of CRP with the aim to monomerize CRP in vivo may be an effective approach to capture modified forms of LDL. PMID:19545552

  14. RFP for the italien satellite AGILE

    DEFF Research Database (Denmark)

    Madsen, Peter Buch; Jørgensen, John Leif; Riis, Troels

    1999-01-01

    The document descibes the ASC Star Tracker (performance, functionality, requirements etc.) to the Italian satellite AGILE.......The document descibes the ASC Star Tracker (performance, functionality, requirements etc.) to the Italian satellite AGILE....

  15. RFP for the Comet Nuclei Tour (CONTOUR)

    DEFF Research Database (Denmark)

    Jørgensen, John Leif; Madsen, Peter Buch; Betto, Maurizio

    1999-01-01

    This document describes the ASC Star Tracker (performance, functionality, requirements etc.) to The Johns Hopkins University - Applied Physics Laboratory for their Comet Nuclei TOUR (CONTOUR) Program.......This document describes the ASC Star Tracker (performance, functionality, requirements etc.) to The Johns Hopkins University - Applied Physics Laboratory for their Comet Nuclei TOUR (CONTOUR) Program....

  16. RFP for CNES micro satellite program

    DEFF Research Database (Denmark)

    Jørgensen, John Leif; Jørgensen, Finn E; Betto, Maurizio

    1999-01-01

    This document descibes the ASC STAR TRACKER (performance, functionality, requirements etc.) to the Centre National d'Etudes Spatiales (CNES) for their micro Satellites.......This document descibes the ASC STAR TRACKER (performance, functionality, requirements etc.) to the Centre National d'Etudes Spatiales (CNES) for their micro Satellites....

  17. Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry.

    Science.gov (United States)

    Moss, Ryan; Mao, Qinyong; Taylor, Dennis; Saucier, Cédric

    2013-08-30

    Stilbenoids are secondary plant metabolites responsible for the protection of multiple plant species including grape vine from bacterial and fungal infection. Red wine has been shown to be a major source of these compounds in the human diet, where they display an array of health benefits. Providing a more complete profile of the stilbenoids present in red wine, this study detects 41 stilbenoid compounds, 23 of which have never before been detected in red wine. Red wine extracts were scanned using an ultra-high-performance liquid chromatograph coupled to a hybrid quadrupole time-of-flight mass analyzer. Multiple targeted MS/MS precursor ion scan experiments were performed using electrospray ionization operated in negative mode. Precursor ion masses were scanned for the monomeric and oligomeric stilbenoids, as well as modifications such as O-glycosylation, methoxylation and oxidation products of these compounds. Accurate mass precursor and characteristic product ions afforded partial structural elucidation and assignment of these compounds. A total of 41 (both known and novel) stilbenoids were detected in extracted red wine. In addition to the well-known monomeric stilbenes, several resveratrol-resveratrol homodimers (m/z 453.1344), resveratrol-piceatannol heterodimers (m/z 469.1293) and piceatannol-piceatannol homodimers (m/z 485.1236) were detected. Modified dimers of resveratrol including O-glycosylated (m/z 615.1872), methoxylated (m/z 485.1606) and oxidized (m/z 471.1449) dimers were also detected. Multiple trimers of resveratrol (m/z 679.1978) were detected for the first time in red wine, as well as some known and some novel stilbenoid tetramers (m/z 905.2604). In summary, 41 stilbenoids were detected in red wine, 23 for the first time. Both monomeric and oligomeric stilbenoids were partially identified and assigned by their accurate mass precursor ions and characteristic stilbenoid fragmentation patterns. Knowledge gained from these experiments contributes to

  18. Evaluating the effect of wood ultrastructural changes from mechanical treatment on kinetics of monomeric sugars and chemicals production in acid bisulfite treatment.

    Science.gov (United States)

    Liu, Yalan; Wang, Jinwu; Wolcott, Michael P

    2017-02-01

    Currently, various chemical-mechanical treatments were widely used in biofuel production to achieve high total sugar yields. However, the interaction between two treatments was scarcely investigated. In this study, we employed a ball milling process to create ultrastructural changes for Douglas-fir (Pseudotsuga menziesii) micronized wood powders. The 0, 30, and 60min ball milled wood powders resulted in a crystallinity index of 0.41, 0.21, and 0.10 respectively. It was found that the ultrastructural changes accelerate monomeric sugars production without influencing the yield of sugar degradation products. The optimal acid bisulfite treatment time was substantially decreased from 120min to 40min as the cellulose crystallinity decreased. Meanwhile, total sugar yield increased from 65% to 92% and had a linear relation with a decrease of the cellulose crystallinity.

  19. Corrosion inhibition of aluminum with a series of aniline monomeric surfactants and their analog polymers in 0.5 M HCl solution

    Directory of Open Access Journals (Sweden)

    M.M. El-Deeb

    2015-07-01

    Full Text Available The inhibition effect of 3-(12-sodiumsulfonate dodecyloxy aniline monomeric surfactant (MC12 and its analog polymer Poly 3-(dodecyloxy sulfonic acid aniline (PC12 on the corrosion of aluminum in 0.5 M HCl solution was investigated using weight loss and potentiodynamic polarization techniques. The presence of these two compounds in 0.5 M HCl inhibits the corrosion of aluminum without modifying the mechanism of corrosion process. It was found that these inhibitors act as mixed-type inhibitors with anodic predominance as well as the inhibition efficiency increases with increasing inhibitor concentration, but decreases with raising temperature. Langmuir and Frumkin adsorption isotherms fit well with the experimental data. Thermodynamic functions for both dissolution and adsorption processes were determined. The obtained results from weight loss and potentiodynamic polarization techniques are in good agreement with contact angle measurements.

  20. Plant small monomeric G-proteins (RAC/ROPs) of barley are common elements of susceptibility to fungal leaf pathogens, cell expansion and stomata development.

    Science.gov (United States)

    Pathuri, Indira Priyadarshini; Eichmann, Ruth; Hückelhoven, Ralph

    2009-02-01

    Small monomeric RAC/ROP GTPases act as molecular switches in signal transduction processes of plant development and stress responses. They emerged as crucial players in plant-pathogen interactions either by supporting susceptibility or resistance. In a recent publication, we showed that constitutively activated (CA) mutants of different barley (Hordeum vulgare) RAC/ROPs regulate susceptibility to barley fungal leaf pathogens of different life style in a contrasting way. This illustrates the distinctive signalling roles of RAC/ROPs for different plant-pathogen combinations. We also reported the involvement of RAC/ROPs in plant epidermis development in a monocotyledonous plant. Here we further discuss a failure of CA HvRAC/ROP-expressing barley to normally develop stomata.

  1. PENGARUH PENGOLAHAN PANAS TERHADAP KONSENTRASI ANTOSIANIN MONOMERIK UBI JALAR UNGU (Ipomoea batatas L (Efect of Heat Processing on Monomeric Anthocyanin of Purple Sweet Potato (Ipomoea batatas L

    Directory of Open Access Journals (Sweden)

    Ai Mahmudatussa'adah

    2015-09-01

    content in PSP during heat processing of flakes. The other purpose of this reseach was to observe the order kinetics model of effect temperature and time baking on total anthocyanin monomeric of fresh PSP and rehydration PSP flakes. The experimental applied a completely randomized design with three replications. The color and amount of anthocyanin (L * = 23.38 ± 0.71, C = 9.84 ± 0.98, Hue = 12.25 ± 1.61. Total monomeric anthocyanin in fresh PSP was 1.45 ± 0.00 mg cyanidin equivalent (CyE/g dry basis (db. In general, the color and the amount of PSP anthocyanin changed during the flakes processing. Steamed PSP for 7 minutes turned its color into a bright purple (L * = 25.88 ± 0.47, C = 24.64 ± 0.25, Hue = 348.83 ± 0.33 with the amount of monomeric anthocyanin increased to 3.76 ± 0.01 CyE mg/g db. Flakes PSP was very bright purple (L * = 36.12 ± 0.11, C = 9.97 ± 0.18, Hue = 359.29 ± 0.31 and the amount of monomericanthocyanin was slightly lower than that of steamed sweet potato (3.19 ± 0.12 mg CyE / g db. Total monomeric anthocyanin of fresh PSP and rehydration flakes PSP decrease during baking time. Keywords: Color, anthocyanin, purple sweet potato, flakes, degradation kinetics   ABSTRAK Antosianin merupakan salah satu kelompok zat warna alami yang terdapat pada tanaman, seperti daun, bunga, umbi, buah atau sayur. Salah satu sumber antosianin pada tanaman adalah ubi jalar ungu (UJU yang mengandung lebih dari 98% antosianin terasilasi dari konsentrasi antosianin umbi. Warna antosianin bervariasi mulai dari merah, ungu, biru, sampai kuning. Warna dan konsentrasi antosianin dapat berubah karena pengaruh panas. Penelitian ini bertujuan untuk mempelajari perubahan warna dan konsentrasi antosianin monomerik sebagai akibat proses pengolahan dalam pembuatan  UJU. Penelitian ini mengkaji juga mengenai model kinetika reaksi pengaruh suhu dan waktu panggang terhadap konsentrasi antosianin UJU segar dan  rehidrasi. Penelitian menggunakan rancangan acak lengkap

  2. Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins.

    Science.gov (United States)

    Gioannini, Theresa L; Teghanemt, Athmane; Zhang, DeSheng; Esparza, Gregory; Yu, Liping; Weiss, Jerrold

    2014-08-01

    A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

  3. Optimization of ultrasound-assisted extraction of total monomeric anthocyanin (TMA) and total phenolic content (TPC) from eggplant (Solanum melongena L.) peel.

    Science.gov (United States)

    Dranca, Florina; Oroian, Mircea

    2016-07-01

    The present study describes the extraction of total monomeric anthocyanin (TMA) and total phenolic content (TPC) from eggplant peel using ultrasonic treatments and methanol and 2-propanol as extraction solvents. The extraction yields were optimized by varying the solvent concentration, ultrasonic frequency, temperature and time of ultrasonic treatment. Box-Behnken design was used to investigate the effect of process variables on the ultrasound-assisted extraction. The results showed that for TPC extraction the optimal condition were obtained with a methanol concentration of 76.6%, 33.88 kHz ultrasonic frequency, a temperature of 69.4 °C and 57.5 min extraction time. For TMA the optimal condition were the following: 54.4% methanol concentration, 37 kHz, 55.1 °C and process time of 44.85 min. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis.

    Science.gov (United States)

    García-García, Jorge D; Girard, Lourdes; Hernández, Georgina; Saavedra, Emma; Pardo, Juan P; Rodríguez-Zavala, José S; Encalada, Rusely; Reyes-Prieto, Adrián; Mendoza-Cózatl, David G; Moreno-Sánchez, Rafael

    2014-03-01

    The phytochelatin synthase from photosynthetic Euglena gracilis (EgPCS) was analyzed at the transcriptional, kinetic, functional, and phylogenetic levels. Recombinant EgPCS was a monomeric enzyme able to synthesize, in the presence of Zn(2+) or Cd(2+), phytochelatin2-phytochelatin4 (PC2-PC4) using GSH or S-methyl-GS (S-methyl-glutathione), but not γ-glutamylcysteine or PC2 as a substrate. Kinetic analysis of EgPCS firmly established a two-substrate reaction mechanism for PC2 synthesis with Km values of 14-22 mM for GSH and 1.6-2.5 μM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and catalytic efficiency with Zn-(GS)2, and was inactivated by peroxides. The EgPCS N-terminal domain showed high similarity to that of other PCSases, in which the typical catalytic core (Cys-70, His-179 and Asp-197) was identified. In contrast, the C-terminal domain showed no similarity to other PCSases. An EgPCS mutant comprising only the N-terminal 235 amino acid residues was inactive, suggesting that the C-terminal domain is essential for activity/stability. EgPCS transcription in Euglena cells was not modified by Cd(2+), whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd(2+) stress. Phylogenetic analysis of the N-terminal domain showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. Although EgPCS showed typical features of PCSases (constitutive expression; conserved N-terminal domain; kinetic mechanism), it also exhibited distinct characteristics such as preference for Zn-(GS)2 over Cd-(GS)2 as a co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs, which may be involved in conferring enhanced heavy-metal resistance.

  5. Resolution of two native monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, Philippa J.L. [School of Chemistry, University of Sydney, NSW 2006 (Australia); McKinzie, Audra A. [School of Medical Sciences (Pharmacology) and Bosch Institute, University of Sydney, NSW 2006 (Australia); Codd, Rachel, E-mail: rachel.codd@sydney.edu.au [School of Chemistry, University of Sydney, NSW 2006 (Australia); School of Medical Sciences (Pharmacology) and Bosch Institute, University of Sydney, NSW 2006 (Australia)

    2010-07-16

    Research highlights: {yields} Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. {yields} Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. {yields} Isolation of NAP as NapA or NapAB correlated with NapA P47E amino acid substitution. -- Abstract: The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90 kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90 kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90 kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90 kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  6. Salt anions promote the conversion of HypF-N into amyloid-like oligomers and modulate the structure of the oligomers and the monomeric precursor state.

    Science.gov (United States)

    Campioni, Silvia; Mannini, Benedetta; López-Alonso, Jorge P; Shalova, Irina N; Penco, Amanda; Mulvihill, Estefania; Laurents, Douglas V; Relini, Annalisa; Chiti, Fabrizio

    2012-12-07

    An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichiacoli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO(4), NaI, Na(2)SO(4)) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO(4)(2-)≥ ClO(4)(-)>I(-)>Cl(-)). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes.

  7. Calcitonina monomérica plasmática e hipercalcemia em pacientes portadores de neoplasia pulmonar Monomeric plasmatic calcitonin and hypercalcemia in lung cancer patients

    Directory of Open Access Journals (Sweden)

    R. Coifman

    1997-06-01

    lung cancer, and its correlation with hypercalcemia, a very common complication in these tumors. METHOD. Blood were sampled from 56 patients with malignant lung disease for the CT and ionized calcium determinations. Calcitonin was measured using a specific radioimmunoassay for the monomeric form of the molecule, in a previous silica extracted serum probe. RESULTS. We did not find elevated levels of monomeric CT in lung cancer. Only 3 patients had mild elevated levels, while in the others CT was normal or undetectable. Hypercalcemia was found in 21.4% of these patients, but only one with supranormal CT levels. CONCLUSION. Monomeric CT serum levels are normal in lung cancer, what makes the latter use an unreliable tumor marker.

  8. The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution

    Energy Technology Data Exchange (ETDEWEB)

    Sarowar, Samema; Hu, Olivia J.; Werneburg, Glenn T.; Thanassi, David G.; Li, Huilin; Christie, P. J.

    2016-06-27

    ABSTRACT

    The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains ofEscherichia coliuse this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 β-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function.

    IMPORTANCEPili are critical virulence factors for many bacterial pathogens. UropathogenicE. colirelies on P and type 1 pili assembled by the chaperone/usher pathway to

  9. Folded-back solution structure of monomeric factor H of human complement by synchrotron X-ray and neutron scattering, analytical ultracentrifugation and constrained molecular modelling.

    Science.gov (United States)

    Aslam, M; Perkins, S J

    2001-06-22

    Factor H (FH) is a regulatory cofactor for the protease factor I in the breakdown of C3b in the complement system of immune defence, and binds to heparin and other polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains, for which the overall arrangement in solution is unknown. As previous studies had shown that FH can form monomeric or dimeric structures, X-ray and neutron scattering was accordingly performed with FH in the concentration range between 0.7 and 14 mg ml(-1). The radius of gyration of FH was determined to be 11.1-11.3 nm by both methods, and the radii of gyration of the cross-section were 4.4 nm and 1.7 nm. The distance distribution function P(r) showed that the overall length of FH was 38 nm. The neutron data showed that FH was monomeric with a molecular mass of 165,000(+/-17,000) Da. Analytical ultracentrifugation data confirmed this, where sedimentation equilibrium curve fits gave a mean molecular mass of 155,000(+/-3,000) Da. Sedimentation velocity experiments using the g*(s) derivative method showed that FH was monodisperse and had a sedimentation coefficient of 5.3(+/-0.1) S. In order to construct a full model of FH for scattering curve and sedimentation coefficient fits, homology models were constructed for 17 of the 20 SCR domains using knowledge of the NMR structures for FH SCR-5, SCR-15 and SCR-16, and vaccinia coat protein SCR-3 and SCR-4. Molecular dynamics simulations were used to generate a large conformational library for each of the 19 SCR-SCR linker peptides. Peptides from these libraries were combined with the 20 SCR structures in order to generate stereochemically complete models for the FH structure. Using an automated constrained fit procedure, the analysis of 16,752 possible FH models showed that only those models in which the 20 SCR domains were bent back upon themselves were able to account for the scattering and sedimentation data. The best-fit models showed that FH had an overall length

  10. Salt bridges regulate both dimer formation and monomeric flexibility in HdeB and may have a role in periplasmic chaperone function.

    Science.gov (United States)

    Wang, Wenjian; Rasmussen, Tim; Harding, Amanda J; Booth, Nuala A; Booth, Ian R; Naismith, James H

    2012-01-20

    Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility.

  11. Salt Bridges Regulate Both Dimer Formation and Monomeric Flexibility in HdeB and May Have a Role in Periplasmic Chaperone Function

    Science.gov (United States)

    Wang, Wenjian; Rasmussen, Tim; Harding, Amanda J.; Booth, Nuala A.; Booth, Ian R.; Naismith, James H.

    2012-01-01

    Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility. PMID:22138344

  12. The sigma-1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands

    Science.gov (United States)

    Singh, Deo R.; Biener, Gabriel; Yang, Jay; Oliver, Julie A.; Ruoho, Arnold; Raicu, Valerică

    2015-01-01

    The sigma-1 receptor (S1R) is a 223-amino-acid membrane protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by various synthetic molecules including (+)-pentazocine, cocaine and haloperidol and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand-regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion channels and G-protein coupled receptors (GPCRs). When S1R constructs containing C-terminal monomeric GFP2 and YFP fusions were co-expressed in COS-7 cells and subjected to FRET spectrometry analysis, monomers, dimers and higher oligomeric forms of S1R were identified under non-liganded conditions. In the presence of the prototypic S1R agonist, (+)-pentazocine, however, monomers and dimers were the prevailing forms of S1R. The prototypic antagonist, haloperidol, on the other hand, favoured higher order S1R oligomers. These data, in sum, indicate that heterologously expressed S1Rs occur in vivo in COS-7 cells in multiple oligomeric forms and that S1R ligands alter these oligomeric structures. We suggest that the S1R oligomerization states may regulate its function(s). PMID:25510962

  13. Structural and enzymatic insights into Lambda glutathione transferases from Populus trichocarpa, monomeric enzymes constituting an early divergent class specific to terrestrial plants.

    Science.gov (United States)

    Lallement, Pierre-Alexandre; Meux, Edgar; Gualberto, José M; Prosper, Pascalita; Didierjean, Claude; Saul, Frederick; Haouz, Ahmed; Rouhier, Nicolas; Hecker, Arnaud

    2014-08-15

    GSTs represent a superfamily of multifunctional proteins which play crucial roles in detoxification processes and secondary metabolism. Instead of promoting the conjugation of glutathione to acceptor molecules as do most GSTs, members of the Lambda class (GSTLs) catalyse deglutathionylation reactions via a catalytic cysteine residue. Three GSTL genes (Pt-GSTL1, Pt-GSTL2 and Pt-GSTL3) are present in Populus trichocarpa, but two transcripts, differing in their 5' extremities, were identified for Pt-GSTL3. Transcripts for these genes were primarily found in flowers, fruits, petioles and buds, but not in leaves and roots, suggesting roles associated with secondary metabolism in these organs. The expression of GFP-fusion proteins in tobacco showed that Pt-GSTL1 is localized in plastids, whereas Pt-GSTL2 and Pt-GSTL3A and Pt-GSTL3B are found in both the cytoplasm and the nucleus. The resolution of Pt-GSTL1 and Pt-GSTL3 structures by X-ray crystallography indicated that, although these proteins adopt a canonical GST fold quite similar to that found in dimeric Omega GSTs, their non-plant counterparts, they are strictly monomeric. This might explain some differences in the enzymatic properties of both enzyme types. Finally, from competition experiments between aromatic substrates and a fluorescent probe, we determined that the recognition of glutathionylated substrates is favoured over non-glutathionylated forms.

  14. Solvent exposure of Tyr10 as a probe of structural differences between monomeric and aggregated forms of the amyloid-β peptide

    Science.gov (United States)

    Aran Terol, Pablo; Kumita, Janet R.; Hook, Sharon C.; Dobson, Christopher M.; Esbjörner, Elin K.

    2015-01-01

    Aggregation of amyloid-β (Aβ) peptides is a characteristic pathological feature of Alzheimer's disease. We have exploited the relationship between solvent exposure and intrinsic fluorescence of a single tyrosine residue, Tyr10, in the Aβ sequence to probe structural features of the monomeric, oligomeric and fibrillar forms of the 42-residue Aβ1-42. By monitoring the quenching of Tyr10 fluorescence upon addition of water-soluble acrylamide, we show that in Aβ1-42 oligomers this residue is solvent-exposed to a similar extent to that found in the unfolded monomer. By contrast, Tyr10 is significantly shielded from acrylamide quenching in Aβ1-42 fibrils, consistent with its proximity to the fibrillar cross-β core. Furthermore, circular dichroism measurements reveal that Aβ1-42 oligomers have a considerably lower β-sheet content than the Aβ1-42 fibrils, indicative of a less ordered molecular arrangement in the former. Taken together these findings suggest significant differences in the structural assembly of oligomers and fibrils that are consistent with differences in their biological effects. PMID:26551456

  15. Novel DDR Processing of Corn Stover Achieves High Monomeric Sugar Concentrations from Enzymatic Hydrolysis (230 g/L) and High Ethanol Concentration (10% v/v) During Fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiaowen; Jennings, Ed; Shekiro, Joe; Kuhn, Erik M.; O' Brien, Marykate; Wang, Wei; Schell, Daniel J.; Himmel, Mike; Elander, Richard T.; Tucker, Melvin P.

    2015-04-03

    Distilling and purifying ethanol, butanol, and other products from second and later generation lignocellulosic biorefineries adds significant capital and operating cost for biofuels production. The energy costs associated with distillation affects plant gate and life cycle analysis costs. Lower titers in fermentation due to lower sugar concentrations from pretreatment increase both energy and production costs. In addition, higher titers decrease the volumes required for enzymatic hydrolysis and fermentation vessels. Therefore, increasing biofuels titers has been a research focus in renewable biofuels production for several decades. In this work, we achieved over 200 g/L of monomeric sugars after high solids enzymatic hydrolysis using the novel deacetylation and disc refining (DDR) process on corn stover. The high sugar concentrations and low chemical inhibitor concentrations from the DDR process allowed ethanol titers as high as 82 g/L in 22 hours, which translates into approximately 10 vol% ethanol. To our knowledge, this is the first time that 10 vol% ethanol in fermentation derived from corn stover without any sugar concentration or purification steps has been reported. Techno-economic analysis shows the higher titer ethanol achieved from the DDR process could significantly reduce the minimum ethanol selling price from cellulosic biomass.

  16. Analysis of amino acid residues involved in cold activity of monomeric isocitrate dehydrogenase from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea.

    Science.gov (United States)

    Yasuda, Wataru; Kobayashi, Miyuki; Takada, Yasuhiro

    2013-11-01

    Monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH-II and CpIDH-M, respectively) are cold-adapted enzymes and show a high degree of amino acid sequential identity to each other (77%). However, maximum activity of CpIDH-M at optimum temperature is much less than that of CmIDH-II. In the C-terminal region 3 of these enzymes, which was suggested from previous study to be responsible for their distinct catalytic ability, several sequential differences of amino acid residue are present. Among them, ten amino acid residues were exchanged between them by site-directed mutagenesis and several properties of the mutated enzymes were examined in this study. The mutated enzymes of CmIDH-II substituted its Gln671, Leu724 and Phe735 residues with the corresponding residues of CpIDH-M (termed Q671K, L724Q and F735L, respectively) showed lower specific activity and thermostability for activity than the wild-type enzyme. Furthermore, the decreased specific activity was also observed in L693F. In contrast, the corresponding mutants of CpIDH-M, F693L, Q724L and L735F, showed the increased specific activity and thermostability for activity. The catalytic efficiency (k(cat)/K(m)) values of these mutated CmIDH-II and CpIDH-M were lower and higher than those of their wild-type IDHs, respectively. These results suggest that the Gln671, Leu693, Leu724 and Phe735 residues of CmIDH-II are important for exerting its high catalytic ability.

  17. Effect of the disulfide bond on the monomeric structure of human amylin studied by combined Hamiltonian and temperature replica exchange molecular dynamics simulations.

    Science.gov (United States)

    Laghaei, Rozita; Mousseau, Normand; Wei, Guanghong

    2010-05-27

    The human Islet amyloid polypeptide (hIAPP or amylin) is a 37-residue peptide hormone that is normally cosecreted with insulin by the pancreatic beta-cells. In patients with type 2 diabetes, hIAPP deposits as amyloid fibrils in the extracellular spaces of the pancreatic islets. Recent experimental studies show that the intramolecular disulfide bond between Cys2 and Cys7 plays a central role in the process of fibril formation. However, the effect of the disulfide bond on the intrinsic structural properties of monomeric hIAPP is yet to be determined. In this study, we characterize the atomic structure and the thermodynamics of full-length hIAPP in the presence and absence of a disulfide bond using extensive combined Hamiltonian and temperature replica exchange molecular dynamics simulations (HT-REMD) with a coarse grained protein force field. Our simulations show that HT-REMD is more efficient in sampling than temperature REMD. On the basis of a total simulation time of 28 mus, we find that, although native hIAPP (in the presence of a disulfide bond) essentially adopts a disordered conformation in solution, consistent with the signal measured by ultraviolet-circular dichroism (UV-CD) spectroscopy, it also transiently samples alpha-helical structure for residues 5-16. In comparison with the N-terminal region, the C-terminal region is highly disordered and populates a much lesser content of isolated beta-strand conformation for residues 22-26 and 30-35. Moreover, the absence of the disulfide bond greatly decreases the extent of helix formed throughout residues 5-9 in favor of random coil and beta-sheet structure. Implications of the stabilization of N-terminal helical structure by disulfide bond on the initialization of hIAPP amyloid formation are discussed.

  18. Monomeric Immunoglobulin A from Plasma Inhibits Human Th17 Responses In Vitro Independent of FcαRI and DC-SIGN

    Science.gov (United States)

    Saha, Chaitrali; Das, Mrinmoy; Patil, Veerupaxagouda; Stephen-Victor, Emmanuel; Sharma, Meenu; Wymann, Sandra; Jordi, Monika; Vonarburg, Cédric; Kaveri, Srini V.; Bayry, Jagadeesh

    2017-01-01

    Circulating immunoglobulins including immunoglobulin G (IgG) and IgM play a critical role in the immune homeostasis by modulating functions of immune cells. These functions are mediated in part by natural antibodies. However, despite being second most abundant antibody in the circulation, the immunoregulatory function of IgA is relatively unexplored. As Th17 cells are the key mediators of a variety of autoimmune, inflammatory, and allergic diseases, we investigated the ability of monomeric IgA (mIgA) isolated from pooled plasma of healthy donors to modulate human Th17 cells. We show that mIgA inhibits differentiation and amplification of human Th17 cells and the production of their effector cytokine IL-17A. mIgA also suppresses IFN-γ responses under these experimental conditions. Suppressive effect of mIgA on Th17 responses is associated with reciprocal expansion of FoxP3-positive regulatory T cells. The effect of mIgA on Th17 cells is dependent on F(ab′)2 fragments and independent of FcαRI (CD89) and DC-SIGN. Mechanistically, the modulatory effect of mIgA on Th17 cells implicates suppression of phosphorylation of signal transducer and activator of transcription 3. Furthermore, mIgA binds to CD4+ T cells and recognizes in a dose-dependent manner the receptors for cytokines (IL-6Rα and IL-1RI) that mediate Th17 responses. Our findings thus reveal novel anti-inflammatory functions of IgA and suggest potential therapeutic utility of mIgA in autoimmune and inflammatory diseases that implicate Th17 cells. PMID:28352269

  19. Monomeric TonB and the Ton box are required for the Formation of a High-Affinity Transporter-TonB Complex†

    Science.gov (United States)

    Freed, Daniel M.; Lukasik, Stephen M.; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S.

    2013-01-01

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In the present study, a soluble construct of Escherichia coli TonB (residues 33–239) was used to determine the affinity of TonB to the outer membrane transporters BtuB, FecA and FhuA. Using fluorescence anisotropy, TonB(33–239) was found to bind with high-affinity (tens of nM) to both BtuB and FhuA; however, no high-affinity binding was observed to FecA. In BtuB, the high affinity binding of TonB(33–239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33–239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33–239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When bound to the outer membrane transporter, DEER shows that the TonB(33–239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle. PMID:23517233

  20. Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

    Science.gov (United States)

    Freed, Daniel M; Lukasik, Stephen M; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S

    2013-04-16

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.

  1. Resolution of two native monomeric 90kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes.

    Science.gov (United States)

    Simpson, Philippa J L; McKinzie, Audra A; Codd, Rachel

    2010-07-16

    The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  2. X-ray Crystal Structures of Monomeric and Dimeric Peptide Inhibitors in Complex with the Human Neonatal Fc Receptor, FcRn

    Energy Technology Data Exchange (ETDEWEB)

    Mezo, Adam R.; Sridhar, Vandana; Badger, John; Sakorafas, Paul; Nienaber, Vicki (Zenobia); (Biogen)

    2010-10-28

    The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337-2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 {angstrom} resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.

  3. No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids.

    Science.gov (United States)

    Permyakov, Serge E; Pershikova, Irina V; Khokhlova, Tatyana I; Uversky, Vladimir N; Permyakov, Eugene A

    2004-05-18

    The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.

  4. The photophysics of monomeric bacteriochlorophylls c and d and their derivatives: properties of the triplet state and singlet oxygen photogeneration and quenching

    Science.gov (United States)

    Krasnovsky, A. A. Jr; Cheng, P.; Blankenship, R. E.; Moore, T. A.; Gust, D.

    1993-01-01

    Measurements of pigment triplet-triplet absorption, pigment phosphorescence and photosensitized singlet oxygen luminescence were carried out on solutions containing monomeric bacteriochlorophylls (Bchl) c and d, isolated from green photosynthetic bacteria, and their magnesium-free and farnesyl-free analogs. The energies of the pigment triplet states fell in the range 1.29-1.34 eV. The triplet lifetimes in aerobic solutions were 200-250 ns; they increased to 280 +/- 70 microseconds after nitrogen purging in liquid solutions and to 0.7-2.1 ms in a solid matrix at ambient or liquid nitrogen temperatures. Rate constants for quenching of the pigment triplet state by oxygen were (2.0-2.5) x 10(9) M-1 s-1, which is close to 1/9 of the rate constant for diffusion-controlled reactions. This quenching was accompanied by singlet oxygen formation. The quantum yields for the triplet state formation and singlet oxygen production were 55-75% in air-saturated solutions. Singlet oxygen quenching by ground-state pigment molecules was observed. Quenching was the most efficient for magnesium-containing pigments, kq = (0.31-1.2) x 10(9) M-1 s-1. It is caused mainly by a physical process of singlet oxygen (1O2) deactivation. Thus, Bchl c and d and their derivatives, as well as chlorophyll and Bchl a, combine a high efficiency of singlet oxygen production with the ability to protect photochemical and photobiological systems against damage by singlet oxygen.

  5. Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

    Directory of Open Access Journals (Sweden)

    Davidson Michael W

    2008-03-01

    Full Text Available Abstract Background In the 15 years that have passed since the cloning of Aequorea victoria green fluorescent protein (avGFP, the expanding set of fluorescent protein (FP variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1, a bright and photostable FP derived from Clavularia cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra. Results Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His. Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP. To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins. Conclusion Based on the results of our

  6. Diffusion of magnetic field lines in a confined RFP plasma

    Energy Technology Data Exchange (ETDEWEB)

    Bazzani, A. [Bologna Univ. (Italy). Dip. di Fisica; INFN, Istituto Nazionale di Fisica Nucleare, Bologna (Italy); Lab. di Tecnologia dei Materiali, Bologna (Italy); Di Sebastiano, A. [Bologna Univ. (Italy). Dip. di Fisica; Bologna Univ. (Italy). Dip. di Matematica; Turchetti, G. [Bologna Univ. (Italy). Dip. di Fisica

    1998-12-01

    A volume-preserving symplectic map is proposed to describe the magnetic field lines when the Taylor equilibrium is perturbed in a generic way. The standard scenario is observed by varying the perturbation strength, but the statistical properties in the chaotic regions are not simple due to the presence of boundaries and remnants of invariant structures. Simpler models of volume-preserving maps are proposed. The slowly modulated standard map captures the basic topological and statistical features. The diffusion is analytically described for large perturbations in terms of correlation functions and for small perturbations using the adiabatic theory, provided that the modulation is sufficiently slow.

  7. Expanded Ion Energy Distribution Measurements on MST RFP Plasmas

    Science.gov (United States)

    Clark, Jerry; Titus, J. B.; Mezonlin, E. D.; Anderson, J. K.; Almagri, A. F.

    2016-10-01

    The Compact Neutral Particle Analyzer (CNPA) is a low energy (0.34 - 5.2 keV), high energy resolution (25 channels) neutral particle analyzer for ion energy distribution and temperature measurements on the Madison Symmetric Torus (MST). In MST plasmas during neutral beam injection, deuterium ions are known to have energies out to 40 keV. A retarding potential was built, installed, and calibrated to allow CNPA measurements to explore this region with high energy resolution, expanding ion energy distribution measurements, allowing us to better understand the dynamics of the bulk and fast ion populations during global magnetic reconnection events. Work supported by US DOE and NSF.

  8. Ion temperature gradient turbulence in helical and axisymmetric RFP plasmas

    CERN Document Server

    Predebon, I

    2015-01-01

    Turbulence induced by the ion temperature gradient (ITG) is investigated in the helical and axisymmetric plasma states of a reversed field pinch device by means of gyrokinetic calculations. The two magnetic configurations are systematically compared, both linearly and nonlinearly, in order to evaluate the impact of the geometry on the instability and its ensuing transport, as well as on the production of zonal flows. Despite its enhanced confinement, the high-current helical state demonstrates a lower ITG stability threshold compared to the axisymmetric state, and ITG turbulence is expected to become an important contributor to the total heat transport.

  9. A collimated neutron detector for RFP plasmas in MST

    Science.gov (United States)

    Capecchi, W. J.; Anderson, J. K.; Bonofiglo, P. J.; Kim, J.; Sears, S.

    2016-11-01

    The neutron emissivity profile in the Madison Symmetric Torus is being reconstructed through the use of a collimated neutron detector. A scintillator-photomultiplier tube (PMT) system is employed to detect the fusion neutrons with the plasma viewing volume defined by a 55 cm deep, 5 cm diameter aperture. Effective detection of neutrons from the viewing volume is achieved through neutron moderation using 1300 lbs of high density polyethylene shielding, which modeling predicts attenuates the penetrating flux by a factor of 104 or more. A broad spectrum of gamma radiation is also present due to the unconfined fusion proton bombardment of the thick aluminum vacuum vessel. A 15 cm cylindrical liquid scintillator of 3.8 cm diameter is used to further increase directional sensitivity. A fast (5 ns rise time) preamplifier and digitization at 500 MHz prevent pulse pile-up even at high count rates (˜104/s). The entire neutron camera system is situated on an adjustable inclining base which provides the differing plasma viewing volumes necessary for reconstruction of the neutron emissivity profile. This profile, directly related to the fast-ion population, allows for an investigation of the critical fast-ion pressure gradient required to destabilize a neutral beam driven Alfvénic mode which has been shown to transport fast ions.

  10. Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine.

    Science.gov (United States)

    Cai, Chenggu; Jiang, Hua; Li, Lei; Liu, Tianming; Song, Xuejie; Liu, Bo

    2016-01-01

    Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species.

  11. Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine

    Science.gov (United States)

    Cai, Chenggu; Jiang, Hua; Li, Lei; Liu, Tianming; Song, Xuejie; Liu, Bo

    2016-01-01

    Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species. PMID:27479072

  12. Crystal structure and photoluminescence properties of a new monomeric copper(II) complex: bis(3-{[(3-hydroxypropyl)imino]methyl}-4-nitrophenolato-κ(3)O,N,O')copper(II).

    Science.gov (United States)

    Kocak, Cagdas; Oylumluoglu, Gorkem; Donmez, Adem; Coban, M Burak; Erkarslan, Ugur; Aygun, Muhittin; Kara, Hulya

    2017-05-01

    Copper(II)-Schiff base complexes have attracted extensive interest due to their structural, electronic, magnetic and luminescence properties. The title novel monomeric Cu(II) complex, [Cu(C10H11N2O4)2], has been synthesized by the reaction of 3-{[(3-hydroxypropyl)imino]methyl}-4-nitrophenol (H2L) and copper(II) acetate monohydrate in methanol, and was characterized by elemental analysis, UV and IR spectroscopies, single-crystal X-ray diffraction analysis and a photoluminescence study. The Cu(II) atom is located on a centre of inversion and is coordinated by two imine N atoms, two phenoxy O atoms in a mutual trans disposition and two hydroxy O atoms in axial positions, forming an elongated octahedral geometry. In the crystal, intermolecular O-H...O hydrogen bonds link the molecules to form a one-dimensional chain structure and π-π contacts also connect the molecules to form a three-dimensional structure. The solid-state photoluminescence properties of the complex and free H2L have been investigated at room temperature in the visible region. When the complex and H2L are excited under UV light at 349 nm, the complex displays a strong green emission at 520 nm and H2L displays a blue emission at 480 nm.

  13. A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect alpha-amylase: cDNA cloning and chromosomal location of the gene.

    Science.gov (United States)

    Mena, M; Sanchez-Monge, R; Gomez, L; Salcedo, G; Carbonero, P

    1992-11-01

    A 14.5 kDa barley endosperm protein that is a major allergen in baker's asthma disease, as previously shown by both in vitro (IgE binding) and in vivo tests, has been identified as a glycosylated monomeric member of the multigene family of inhibitors of alpha-amylase/trypsin from cereals. A cDNA encoding this allergen (renamed BMAI-1) has been isolated and characterized. The deduced sequence for the mature protein, which is 132 residues long, is identical in its N-terminal end to the 20 amino acid partial sequence previously determined from the purified allergen, and fully confirms that it is a member of the multigene family of cereal inhibitors. Southern-blot analysis of wheat/barley addition lines using the insert in the BMAI-1 cDNA clone as a probe, has led to the location of the allergen gene (Iam1) in barley chromosome 2, while another related member of this protein family, the barley dimeric alpha-amylase inhibitor BDAI-1 gene (Iad1) has been located in chromosome 6. Iam1 is the first member of this inhibitor family in cereals to be assigned to chromosome group 2, thus extending the dispersion of genes in the family to five out of the seven homology groups of chromosomes in wheat and barley (chromosomes 2, 3, 4, 6 and 7).

  14. Stereospecific approach to the synthesis of ring-A oxygenated sarpagine indole alkaloids. Total synthesis of the dimeric indole alkaloid P-(+)-dispegatrine and six other monomeric indole alkaloids.

    Science.gov (United States)

    Edwankar, Chitra R; Edwankar, Rahul V; Namjoshi, Ojas A; Liao, Xuebin; Cook, James M

    2013-07-05

    The first regio- and stereocontrolled total synthesis of the bisphenolic, bisquaternary alkaloid (+)-dispegatrine (1) has been accomplished in an overall yield of 8.3% (12 reaction vessels) from 5-methoxy-d-tryptophan ethyl ester (17). A crucial late-stage thallium(III) mediated intermolecular oxidative dehydrodimerization was employed in the formation of the C9-C9' biaryl axis in 1. The complete stereocontrol observed in this key biaryl coupling step is due to the asymmetric induction by the natural sarpagine configuration of the monomer lochnerine (6) and was confirmed by both the Suzuki and the oxidative dehydrodimerization model studies on the tetrahydro β-carboline (35). The axial chirality of the lochnerine dimer (40) and in turn dispegatrine (1) was established by X-ray crystallography and was determined to be P(S). Additionally, the first total synthesis of the monomeric indole alkaloids (+)-spegatrine (2), (+)-10-methoxyvellosimine (5), (+)-lochnerine (6), lochvinerine (7), (+)-sarpagine (8), and (+)-lochneram (11) were also achieved via the common pentacyclic intermediate 16.

  15. Development of Broadly Neutralizing Antibodies and Their Mapping by Monomeric gp120 in Human Immunodeficiency Virus Type 1-Infected Humans and Simian-Human Immunodeficiency Virus SHIVSF162P3N-Infected Macaques

    Science.gov (United States)

    Jia, Manxue; Lu, Hong; Markowitz, Martin; Cheng-Mayer, Cecilia

    2016-01-01

    ABSTRACT To improve our understanding of the similarities and differences between neutralizing antibodies elicited by simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and human immunodeficiency virus type 1 (HIV-1)-infected humans, we examined the plasma of 13 viremic macaques infected with SHIVSF162P3N and 85 HIV-1-infected humans with known times of infection. We identified 5 macaques (38%) from 1 to 2 years postinfection (p.i.) with broadly neutralizing antibodies (bnAbs) against tier 2 HIV-1. In comparison, only 2 out of 42 (5%) human plasma samples collected in a similar time frame of 1 to 3 years p.i. exhibited comparable neutralizing breadths and potencies, with the number increasing to 7 out of 21 (30%) after 3 years p.i. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contained gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasma samples, with most of them recognizing the envelope trimer (including gp41) rather than the gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (MAbs) isolated from a human subject (AD358) and a Chinese rhesus macaque (GB40) displayed no or limited neutralizing activity against tier 2 strains. These isolated MAbs, mapped to the CD4-binding site, the V3 loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed MAbs. These results also support the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs. IMPORTANCE HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques, commonly used to assess vaccine immunogenicity and efficacy. Here, we examined 85 HIV-1-infected humans and 13 SHIVSF162P3N-infected macaques for bnAbs and found that, similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare

  16. Influence of osmolarity of contrast medium and saline flush on computed tomography angiography: Comparison of monomeric and dimeric iodinated contrast media with different iodine concentrations at an identical iodine delivery rate

    Energy Technology Data Exchange (ETDEWEB)

    Kishimoto, Miori, E-mail: miori@mx6.et.tiki.ne.j [Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada-cho, Obihiro 080-8555 (Japan); Doi, Shoko, E-mail: s16024@st.obihiro.ac.j [Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada-cho, Obihiro 080-8555 (Japan); Shimizu, Junichiro, E-mail: s01163@st.obihiro.ac.j [Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada-cho, Obihiro 080-8555 (Japan); Lee, Ki-Ja, E-mail: s01173@st.obihiro.ac.j [Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada-cho, Obihiro 080-8555 (Japan); Iwasaki, Toshiroh, E-mail: bpag2180@cc.tuat.ac.j [Department of Veterinary Internal Medicine, Tokyo University of Agriculture and Technology, Saiwai-cho, 3-5-8, Fuchu 183-8509 (Japan); Miyake, Yoh-Ichi, E-mail: miyake@obihiro.ac.j [Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada-cho, Obihiro 080-8555 (Japan); Yamada, Kazutaka, E-mail: kyamada@obihiro.ac.j [Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-11 Inada-cho, Obihiro 080-8555 (Japan)

    2010-10-15

    Purpose: To evaluate the influence of osmolarity of iodinated contrast media and saline flush on the contrast effect in thoracic computed tomography angiography (CTA) at an identical iodine delivery rate (IDR). Materials and methods: Seven beagles were used in a cross-over experiment. The contrast media used were iohexol 350 mgI/ml (IOH350; osmolarity 844 mmol/kg) and iodixanol 320 mgI/ml (IDX320; osmolarity 290 mmol/kg). Each contrast medium was administered to groups with and without saline flush at 40.0 mgI/kg/s for all experiments. Dynamic CT scanning was performed at the ninth thoracic vertebra level. The peak value, area under the curve (AUC), and time to peak (TTP) were calculated from the time attenuation curves of the pulmonary artery and aorta. Results: There was no significant difference between IOH350 and IDX320 with or without saline flush in the peak values for the pulmonary artery and aorta. AUC was significantly higher in groups with saline flush for both contrast media and arteries (p < 0.05) with no significant difference between contrast media. TTP was significantly longer in groups with saline flush than without saline flush for both contrast media and arteries (p < 0.05), with no significant difference between contrast media. Conclusions: There were no significant differences in the contrast effects of monomeric IOH350 and dimeric IDX320 in thoracic CTA when used at an identical IDR. Moreover, saline flush prolonged the peak duration at 600 mgI/kg.

  17. Hydrogen for fluorine exchange in C6F6 and C6F5H by monomeric [1,3,4-(Me3C)3C5H2]2CeH: experimental and computational studies.

    Science.gov (United States)

    Maron, Laurent; Werkema, Evan L; Perrin, Lionel; Eisenstein, Odile; Andersen, Richard A

    2005-01-12

    The net reaction of monomeric Cp'(2)CeH [Cp' = 1,3,4-(Me(3)C)(3)(C(5)H(2))] in C(6)D(6) with C(6)F(6) is Cp'(2)CeF, H(2), and tetrafluorobenzyne. The pentafluorophenylmetallocene, Cp'(2)Ce(C(6)F(5)), is formed as an intermediate that decomposes slowly to Cp'(2)CeF and C(6)F(4) (tetrafluorobenzyne), and the latter is trapped by the solvent C(6)D(6) as a [2+4] cycloadduct. In C(6)F(5)H, the final products are also Cp'(2)CeF and H(2), which are formed from the intermediates Cp'(2)Ce(C(6)F(5)) and Cp'(2)Ce(2,3,5,6-C(6)F(4)H) and from an unidentified metallocene of cerium and the [2+4] cycloadducts of tetra- and trifluorobenzyne with C(6)D(6). The hydride, fluoride, and pentafluorophenylmetallocenes are isolated and characterized by X-ray crystallography. DFT(B3PW91) calculations have been used to explore the pathways leading to the observed products of the exergonic reactions. A key step is a H/F exchange reaction which transforms C(6)F(6) and the cerium hydride into C(6)F(5)H and Cp'(2)CeF. This reaction starts by an eta(1)-F-C(6)F(5) interaction, which serves as a hook. The reaction proceeds via a sigma bond metathesis where the fluorine ortho to the hook migrates toward H with a relatively low activation energy. All products observed experimentally are accommodated by pathways that involve C-F and C-H bond cleavages.

  18. Zipper-mediated oligomerization of the mixed lineage kinase SPRK/MLK-3 is not required for its activation by the GTPase cdc 42 but Is necessary for its activation of the JNK pathway. Monomeric SPRK L410P does not catalyze the activating phosphorylation of Thr258 of murine MITOGEN-ACTIVATED protein kinase kinase 4.

    Science.gov (United States)

    Vacratsis, P O; Gallo, K A

    2000-09-08

    Src homology 3 domain-containing proline-rich kinase (SPRK)/mixed lineage kinase-3 is a serine/threonine kinase that has been identified as an upstream activator of the c-Jun NH(2)-terminal kinase (JNK) pathway. SPRK is capable of activating MKK4 by phosphorylation of serine and threonine residues, and mutant forms of MKK4 that lack the phosphorylation sites Ser(254) and Thr(258) block SPRK-induced JNK activation. A region of 63 amino acids following the kinase domain of SPRK is predicted to form a leucine zipper. The leucine zipper domain of SPRK has been shown to be necessary and sufficient for SPRK oligomerization, but its role in regulating activation of SPRK and downstream signaling remains unclear. In this study, we substituted a proposed stabilizing leucine residue in the zipper domain with a helix-disrupting proline to abrogate zipper-mediated SPRK oligomerization. We demonstrate that constitutively activated Cdc42 fully activates this monomeric SPRK mutant in terms of both autophosphorylation and histone phosphorylation activity and induces the same in vivo phosphorylation pattern as wild type SPRK. However, this catalytically active SPRK zipper mutant is unable to activate JNK. Our data show that the monomeric SPRK mutant fails to phosphorylate one of the two activating phosphorylation sites, Thr(258), of MKK4. These studies suggest that zipper-mediated SPRK oligomerization is not required for SPRK activation by Cdc42 but instead is critical for proper interaction and phosphorylation of a downstream target, MKK4.

  19. Reactions of Cu(I)Br with aziridine derivatives. Synthesis, characterization and crystal structures of monomeric, dimeric and hexameric aziridine (= az) complexes of the formal type [CuBr(az)2]n (n = 1, 2) and [CuBr(az)]6.

    Science.gov (United States)

    Bobka, Roman; Roedel, J Nicolas; Wirth, Stefan; Lorenz, Ingo-Peter

    2010-11-14

    The first syntheses of monomeric and oligomeric aziridine complexes of copper(I) are described. Cu(I)Br (1) reacts with a series of different aziridine derivatives (C(2)H(3)PhNH (2), C(2)H(2)Me(2)NH (3), C(2)H(2)Me(2)NC(2)H(2)Me(2)NH(2) (4)) to give the neutral dimeric complex [CuBr(C(2)H(3)PhNH)(2)](2) (5) and the ionic hexameric complex [Cu(6)Br(5)(C(2)H(2)Me(2)NH)(6)]Br (6) with terminal bound aziridine ligands as well as the neutral monomeric complex [CuBr(C(2)H(2)Me(2)NC(2)H(2)Me(2)NH(2))] (7) where the dimerized aziridine acts as a N,N'-chelating ligand. After purification, all of the complexes were fully characterized and their IR, (1)H and (13)C NMR spectra are reported and discussed. The single crystal structure analysis revealed distorted tetrahedral geometry for the copper(I) centres in the complexes 5 and 6 and a trigonal planar structure for complex 7. In the oligomers the copper centres are bridged by two μ(2)- (5) or two μ(3)- and three μ(4)-bromido ligands (6), respectively.

  20. From Coordination Polymer to Monomeric Complex: Two Cobalt Complexes from a Single Demko-Sharpless's Tetrazole Synthesis Reaction%从配位聚合物到单分子化合物的转化:Demko-Sharpless四唑合成法制备两个新颖的Co(Ⅱ)配合物

    Institute of Scientific and Technical Information of China (English)

    瞿志荣

    2007-01-01

    The reaction of CoCl2· 6H2O with 4-cyanopyridine N-oxide in the present of NaN3 affords two novel complexes, {[(POTZ)(H2O)2N3]Co(H2O)} (1) and Co(POTZ)2(H2O)4 (2) (POTZ=4-tetrazolyl pyridine N-oxide), which are two different phases yielded at different stages of a single Demko-Sharpless' tetrazole synthesis reaction.Surprisingly the 1D chain coordination polymer 1 is almost completely converted into monomeric complex 2 in this reaction, and, in a separate test, 2 also can be converted into 1. CCDC: 641222, 1; 641223, 2.

  1. [Bi3GaS5]2[Ga3Cl10]2[GaCl4]2·S8 containing heterocubane-type [Bi3GaS5]2+, star-shaped [Ga3Cl10]-, monomeric [GaCl4]- and crown-like S8.

    Science.gov (United States)

    Freudenmann, Dominic; Feldmann, Claus

    2011-01-14

    By reaction of elemental bismuth, sulfur, bismuth(III) chloride and gallium(III) chloride in the ionic liquid (BMIm)Cl (BMIm: 1-butyl-3-methylimidazolium), [Bi(3)GaS(5)](2)[Ga(3)Cl(10)](2)[GaCl(4)](2)·S(8) is obtained as red transparent crystals. According to X-ray structure analysis based on single crystals, the title compound crystallizes with triclinic lattice symmetry and is composed of heterocubane-type [Bi(3)GaS(5)](2+) cations, trimeric star-shaped [Ga(3)Cl(10)](-) anions with three (GaCl(4)) tetrahedra sharing a single central chlorine atom, monomeric [GaCl(4)](-) tetrahedra and neutral, crown-shaped S(8)-rings. Here, the heterocubane [Bi(3)GaS(5)](2+) as well as the star-shaped [Ga(3)Cl(10)](-) are observed as building units for the first time. [Bi(3)GaS(5)](2)[Ga(3)Cl(10)](2)[GaCl(4)](2)·S(8) is further characterized by X-ray powder diffraction as well as by thermogravimetry/differential thermal analysis.

  2. Coorientation Theory and Assessment of the RFP Solution to Client/Service Learner Matchmaking

    Science.gov (United States)

    Rogers, Cathy; Andrews, Valerie

    2013-01-01

    Tensions that result from varying expectations of service learners and clients/community partners are as common as the pedagogical practice of service learning in public relations courses. The matchmaking process between instructors and clients can influence expectations; however, the literature includes little guidance about the process of client…

  3. RFP to work on formation flying capabilities for spacecrafts for the GRACE project

    DEFF Research Database (Denmark)

    Riis, Troels; Thuesen, Gøsta; Kilsgaard, Søren

    1999-01-01

    The National Aeronautics and Space Agency of USA, NASA, are working on formation flying capabilities for spacecrafts, GRACE Project. IAU and JPL are developing the inter spacecraft attitude link to be used on the two spacecrafts.......The National Aeronautics and Space Agency of USA, NASA, are working on formation flying capabilities for spacecrafts, GRACE Project. IAU and JPL are developing the inter spacecraft attitude link to be used on the two spacecrafts....

  4. The pre-start training technique of marksmen-sportsmen in the Olympic exercise RFP

    Directory of Open Access Journals (Sweden)

    Petriv O. S.

    2012-11-01

    Full Text Available It is shown the features of the new method prelaunch athletes in high-speed shooting of a standard pistol at five targets, which appear simultaneously at a distance of 25 meters. It is determined that the complexity of the rapid-fire is in necessity to coordinate the exact aiming at the center of the target with a smooth touch of the trigger. The methodology proposed new materials that clarify a system of forming knowledge and skills in the shooting sports. Practical recommendations containing new, scientifically based information to improve the technical and tactical actions of athletes in high-speed shooting of a standard pistol at five targets simultaneously appear at a distance of 25 meters.

  5. Development of PCR-RFP and DNA barcoding plastic markers for yellow toadflax and Dalmatian toadflax

    Science.gov (United States)

    Andrew Boswell

    2013-01-01

    Yellow toadflax and Dalmatian toadflax are problematic invasive plant species in North America. Yellow toadflax was introduced multiple times to the United States from Europe, beginning in the late 1600s. Dalmatian toadflax has similarly been repeatedly introduced to the United States, starting in 1874. Both species are known to inhabit disturbed areas, competing for...

  6. Study of Opacity Effects on Emission Lines at EXTRAP T2R RFP

    Science.gov (United States)

    Stancalie, Viorica; Rachlew, Elisabeth

    We have investigated the influence of opacity on hydrogen (H-α and Ly-β) and Li-like oxygen emission lines from the EXTRAP T2R reversed field pinch. We used the Atomic Data Analysis System (AzDAS) based on the escape factor approximation for radiative transfer to calculate metastable and excited population densities via a collisional-radiative model. Population escape factor, emergent escape factor and modified line profiles are plotted vs. optical depth. The simulated emission line ratios in the density/temperature plane are in good agreement with experimental data for electron density and temperature measurements.

  7. 3D equilibrum reconstruction for the RFP with V3FIT

    Science.gov (United States)

    Terranova, David; Marrelli, Lionello; Hanson, James; Hirshman, Steven; Marco, Gobbin; Trevisan, Gregorio

    2012-10-01

    Helical states are routinely found in all Reversed Field Pinch experiments and their description requires a 3D equilibrium reconstruction. We present the application of the V3FIT code for the RFX-mod experiment. Magnetic and kinetic diagnostics (Te from Thomson scattering, SXR emissivity, Ne from interferometer) are used in order to properly deal with the problem of degeneracy when only external magnetic measurements are used. A sensitivity study of external measurements on the internal topological structure is also presented, showing a link between external measurements and internal profiles. Fixed-boundary equilibria can be computed by independently calculating vacuum fields. The results provide a good match with experimental data and the obtained equilibria are suitable for both transport and stability analysis.

  8. An integrated data analysis tool for improving measurements on the MST RFP

    Energy Technology Data Exchange (ETDEWEB)

    Reusch, L. M., E-mail: lmmcguire@wisc.edu; Galante, M. E.; Johnson, J. R.; McGarry, M. B.; Den Hartog, D. J. [Physics Department, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Franz, P. [Consorzio RFX, EURATOM-ENEA Association, Padova (Italy); Stephens, H. D. [Physics Department, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Pierce College Fort Steilacoom, Lakewood, Washington 98498 (United States)

    2014-11-15

    Many plasma diagnostics contain complementary information. For example, the double-foil soft x-ray system (SXR) and the Thomson Scattering diagnostic (TS) on the Madison Symmetric Torus both measure electron temperature. The complementary information from these diagnostics can be combined using a systematic method based on integrated data analysis techniques, leading to more accurate and sensitive results. An integrated data analysis tool based on Bayesian probability theory was able to estimate electron temperatures that are consistent with both the SXR and TS diagnostics and more precise than either. A Markov Chain Monte Carlo analysis to increase the flexibility of the tool was implemented and benchmarked against a grid search method.

  9. Charge-to-mass-ratio-dependent ion heating during magnetic reconnection in the MST RFP

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, S. T. A.; Almagri, A. F.; Den Hartog, D. J.; Nornberg, M. D.; Sarff, J. S.; Terry, P. W. [Department of Physics, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Center for Magnetic Self-Organization in Laboratory and Astrophysical Plasmas, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Craig, D. [Wheaton College, Wheaton, Illinois 60187 (United States)

    2013-05-15

    Temperature evolution during magnetic reconnection has been spectroscopically measured for various ion species in a toroidal magnetized plasma. Measurements are made predominantly in the direction parallel to the equilibrium magnetic field. It is found that the increase in parallel ion temperature during magnetic reconnection events increases with the charge-to-mass ratio of the ion species. This trend can be understood if the heating mechanism is anisotropic, favoring heating in the perpendicular degree of freedom, with collisional relaxation of multiple ion species. The charge-to-mass ratio trend for the parallel temperature derives from collisional isotropization. This result emphasizes that collisional isotropization and energy transfer must be carefully modeled when analyzing ion heating measurements and comparing to theoretical predictions.

  10. Classical confinement and outward convection of impurity ions in the MST RFP

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, S. T. A.; Den Hartog, D. J.; Mirnov, V. V.; Eilerman, S.; Nornberg, M.; Reusch, J. A.; Sarff, J. S. [Department of Physics, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Center for Magnetic Self-Organization in Laboratory and Astrophysical Plasmas, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Caspary, K. J.; Chapman, B. E.; Parke, E. [Department of Physics, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Magee, R. M. [Department of Physics, West Virginia University, Morgantown, WV 26506 (United States); Brower, D. L.; Ding, W. X.; Lin, L. [Department of Physics and Astronomy, University of California, Los Angeles, California 90095 (United States); Craig, D. [Physics Department, Wheaton College, Wheaton, Illinois 60187 (United States); Fiksel, G. [Center for Magnetic Self-Organization in Laboratory and Astrophysical Plasmas, University of Wisconsin-Madison, Madison, Wisconsin 53706 (United States); Laboratory for Laser Energetics, University of Rochester, Rochester, New York (United States)

    2012-05-15

    Impurity ion dynamics measured with simultaneously high spatial and temporal resolution reveal classical ion transport in the reversed-field pinch. The boron, carbon, oxygen, and aluminum impurity ion density profiles are obtained in the Madison Symmetric Torus [R. N. Dexter et al., Fusion Technol. 19, 131 (1991)] using a fast, active charge-exchange-recombination-spectroscopy diagnostic. Measurements are made during improved-confinement plasmas obtained using inductive control of tearing instability to mitigate stochastic transport. At the onset of the transition to improved confinement, the impurity ion density profile becomes hollow, with a slow decay in the core region concurrent with an increase in the outer region, implying an outward convection of impurities. Impurity transport from Coulomb collisions in the reversed-field pinch is classical for all collisionality regimes, and analysis shows that the observed hollow profile and outward convection can be explained by the classical temperature screening mechanism. The profile agrees well with classical expectations. Experiments performed with impurity pellet injection provide further evidence for classical impurity ion confinement.

  11. SOS-red fluorescent protein (RFP) bioassay system for monitoring of antigenotoxic activity in plant extracts.

    Science.gov (United States)

    Bartolome, Amelita; Mandap, Katheryn; David, Kevim Jhean; Sevilla, Fortunato; Villanueva, James

    2006-05-15

    An optical antigenotoxicity assay using genetically engineered red fluorescent bacteria is presented. Exposure of Escherichia coli RS4U to genotoxicants [mitomycin C (MMC), nalidixic acid (NA) and hydrogen peroxide (HP)] resulted in phenotypic red fluorescence proportional to the concentration of the inducer. Except for tannic acid (TA), co-treatment of the genotoxicant-activated bacteria with ascorbic acid (AA) and aqueous plant extracts (Mangifera indica, Psidium guajava and Syzygium cumini) afforded protection against all three genotoxicants. TA was effective in suppressing the genotoxic effect of MMC and HP. The antigenotoxic effect is seen as inhibition of the genotoxicant-triggered red fluorescence. The IC50 of the plant extracts and AA varied with the genotoxicant used. Rec assay verified the antigenotoxic activity of the plant extracts. Folin-Ciocalteu test, FeCl3 test and DPPH assay confirmed the presence of polyphenolic compounds and hydrolyzable tannins in the plant extracts and the antioxidant capacity of the plant samples.

  12. 77 FR 54877 - Request for Proposals (RFP): Farm Labor Housing Technical Assistance Grants

    Science.gov (United States)

    2012-09-06

    ... USDA--Rural Housing Service; Attention: Mirna Reyes-Bible, Finance and Loan Analyst, Multi- Family... receipt. FOR FURTHER INFORMATION CONTACT: Mirna Reyes-Bible, Finance and Loan ] Analyst, Multi-Family... corporate felony convictions and corporate federal tax delinquencies. To comply with these provisions, all...

  13. Oligosaccharides and monomeric carbohydrates production from olive tree pruning biomass.

    Science.gov (United States)

    Mateo, Soledad; Puentes, Juan G; Sánchez, Sebastián; Moya, Alberto J

    2013-04-02

    Using the severity factor, it has been possible to study cellulose and hemicellulose fractional conversion, sugar yields change and oligosaccharides variation through olive tree pruning biomass pretreatments with acid or liquid hot water under pressure. The temperatures tested were in the range 180-230°C, operation time varying between 0 and 30min and acid concentration used did not exceed 0.05M. Complete hemicellulose solubilization in autohydrolysis was achieved using severity factors (logR0) close to 3.9 (most sugars are like oligomers), while if sulfuric acid 0.025M is employed, this parameter could be smaller (≥3.4). With these treatments, we have obtained cellulose conversions between 30 and 42% from liquid hot water experiments, 40-51% with sulfuric acid 0.025M and 42-57% when the acid concentration was 0.05M. The best results in terms of maximum yield in total sugars, d-glucose and d-xylose, with a low amount of acetic acid and hydroxymethylfurfural, was obtained at 200°C, 0min (what means that there is no time of temperature maintenance, only heating and cooling) and H2SO4 0.025M.

  14. Ischemic Monomeric Neuropathy in a Woman with Sickle Cell Anaemia

    Directory of Open Access Journals (Sweden)

    Alexandra Agapidou

    2016-01-01

    Full Text Available Sickle cell disease is an inherited haemoglobinopathy that can affect multiple organs and systems. The most common neurological complication in sickle cell disease is stroke and silent cerebral infarcts. Peripheral nervous system involvement has been described but is exceedingly rare. Herein, we describe the case of a young woman who presented with acute flaccid paralysis and sensory loss of the left lower extremity in the context of a painful vasoocclusive crisis which resolved rapidly after receiving an emergency automated red cell exchange transfusion.

  15. Clinical characterization of patients with macroprolactinemia and monomeric hyperprolactinemia

    Directory of Open Access Journals (Sweden)

    Murat Can

    2011-05-01

    Full Text Available Macroprolactinemia is often a cause of misdiagnosis, unnecessary expensive investigation, and unsuitable treatment. The aim of the present study was to investigate the clinical findings and the concentrations of macroprolactin in patients with hyperprolactinemia in our region. Eighty-four female hyperprolactinemic patients were screened for macroprolactinemia. Prolactin was measured by chemiluminesans method on an Immulite 2000 analyzer (Siemens Health Diagnostics, Deerfield, IL, USA. Recoveries less than or equal to 40% after polyethylene glycol precipitation were indicative of macroprolactinemia. Clinical features and biochemical values were compared in true hyperprolactinemic and macroprolactinemic patients. Macroprolactinemia was detected in 31 patients (36.9%, with 84 hyperprolactinemic female patients. There was no difference in frequency of galactorrhea and oligomenorrhea/amenorrhea between the two groups. When we evaluated the clinical features of patients according to prolactin levels, no significant difference was found between the groups. In conclusion, our initial data show that no clinical features could reliably differentiate macroprolactinemic from true hyperprolactinemic patients, but at least one of these symptoms was present in most macroprolactinemic patients.

  16. Micellization of monomeric and poly-ω-methacryloyloxyundecyltrimethylammonium surfactants.

    Science.gov (United States)

    FitzGerald, Paul A; Chatjaroenporn, Khwanrat; Zhang, Xiaoli; Warr, Gregory G

    2011-10-04

    We have used small-angle neutron scattering to study how micelle morphology of the tail-polymerizable surfactants MUTAB and MUTAC (ω-methacryloyloxyundecyltrimethylammonium bromide and chloride) is affected by classic self-assembly modifiers such as temperature changes, salt addition, and counterion exchange, as a function of their conversion from monomer into polymer amphiphile in aqueous solution. Contrary to common assumptions about polymerized surfactants, these systems remain in dynamic equilibrium under all conditions examined and at all conversions (except for a small amount of high-molecular-weight precipitation by MUTAC). Counterintuitively, the polymerized methacrylate backbone has little influence on aggregate morphology, except for the formation of rod-like mixed micelles of polymerized and unpolymerized surfactant at intermediate conversions. The addition of salt produces a transition to rod-like micelles at all conversions except in the unpolymerized surfactant, which has some characteristics of an asymmetric bolaform surfactant and retains its spheroidal geometry under almost all conditions.

  17. Structure of the novel monomeric glyoxalase I from Zea mays.

    Science.gov (United States)

    Turra, Gino L; Agostini, Romina B; Fauguel, Carolina M; Presello, Daniel A; Andreo, Carlos S; González, Javier M; Campos-Bermudez, Valeria A

    2015-10-01

    The glyoxalase system is ubiquitous among all forms of life owing to its central role in relieving the cell from the accumulation of methylglyoxal, a toxic metabolic byproduct. In higher plants, this system is upregulated under diverse metabolic stress conditions, such as in the defence response to infection by pathogenic microorganisms. Despite their proven fundamental role in metabolic stresses, plant glyoxalases have been poorly studied. In this work, glyoxalase I from Zea mays has been characterized both biochemically and structurally, thus reporting the first atomic model of a glyoxalase I available from plants. The results indicate that this enzyme comprises a single polypeptide with two structurally similar domains, giving rise to two lateral concavities, one of which harbours a functional nickel(II)-binding active site. The putative function of the remaining cryptic active site remains to be determined.

  18. Mechanism of Processive Movement of Monomeric and Dimeric Kinesin Molecules

    Directory of Open Access Journals (Sweden)

    Ping Xie

    2010-01-01

    Full Text Available Kinesin molecules are motor proteins capable of moving along microtubule by hydrolyzing ATP. They generally have several forms of construct. This review focuses on two of the most studied forms: monomers such as KIF1A (kinesin-3 family and dimers such as conventional kinesin (kinesin-1 family, both of which can move processively towards the microtubule plus end. There now exist numerous models that try to explain how the kinesin molecules convert the chemical energy of ATP hydrolysis into the mechanical energy to “power” their proceesive movement along microtubule. Here, we attempt to present a comprehensive review of these models. We further propose a new hybrid model for the dimeric kinesin by combining the existing models and provide a framework for future studies in this subject.

  19. Total Monomeric Anthocyanin and Total Flavonoid Content of Processed Purple

    Directory of Open Access Journals (Sweden)

    Potato Florentina Damşa

    2016-01-01

    Full Text Available It is well known that processing change physical and chemical composition of foods, thus affecting the content in bioactive substances. Potatoes are almost always consumed after processing (baked, fried or boiled making it critical to understand the effect of such processing techniques on the containing in bioactive compounds. In order to determine the influence of processing on the content of anthocyanin pigments and flavonoids was achieved the extraction of these compounds from boiled and baked purple potato tuber (Albastru-Violet de Galanesti variety. Also, in order to obtain the maximum amount of anthocyanin pigments and flavonoids from processed potatoes was applied ultrasonic extraction (20 kHz and was performed the mathematical modeling (central composite design using SigmaXL software. The total anthocyanins content were determined spectrophotometrically by the pH differential method and the total flavonoids content were determine colorimetric by AlCl3 method. This study proves that the potato processing decreases the content of anthocyanin pigments and flavonoids.

  20. Monomeric, dimeric and multimeric system of RGD peptides radiolabeled with {sup 177}Lu for tumors therapy that expressing αβ integrin s; Sistema monomerico, dimerico y multimerico de peptidos de RGD radiomarcados con {sup 177}Lu para terapia de tumores que expresan integrinas αβ

    Energy Technology Data Exchange (ETDEWEB)

    Luna G, M. A.

    2014-07-01

    The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target-specific molecular recognition. Peptides based on the cyclic Arg-Gly-Asp (RGD) sequence have been reported as high affinity agents for the α(v)β(3) and α(v)β(5) integrin. The aim of this research was to prepare a multimeric system of {sup 177}Lu-labeled gold nanoparticles conjugated to c[RGDfK(C)] [cyclo(Arg-Gly-Asp-Phe-Lys(Cys)] peptides and to compare the radiation absorbed dose with that of {sup 177}Lu-labeled monomeric and dimeric RGD peptides to α(v)β(3) integrin-positive U87MG tumors in mice, as well as, evaluate the in vitro potential {sup 177}Lu-AuNP-c[RGDfK(C)] as a plasmonic photothermal therapy and targeted radiotherapy system in MCF7 breast cancer cells. DOTA-GGC (1,4,7,10-tetraaza cyclododecane-N,N,N-tetraacetic-Gly-Gly-Cys) and c[RGDfK(C)] peptides were synthesized and conjugated to AuNPs by the spontaneous reaction of the thiol groups. Tem, UV-Vis, XP S, Raman and Far-IR spectroscopy techniques demonstrated that AuNPs were functionalized with the peptides. To obtain {sup 177}Lu-AuNP-c[RGDfK(C)], the {sup 177}Lu-DOTA-GGC radio peptide was first prepared and added to a solution of AuNPs followed by c[RGDfK(C)] (25 μL, 5 μM) at 18 grades C for 15 min. {sup 177}Lu-DOTA-GGC, {sup 177}Lu- DOTA-cRGDfK and {sup 177}Lu-DOTA-E-c(RGDfK){sub 2} were prepared by adding {sup 177}LuCl{sub 3} (370 MBq) to 5 μL (1 mg/ml) of the DOTA derivative diluted with 50 μL of 1 M acetate buffer at ph 5. The mixture was incubated at 90 grades C in a block heater for 30 min. Radiochemical purity was determined by ultrafiltration and HPLC analyses. After laser irradiation, the presence of c[RGDfK(C)]-AuNP in cells caused a significant increase in the temperature of the medium (50.5 grades C, compared to 40.3 grades C without AuNPs) resulting in a significant decrease in MCF7 cell viability down to 9 %. After treatment with {sup 177}Lu

  1. A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.

    Directory of Open Access Journals (Sweden)

    Jianjie Mi

    Full Text Available Filament bundles (rods of cofilin and actin (1:1 form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30-60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.

  2. GPP Webinar: Solar Utilization in Higher Education Networking & Information Sharing Group: RFP, Contract, and Administrative Issues Discussion

    Science.gov (United States)

    This presentation from a Solar Utilization in Higher Education Networking and Information webinar covers contracts, Request for Proposals (RFPs), and administrative issues related to solar project development in the higher education sector.

  3. Fluorine for Hydrogen Exchange in the Hydrofluorobenzene Derivatives C6HxF(6-x), where x = 2, 3, 4 and 5 by Monomeric [1,2,4-(Me3C)3C5H2]2CeH; The Solid State Isomerization of [1,2,4-(Me3C)3C5H2]2Ce(2,3,4,5-C6HF4) to [1,2,4-(Me3C)3C5H2]2Ce(2,3,4,6-C6HF4)

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Richard; Werkema, Evan L.; Andersen, Richard A.

    2008-04-21

    The reaction between monomeric bis(1,2,4-tri-t-butylcyclopentadienyl)cerium hydride, Cp'2CeH, and several hydrofluorobenzene derivatives is described. The aryl derivatives that are the primary products, Cp'2Ce(C6H5-xFx) where x = 1,2,3,4, are thermally stable enough to be isolated in only two cases, since all of them decompose at different rates to Cp'2CeF and a fluorobenzyne; the latter is trapped by either solvent when C6D6 is used or by a Cp'H ring when C6D12 is the solvent. The trapped products are identified by GCMS analysis after hydrolysis. The aryl derivatives are generated cleanly by reaction of the metallacycle, Cp'((Me3C)2C5H2C(Me2)CH2)Ce, with a hydrofluorobenzene and the resulting arylcerium products, in each case, are identified by their 1H and 19F NMR spectra at 20oC. The stereochemical principle that evolves from these studies is that the thermodynamic isomer is the one in which the CeC bond is flanked by two ortho-CF bonds. This orientation is suggested to arise from the negative charge that is localized on the ipso-carbon atom due to Co(delta+)-Fo(delta-) polarization. The preferred regioisomer is determined by thermodynamic rather than kinetic effects; this is illustrated by the quantitative, irreversible solid-state conversion at 25oC over two months of Cp'2Ce(2,3,4,5-C6HF4) to Cp'2Ce(2,3,4,6-C6HF4), an isomerization that involves a CeC(ipso) for C(ortho)F site exchange.

  4. Fluorine for Hydrogen Exchange in the Hydrofluorobenzene Derivatives C6HxF(6-x), where x = 2, 3, 4 and 5 by Monomeric [1,2,4-(Me3C)3C5H2]2CeH; The Solid State Isomerization of [1,2,4-(Me3C)3C5H2]2Ce(2,3,4,5-C6HF4) to [1,2,4-(Me3C)3C5H2]2Ce(2,3,4,6-C6HF4)

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Richard; Werkema, Evan L.; Andersen, Richard A.

    2008-04-21

    The reaction between monomeric bis(1,2,4-tri-t-butylcyclopentadienyl)cerium hydride, Cp'2CeH, and several hydrofluorobenzene derivatives is described. The aryl derivatives that are the primary products, Cp'2Ce(C6H5-xFx) where x = 1,2,3,4, are thermally stable enough to be isolated in only two cases, since all of them decompose at different rates to Cp'2CeF and a fluorobenzyne; the latter is trapped by either solvent when C6D6 is used or by a Cp'H ring when C6D12 is the solvent. The trapped products are identified by GCMS analysis after hydrolysis. The aryl derivatives are generated cleanly by reaction of the metallacycle, Cp'((Me3C)2C5H2C(Me2)CH2)Ce, with a hydrofluorobenzene and the resulting arylcerium products, in each case, are identified by their 1H and 19F NMR spectra at 20oC. The stereochemical principle that evolves from these studies is that the thermodynamic isomer is the one in which the CeC bond is flanked by two ortho-CF bonds. This orientation is suggested to arise from the negative charge that is localized on the ipso-carbon atom due to Co(delta+)-Fo(delta-) polarization. The preferred regioisomer is determined by thermodynamic rather than kinetic effects; this is illustrated by the quantitative, irreversible solid-state conversion at 25oC over two months of Cp'2Ce(2,3,4,5-C6HF4) to Cp'2Ce(2,3,4,6-C6HF4), an isomerization that involves a CeC(ipso) for C(ortho)F site exchange.

  5. Immobilization of homogeneous monomeric, oligomeric and fibrillar Aβ species for reliable SPR measurements.

    Science.gov (United States)

    Frenzel, Daniel; Glück, Julian M; Brener, Oleksandr; Oesterhelt, Filipp; Nagel-Steger, Luitgard; Willbold, Dieter

    2014-01-01

    There is strong evidence that the amyloid-beta peptide (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). In this context, a detailed quantitative description of the interactions with different Aβ species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aβ in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aβ conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aβ allows reliable SPR measurements and quality control of the immobilized Aβ aggregate species at any step throughout the experiment.

  6. Immobilization of homogeneous monomeric, oligomeric and fibrillar Aβ species for reliable SPR measurements.

    Directory of Open Access Journals (Sweden)

    Daniel Frenzel

    Full Text Available There is strong evidence that the amyloid-beta peptide (Aβ plays a central role in the pathogenesis of Alzheimer's disease (AD. In this context, a detailed quantitative description of the interactions with different Aβ species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aβ in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR studies with homogeneous Aβ conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC for sample purification and the use of a single chain variable fragment (scFv of a monoclonal antibody directed against the amino-terminus of Aβ allows reliable SPR measurements and quality control of the immobilized Aβ aggregate species at any step throughout the experiment.

  7. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

    NARCIS (Netherlands)

    Shaner, Nathan C; Campbell, Robert E; Steinbach, Paul A; Giepmans, Ben N G; Palmer, Amy E; Tsien, Roger Y

    2004-01-01

    Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red

  8. Development and validation of analytical methods for monomeric and oligomeric migrants from nylon 12 packaging materials.

    NARCIS (Netherlands)

    Stoffers, N.H.; Brandl, F.; Linssen, J.P.H.; Franz, R.

    2003-01-01

    Analytical methods for the determination of laurolactam-the monomer of nylon 12-as well as the cyclic dimer and trimer were established. High performance liquid chromatography using ultraviolet (HPLC-UV) and mass spectrometric detection (HPLC-MS) were both found suitable to identify and quantify mon

  9. MONOMERIC ANTHOCYANIN COMPOSITION OF FIVE PHENOTYPES OF COLORED CORN (Zea mays FROM CENTRAL REGION OF COLOMBIA

    Directory of Open Access Journals (Sweden)

    Roberto Quiñones

    2015-01-01

    Full Text Available Colombia cuenta con variedades nativas de maíz (Zea mays L. que lentamente se han ido perdiendo, a pesar de su interés potencial para programas de mejoramiento genético y uso nutracéutico. En Colombia, el maíz coloreado es poco conocido y escasamente aprovechado, el cual si ha sido investigado en otros países. Como parte de nuestros estudios del aprovechamiento de materiales vegetales nativos, cinco accesiones locales de maíz coloreado, provenientes de la región cundiboyacense colombiana, fueron recuperadas y cultivadas en condiciones agroecológicas. Se caracterizaron los extractos metanólicos obtenidos de sus granos, evaluando el contenido de antocianinas totales y la capacidad captadora de radicales DPPH•, cuyos valores estuvieron entre 21-168 mg eq cianidina 3-glucósido/kg grano seco y 195-412 µM Trolox/g grano seco, respectivamente. Se determinaron los perfiles de antocianinas de cada fenotipo mediante HPLC-DAD-MS. Se identificaron tentativamente nueve antocianinas, cuatro de ellas presentes en todos los fenotipos. Tales perfiles permitieron realizar una discriminación de los cinco fenotipos a partir de análisis multivariado. La información estructural se apoyó en el aislamiento y elucidación de cuatro de las antocianinas a partir del extracto enriquecido del fenotipo púrpura.

  10. A de novo designed monomeric, compact three helix bundle protein on a carbohydrate template

    DEFF Research Database (Denmark)

    Malik, Leila; Nygård, Jesper; Christensen, Niels Johan

    2015-01-01

    De novo design and chemical synthesis of proteins and of other artificial structures, which mimic them, is a central strategy for understanding protein folding and for accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial...... proteins, so called carboproteins. The hypothesis is that the template pre-organizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from...

  11. Monomeric Amyloid Beta Peptide in Hexafluoroisopropanol Detected by Small Angle Neutron Scattering.

    Directory of Open Access Journals (Sweden)

    Bo Zhang-Haagen

    Full Text Available Small proteins like amyloid beta (Aβ monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP by using SANS and dynamic light scattering (DLS. We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ1-40 and 1.6±0.1 nm for Aβ1-42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ1-40 and 3.2±0.4 nm for Aβ1-42 including a surface layer of dHFIP solvent molecules.

  12. The effect of proteolysis on the induction of cell death by monomeric alpha-lactalbumin.

    Science.gov (United States)

    Brück, Wolfram M; Gibson, Glenn R; Brück, Thomas B

    2014-02-01

    α-Lactalbumin (α-la) is a major whey protein found in milk. Previous data suggested that α-la has antiproliferative effects in human adenocarcinoma cell lines such as Caco-2 and HT-29. However, the cell death inducing α-la was not a naturally occurring monomer but either a multimeric variant or an α-la:oleic acid complex (HAMLET/BAMLET). Proteolysis showed that both human and bovine α-la are susceptible to digestion. ELISA assays assessing cell death with the native undigested α-la fractions showed that undigested protein fractions did have a significant cell death effect on CaCo-2 cells. Bovine α-la was also more effective than human α-la. A reduction in activity corresponded with lower concentrations of the protein and partial digestion and fragmentation of the protein using trypsin and pepsin. This suggests that the tertiary structure is vital for the apoptotic effect.

  13. Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge

    CSIR Research Space (South Africa)

    Mallajosyula, JK

    2014-03-01

    Full Text Available Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low...

  14. Effects of actin-binding proteins on the thermal stability of monomeric actin.

    Science.gov (United States)

    Pivovarova, Anastasia V; Chebotareva, Natalia A; Kremneva, Elena V; Lappalainen, Pekka; Levitsky, Dmitrii I

    2013-01-08

    Differential scanning calorimetry (DSC) was applied to investigate the thermal unfolding of rabbit skeletal muscle G-actin in its complexes with actin-binding proteins, cofilin, twinfilin, and profilin. The results show that the effects of these proteins on the thermal stability of G-actin depend on the nucleotide, ATP or ADP, bound in the nucleotide-binding cleft between actin subdomains 2 and 4. Interestingly, cofilin binding stabilizes both ATP-G-actin and ADP-G-actin, whereas twinfilin increases the thermal stability of the ADP-G-actin but not that of the ATP-G-actin. By contrast, profilin strongly decreases the thermal stability of the ATP-G-actin but has no appreciable effect on the ADP-G-actin. Comparison of these DSC results with literature data reveals a relationship between the effects of actin-binding proteins on the thermal unfolding of G-actin, stabilization or destabilization, and their effects on the rate of nucleotide exchange in the nucleotide-binding cleft, decrease or increase. These results suggest that the thermal stability of G-actin depends, at least partially, on the conformation of the nucleotide-binding cleft: the actin molecule is more stable when the cleft is closed, while an opening of the cleft leads to significant destabilization of G-actin. Thus, DSC studies of the thermal unfolding of G-actin can provide new valuable information about the conformational changes induced by actin-binding proteins in the actin molecule.

  15. Spin Equilibria in Monomeric Manganocenes: Solid State Magnetic and EXAFS Studies

    Energy Technology Data Exchange (ETDEWEB)

    Walter, M. D.; Sofield, C. D.; Booth, C. H.; Andersen, R. A.

    2009-02-09

    Magnetic susceptibility measurements and X-ray data confirm that tert-butyl-substituted manganocenes [(Me{sub 3}C){sub n}C{sub 5}H{sub 5?n}]{sub 2}Mn (n = 1, 2) follow the trend previously observed with the methylated manganocenes; that is, electron-donating groups attached to the Cp ring stabilize the low-spin (LS) electronic ground state relative to Cp{sub 2}Mn and exhibit higher spin-crossover (SCO) temperatures. However, introducing three CMe{sub 3} groups on each ring gives a temperature-invariant high-spin (HS) state manganocene. The origin of the high-spin state in [1,2,4-(Me{sub 3}C){sub 3}C{sub 5}H{sub 2}]{sub 2}Mn is due to the significant bulk of the [1,2,4-(Me{sub 3}C){sub 3}C{sub 5}H{sub 2}]{sup -} ligand, which is sufficient to generate severe inter-ring steric strain that prevents the realization of the low-spin state. Interestingly, the spin transition in [1,3-(Me{sub 3}C){sub 2}C{sub 5}H{sub 3}]{sub 2}Mn is accompanied by a phase transition resulting in a significant irreversible hysteresis ({Delta}T{sub c} = 16 K). This structural transition was also observed by extended X-ray absorption fine-structure (EXAFS) measurements. Magnetic susceptibility studies and X-ray diffraction data on SiMe{sub 3}-substituted manganocenes [(Me{sub 3}Si){sub n}C{sub 5}H{sub 5-n}]{sub 2}Mn (n = 1, 2, 3) show high-spin configurations in these cases. Although tetra- and hexasubstituted manganocenes are high-spin at all accessible temperatures, the disubstituted manganocenes exhibit a small low-spin admixture at low temperature. In this respect it behaves similarly to [(Me{sub 3}C)(Me{sub 3}Si)C{sub 5}H{sub 3}]{sub 2}Mn, which has a constant low-spin admixture up to 90 K and then gradually converts to high-spin. Thermal spin-trapping can be observed for [(Me{sub 3}C)(Me{sub 3}Si)C{sub 5}H{sub 3}]{sub 2}Mn on rapid cooling.

  16. Sequence determination and modeling of structural motifs for the smallest monomeric aminoacyl-tRNA synthetase.

    OpenAIRE

    Hou, Y M; Shiba, K; Mottes, C; Schimmel, P.

    1991-01-01

    Polypeptide chains of 19 previously studied Escherichia coli aminoacyl-tRNA synthetases are as large as 951 amino acids and, depending on the enzyme, have quaternary structures of alpha, alpha 2, alpha 2 beta 2, and alpha 4. These enzymes have been organized into two classes which are defined by sequence motifs that are associated with specific three-dimensional structures. We isolated, cloned, and sequenced the previously uncharacterized gene for E. coli cysteine-tRNA synthetase (EC 6.1.1.16...

  17. Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins

    Directory of Open Access Journals (Sweden)

    Koga Yuichi

    2010-07-01

    Full Text Available Abstract Background The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI. Results To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII and Aquifex aeolicus (Aa-RNase HII and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI. These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins. Conclusions These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

  18. Order-disorder transitions govern kinetic cooperativity and allostery of monomeric human glucokinase.

    Directory of Open Access Journals (Sweden)

    Mioara Larion

    Full Text Available Glucokinase (GCK catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity-onset diabetes of the young type II (MODY-II and persistent hypoglycemic hyperinsulinemia of infancy (PHHI. GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder-order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder-order cycle of the small domain as a "time-delay loop," which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.

  19. A monomeric protein with hemagglutinating activity from seeds of Vigna mungo (Phaseolus mungo).

    Science.gov (United States)

    Singh, S S; Rao, S L

    1991-01-01

    Black gram (Vigna mungo) seeds are shown to contain a lectin with certain unusual features. The lectin agglutinates only trypsinized red cells, and its sugar specificity is complex as none of the common sugars, oligosaccharides or complex polysaccharides exhibit any affinity for the lectin. The purified lectin has a molecular weight of 58 kDa and is a monomer. Unlike other plant lectins, antibodies to the P. mungo lectin do not exhibit any immunological cross reactivity. The clot forming ability of the lectin is unusual in that the clot once formed is rapidly disaggregated indicated that it induces, as yet undefined, certain membrane alterations.

  20. Crystal structure of monomeric photosystem II from Thermosynechococcus elongatus at 3.6-a resolution

    NARCIS (Netherlands)

    Broser, Matthias; Gabdulkhakov, Azat; Kern, Jan; Guskov, Albert; Müh, Frank; Saenger, Wolfram; Zouni, Athina

    2010-01-01

    The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first c

  1. Who is Mr. HAMLET? Interaction of human alpha-lactalbumin with monomeric oleic acid.

    Science.gov (United States)

    Knyazeva, Ekaterina L; Grishchenko, Valery M; Fadeev, Roman S; Akatov, Vladimir S; Permyakov, Sergei E; Permyakov, Eugene A

    2008-12-09

    A specific state of the human milk Ca(2+) binding protein alpha-lactalbumin (hLA) complexed with oleic acid (OA) prepared using an OA-pretreated ion-exchange column (HAMLET) triggers several cell death pathways in various tumor cells. The possibility of preparing a hLA-OA complex with structural and cytotoxic properties similar to those of the HAMLET but under solution conditions has been explored. The complex was formed by titration of hLA by OA at pH 8.3 up to OA critical micelle concentration. We have shown that complex formation strongly depends on calcium, ionic strength, and temperature; the optimal conditions were established. The spectrofluorimetrically estimated number of OA molecules irreversibly bound per hLA molecule (after dialysis of the OA-loaded preparation against water followed by lyophilization) depends upon temperature: 2.9 at 17 degrees C (native apo-hLA; resulting complex referred to as LA-OA-17 state) and 9 at 45 degrees C (thermally unfolded apo-hLA; LA-OA-45). Intrinsic tryptophan fluorescence measurements revealed substantially decreased thermal stability of Ca(2+)-free forms of HAMLET, LA-OA-45, and OA-saturated protein. The irreversibly bound OA does not affect the Ca(2+) association constant of the protein. Phase plot analysis of fluorimetric and CD data indicates that the OA binding process involves several hLA intermediates. The effective pseudoequilibrium OA association constants for Ca(2+)-free hLA were estimated. The far-UV CD spectra of Ca(2+)-free hLA show that all OA-bound forms of the protein are characterized by elevated content of alpha-helical structure. The various hLA-OA complexes possess similar cytotoxic activities against human epidermoid larynx carcinoma cells. Overall, the LA-OA-45 complex possesses physicochemical, structural, and cytotoxic properties closely resembling those of HAMLET. The fact that the HAMLET-like complex can be formed in aqueous solution makes the process of its preparation more transparent and controllable, opening up opportunities for formation of active complexes with specific properties.

  2. Monomeric α-synuclein binds Congo Red micelles in a disordered manner.

    Science.gov (United States)

    Maltsev, Alexander S; Grishaev, Alexander; Bax, Ad

    2012-01-17

    The histological dye Congo Red (CR) previously has been shown to inhibit α-synuclein (aS) fibrillation, but the mode of this inhibition remained unclear. Because of favorable exchange kinetics, interaction between CR and aS lends itself to a detailed nuclear magnetic resonance study, and relaxation dispersion measurements yield the bound fraction and time scales for the interaction of aS with CR. We find that at pH 6, CR exists as a micelle, and at a CR:aS molar ratio of ~1, only a small fraction of aS (~2%) is bound to these micelles. Rapid exchange (k(ex) ~ 3000 s(-1)) between the free and CR-bound states broadens and strongly attenuates resonances of aS by two processes: a magnetic field-dependent contribution, caused by the chemical shift difference between the two states, and a nearly field-independent contribution caused by slower tumbling of aS bound to the CR micelle. The salt dependence of the interaction suggests a predominantly electrostatic mechanism for the 60 N-terminal residues, while the weaker interaction between residues 61-100 and CR is mostly hydrophobic. Chemical shift and transferred NOE data indicate that aS becomes slightly more helical but remains largely disordered when bound to CR. Results indicate that inhibition of fibril formation does not result from binding of CR to free aS and, therefore, must result from interaction of aS fibrils or protofibrils with CR micelles.

  3. Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography

    Science.gov (United States)

    Kuehne, Christian; Wedepohl, Stefanie; Dernedde, Jens

    2017-01-01

    l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin. PMID:28125045

  4. Monomeric Chiral and Achiral Basket-Handle Porphyrins: Synthesis, Structural Features, and Arrested Tautomerism.

    Science.gov (United States)

    Gehrold, Andreas C; Bruhn, Torsten; Schneider, Heidi; Radius, Udo; Bringmann, Gerhard

    2015-12-18

    Chiral and achiral basket-handle porphyrins (BHPs) with different p-xylene straps and peripheral solubilizing groups were synthesized using a previously established synthetic approach. Subsequent modification, functionalization, and metalation of the tetrapyrrolic macrocycle yielded more than 80 BHPs. The chiral representatives were resolved into their enantiomers, whose absolute configurations were determined by comparison of their ECD spectra with other experimental or quantum chemically calculated spectra. NMR studies and coupled-cluster calculations proved that the free base BHPs, although highly symmetric, exhibited the phenomenon of "arrested tautomerism". Comparison of the solid-state structures of three metalated BHPs offered detailed insight into their three-dimensional shape. Finally, directly linked dimeric porphyrins with a BHP subunit were synthesized from functionalized BHPs to prove their value as synthetic building blocks.

  5. Spectroscopic Characterization of a Monomeric, Cyclopentadienyl-Based Rhenium(V) Dioxo Complex

    DEFF Research Database (Denmark)

    Raju, Suresh; Jastrzebski, Johann T. B. H.; Lutz, Martin

    2015-01-01

    spectrometry. (Cpttt)ReO2 is shown to be the primary product of reduction of the rhenium(VII) complex (Cpttt)ReO3 with PPh3 and demonstrated to react with ethylene glycol significantly faster than its dimeric counterpart, supporting its role as an intermediate in rhenium-catalyzed deoxydehydration reactions....

  6. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

    Science.gov (United States)

    Cui, Boyu; Zhang, Lifeng; Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

  7. Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin.

    Science.gov (United States)

    Phang, Juanita M; Harrop, Stephen J; Duff, Anthony P; Sokolova, Anna V; Crossett, Ben; Walsh, James C; Beckham, Simone A; Nguyen, Cuong D; Davies, Roberta B; Glöckner, Carina; Bromley, Elizabeth H C; Wilk, Krystyna E; Curmi, Paul M G

    2016-09-15

    Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

  8. The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica.

    Science.gov (United States)

    Verma, Kuldeep; Datta, Sunando

    2017-03-24

    One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Conformational equilibria in monomeric alpha-synuclein at the single molecule level

    CERN Document Server

    Sandal, Massimo; Tessari, Isabella; Mammi, Stefano; Bergantino, Elisabetta; Musiani, Francesco; Brucale, Marco; Bubacco, Luigi; Samori', Bruno

    2007-01-01

    Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn ag...

  10. Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation.

    Directory of Open Access Journals (Sweden)

    Wahyu Surya

    Full Text Available The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

  11. Purification and properties of a monomeric lactate dehydrogenase from yak Hypoderma sinense larva.

    Science.gov (United States)

    Li, Pengfei; Jin, Suyu; Huang, Lin; Liu, Haohao; Huang, Zhihong; Lin, Yaqiu; Zheng, Yucai

    2013-06-01

    The objective of the present study was to study the characteristics of lactate dehydrogenase (LDH) from Hypoderma sinense larva. H. sinense larvae were collected from yak (Bos grunniens) and identified by a PCR-RFLP method. Analysis of LDH activity showed that the total LDH activity in H. sinense larva was negatively correlated with the length of larva. Polyacrylamide gel electrophoresis of the extracts of H. sinense larvae revealed one band of LDH, which was then purified by affinity chromatography and gel filtration. This enzyme showed an approximately 36 kDa band on SDS-gel under both reducing and non-reducing conditions, in addition, size exclusion chromatography analysis showed that its molecular weight was smaller than bovine serum albumin (67 kDa), indicating that it contains only one subunit. Michaelis constants (Km) values assay revealed that LDH from H. sinense larva showed significantly lower Km for lactate than other animals. LDH of H. sinense larva was stable at 60 °C for 15 min, and also exhibited high catalytic efficiency in a wide range of pH. HgCl₂ at the concentration of 0.1mM significantly decreased the activity of LDH from H. sinense larva but not at the concentration of 0.01 mM. The results of the present study demonstrate that LDH from H. sinense larva is a thermal stable and pH insensitive enzyme suitable for catalyzing both forward and reverse reactions.

  12. Ultrafast polarized fluorescence measurements on monomeric and self-associated melittin

    NARCIS (Netherlands)

    Pandit, A.; Larsen, O.F.A.; Stokkum, van I.H.M.; Grondelle, van R.; Kraayenhof, R.; Amerongen, van H.

    2003-01-01

    The anisotropic and magic-angle fluorescence decay of the single tryptophan (Trp) residue of melittin, a bee venom peptide, was investigated by time-resolved fluorescence anisotropy using a streak camera setup. The peptide was dissolved either in distilled water or in Hepes/NaOH buffer containing lo

  13. Stapling monomeric GCN4 peptides allows for DNA binding and enhanced cellular uptake.

    Science.gov (United States)

    Iyer, Abhishek; Van Lysebetten, Dorien; Ruiz García, Yara; Louage, Benoit; De Geest, Bruno G; Madder, Annemieke

    2015-04-01

    The basic DNA recognition region of the GCN4 protein comprising 23 amino acids has been modified to contain two optimally positioned cysteines which have been linked and stapled using cross-linkers of suitable lengths. This results in stapled peptides with a stabilized α-helical conformation which allows for DNA binding and concurrent enhancement of cellular uptake.

  14. The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids

    Directory of Open Access Journals (Sweden)

    Graham H Cowan

    2012-12-01

    Full Text Available The potato mop-top virus (PMTV triple gene block 2 (TGB2 movement protein fused to monomeric red fluorescent protein (mRFP-TGB2 was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localisations and interactions of mRFP-TGB2 were investigated using confocal imaging (CLSM and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum, mobile granules, small round structures (1-2 µm in diameter and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labelled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein, genomic RNA and fluorescently-labelled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localised to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.

  15. The main differences of accomplishment conditions of shooting exercise RFP in the training process for the XXIX and XXX Olympic Games.

    Directory of Open Access Journals (Sweden)

    Petriv O. S.

    2012-10-01

    Full Text Available Main changes and terms of implementation of the speed firing an are certain standard pistol on five targets which simultaneously appear on distance 25 meters. The features of such firing are analysed. The changes of cartridges, pull of trigger hook, new features of firing in a finale, are described. Assertion is grounded, that complication of implementation of the speed firing consists in exact co-ordination of aiming in the center of target with smooth pressure on a trigger hook. The necessity of development of method of prelaunch training of sportsmen is certain. The necessity of perfection of facilities and methods of prelaunch limbering-up is well-proven before implementation of olympic exercise the «Speed firing a pistol». Directions the correction of training of sportsmen are recommended in accordance with cardinal changes in order of leadthrough of finale.

  16. A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

    Science.gov (United States)

    Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled u...

  17. Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine

    Directory of Open Access Journals (Sweden)

    Wu Chaoqun

    2009-04-01

    Full Text Available Abstract Background Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB transposon and polyethylenimine (PEI for long-term transgene expression in mouse ovarian tumors. Methods A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk and the monomeric red fluorescent protein (mRFP1 under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Results Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Conclusion Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used

  18. Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host.

    Directory of Open Access Journals (Sweden)

    Keith W Jarosinski

    Full Text Available Hosts can be infected with multiple herpesviruses, known as superinfection; however, superinfection of cells is rare due to the phenomenon known as superinfection inhibition. It is believed that dual infection of cells occurs in nature, based on studies examining genetic exchange between homologous alphaherpesviruses in the host, but to date, this has not been directly shown in a natural model. In this report, gallid herpesvirus 2 (GaHV-2, better known as Marek's disease virus (MDV, was used in its natural host, the chicken, to determine whether two homologous alphaherpesviruses can infect the same cells in vivo. MDV shares close similarities with the human alphaherpesvirus, varicella zoster virus (VZV, with respect to replication in the skin and exit from the host. Recombinant MDVs were generated that express either the enhanced GFP (eGFP or monomeric RFP (mRFP fused to the UL47 (VP13/14 herpesvirus tegument protein. These viruses exhibited no alteration in pathogenic potential and expressed abundant UL47-eGFP or -mRFP in feather follicle epithelial cells in vivo. Using laser scanning confocal microscopy, it was evident that these two similar, but distinguishable, viruses were able to replicate within the same cells of their natural host. Evidence of superinfection inhibition was also observed. These results have important implications for two reasons. First, these results show that during natural infection, both dual infection of cells and superinfection inhibition can co-occur at the cellular level. Secondly, vaccination against MDV with homologous alphaherpesvirus like attenuated GaHV-2, or non-oncogenic GaHV-3 or meleagrid herpesvirus (MeHV-1 has driven the virus to greater virulence and these results implicate the potential for genetic exchange between homologous avian alphaherpesviruses that could drive increased virulence. Because the live attenuated varicella vaccine is currently being administered to children, who in turn could be

  19. Very bright orange fluorescent plants: endoplasmic reticulum targeting of orange fluorescent proteins as visual reporters in transgenic plants

    Directory of Open Access Journals (Sweden)

    Mann David GJ

    2012-05-01

    Full Text Available Abstract Background The expression of fluorescent protein (FP genes as real-time visual markers, both transiently and stably, has revolutionized plant biotechnology. A palette of colors of FPs is now available for use, but the diversity has generally been underutilized in plant biotechnology. Because of the green and far-red autofluorescent properties of many plant tissues and the FPs themselves, red and orange FPs (RFPs, and OFPs, respectfully appear to be the colors with maximum utility in plant biotechnology. Within the color palette OFPs have emerged as the brightest FP markers in the visible spectra. This study compares several native, near-native and modified OFPs for their “brightness” and fluorescence, therefore, their usability as marker genes in transgenic plant tissues. Results The OFPs DsRed2, tdTomato, mOrange and pporRFP were all expressed under the control of the CaMV 35S promoter in agroinfiltration-mediated transient assays in Nicotiana benthamiana. Each of these, as well as endoplasmic reticulum (ER-targeted versions, were stably expressed in transgenic Nicotiana tabacum and Arabidopsis thaliana. Congruent results were observed between transient and stable assays. Our results demonstrated that there are several adequate OFP genes available for plant transformation, including the new pporRFP, an unaltered tetramer from the hard coral Porites porites. When the tandem dimer tdTomato and the monomeric mOrange were targeted to the ER, dramatic, ca. 3-fold, increase in plant fluorescence was observed. Conclusions From our empirical data, and a search of the literature, it appears that tdTomato-ER and mOrange-ER are the two highest fluorescing FPs available as reporters for transgenic plants. The pporRFP is a brightly fluorescing tetramer, but all tetramer FPs are far less bright than the ER-targeted monomers we report here.

  20. Novel Infectivity-Enhanced Oncolytic Adenovirus with a Capsid-Incorporated Dual-Imaging Moiety for Monitoring Virotherapy in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kristopher J. Kimball

    2009-09-01

    Full Text Available We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for non-invasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk and monomeric red fluorescent protein 1 (mRFP1 into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

  1. pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins.

    Science.gov (United States)

    Gong, Xiaoyan; Hurtado, Oscar; Wang, Baohua; Wu, Congqing; Yi, Mihwa; Giraldo, Martha; Valent, Barbara; Goodin, Michael; Farman, Mark

    2015-02-01

    As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

  2. In vivo modulation and quantification of microRNAs during axolotl tail regeneration.

    Science.gov (United States)

    Erickson, Jami R; Echeverri, Karen

    2015-01-01

    The ability to regenerate diseased, injured, or missing complex tissue is widespread throughout lower vertebrates and invertebrates; however, our knowledge of the molecular mechanisms that regulate this amazing ability is still in its infancy. Many recent papers have shown important roles for microRNAs in regulating regeneration in a number of species. The ability to detect and quantify miRNA expression fluctuations at a single cell level in vivo in different cell types during processes like regeneration is very informative. In this chapter, we describe how to use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time following miRNA mimic injection as well as during regeneration. In this bicistronic vector, the mRFP allows for verification of miRNA expression, while the GFP functions as an internal control to normalize miRNA expression and thus obtain quantitative results. In addition, we demonstrate how this technique revealed dynamic miR-23a expression and function during tail regeneration.

  3. The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique; Sibler, Annie-Paule; Baltzinger, Mireille [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France); Rochel, Natacha [Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, CNRS/INSERM/Université de Strasbourg, rue Laurent Fries, 67404 Illkirch (France); Mély, Yves; Didier, Pascal [Faculté de Pharmacie, UMR 7213, CNRS/Université de Strasbourg, route du Rhin, 67401 Illkirch (France); Weiss, Etienne, E-mail: eweiss@unistra.fr [Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de Strasbourg, boulevard Sébastien Brant, 67412 Illkirch (France)

    2013-04-01

    Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and the ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.

  4. Interfacial polymerization of cyanuric chloride and monomeric amines: pH resistant thin film composite polyamine nanofiltration membranes

    NARCIS (Netherlands)

    Lee, Kah P.; Bargeman, Gerrald; Rooij, de Ralph; Kemperman, Antoine J.B.; Benes, Nieck E.

    2017-01-01

    Polyamine nanofiltration membranes have a high stability at extreme pH conditions. In contrast to polyamides, polyamines do not contain the carbonyl group that is susceptible to nucleophilic attack. A previous study has shown that polyamine membranes can be prepared from the interfacial polymerizati

  5. Molecular orbital study of coordinated dioxygen. I. Structure and bonding of model monomeric Co(II) complexes

    Science.gov (United States)

    Boča, R.

    1980-08-01

    The CNDO—UHF type of MO—LCAO—SCP calculation is carried out for model systems of dioxygen fixation: O 2 CoCl 4L 2- complexes in which L = none and L = NH 3. A geometry variation is performed with respect to 5 internal coordinates describing the degrees of freedom of the Co—O 2 group. The calculated geometry, spin densities and atomic charges agree with available data based on X-ray and ESR measurements of real dioxygen carriers. Structure and bonding of complexes are discussed in more detail.

  6. Interactions between nitric oxide and ethylene in monomeric G-protein activation in relation to food spoilage

    DEFF Research Database (Denmark)

    Hall, M A; moshkov, moshkov; Novikova, G

    2014-01-01

    and it is notable that many are dependent on the production of volatile signals or signals which have volatile derivatives. Ethylene (ET) has long been recognized as an important regulator of development, stress responses, senescence and food spoilage. Our work has focused on the gaseous signal nitric oxide (NO......) and how it interacts with established stress signalling pathways and in particular, those regulated by ET. Using laser photoacoustic detection (LPAD) we have established that NO production overlaps with that of ethylene during plant responses to disease. To examine the interaction of NO and ET signalling...... approach and 2D-electrophoresis (2DE) a series of GTP binding proteins which were activated by both ethylene and SNP were detected and some that exhibited specific activation patterns in response to both signals. These observations underline the close relationship between ET and NO signalling cascades...

  7. Proline substitution of dimer interface β-strand residues as a strategy for the design of functional monomeric proteins.

    Science.gov (United States)

    Joseph, Prem Raj B; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M; Garofalo, Roberto P; Rajarathnam, Krishna

    2013-09-17

    Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Kinetic parameters and monomeric conversion of different dental composites using standard and soft-start photoactivation modes

    Science.gov (United States)

    Denis, A. B.; Viana, R. B.; Plepis, A. M. G.

    2012-06-01

    This paper evaluates the photopolymerization kinetics and degree of conversion of different commercial dental composites when photoactivated by a LED curing unit using two different modes (standard and soft-start mode). The investigation was performed on with RelyX ARC (dual-cured), Filtek Z-350 (Nanocomposite), Filtek Z-250 (Hybrid), and Filtek Z-350flow (Flowable) resin composites. The analysis used was attenuated total reflection with a Fourier transform infrared (ATR-FTIR). The RelyX ARC resin demonstrated the highest degree of conversion with both LED photoactivation modes. For this resin a 28% decrease in maximum rate was observed and the time to reach its highest rate was almost 2.3 times higher than when the soft-start photoactivation light curing was used. Z-350flow resin recorder a higher maximum rate using the soft-start mode rather than the standard mode. In contrast, the Z-250 showed a higher value using the standard mode. Although Z-250 and Z-350 showed a higher total degree of conversion effectiveness using the soft-start mode, RelyX and Z-350flow achieved a higher value using the standard mode.

  9. Structures and Magnetic Properties of Monomeric Copper(II) Bromide Complexes with a Pyridine-Containing Tridentate Schiff Base

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Sung Kwon [Chungnam National Univ., Daejeon (Korea, Republic of); Yong, Soon Jung; Song, Young Kwang; Kim, Young Inn [Pusan National Univ., Busan (Korea, Republic of)

    2013-12-15

    Two novel copper(II) bromide complexes with pyridine containing Schiff base ligands, Cu(pmed)Br{sub 2} and Cu(dpmed)Br{sub 2} where pmed = N'-((pyridin-2-yl)methylene)ethane-1,2-diamine (pmed) and dpmed = N,N-diethyl-N'-((pyridin-2-yl)methylene)ethane-1,2-diamine (dpmed) were synthesized and characterized using Xray single crystal structure analysis, optical and magnetic susceptibility measurements. Crystal structural analysis of Cu(pmed)Br{sub 2} showed that the copper(II) ion has a distorted square-pyramidal geometry with the trigonality index of τ = 0.35 and two intermolecular hydrogen bonds, which result in the formation of two dimensional networks in the ab plane. On the other hand, Cu(dpmed)Br{sub 2} displayed a near square-pyramidal geometry with the value of τ = 0.06. In both compounds, the NNN Schiff base and one Br atom occupy the basal plane, whereas the fifth apical position is occupied by the other Br atom at a greater Cu-Br apical distance. The reported complexes show g{sub Π} > g{sub Τ} > 2.0023 with a d{sub x2-y2} ground state and a penta-coordinated square pyramidal geometry. Variable temperature magnetic susceptibility measurements showed that the developed copper(II) complexes follow the Curie-Weiss law, that is there are no magnetic interactions between the copper(II) ions since the Cu--Cu distance is too far for magnetic contact.

  10. Absorption Mechanism of a Physical Complex of Monomeric Insulin and Deoxycholyl-l-lysyl-methylester in the Small Intestine.

    Science.gov (United States)

    Mahmud, Foyez; Jeon, Ok-Cheol; Al-Hilal, Taslim A; Kweon, Seho; Yang, Victor C; Lee, Dong Soo; Byun, Youngro

    2015-06-01

    Currently, oral administration of insulin still remains the best option to avoid the burden of repeated subcutaneous injections and to improve its pharmacokinetics. The objective of the present investigation was to demonstrate the absorption mechanism of insulin in the physical complexation of deoxycholyl-l-lysyl-methylester (DCK) for oral delivery. The oral insulin/DCK complex was prepared by making a physical complex of insulin aspart with DCK through ion-pair interaction in water. For the cellular uptake study, fluorescein-labeled insulin or DCK were prepared according to a standard protocol and applied to Caco-2 or MDCK cell lines. For the PK/PD studies, we performed intrajejunal administration of different formulation of insulin/DCK complex to diabetic rats. The resulting insulin and DCK complex demonstrated greatly enhanced lipophilicity as well as increased permeation across Caco-2 monolayers. The immunofluorescence study revealed the distribution of the complex in the cytoplasm of Caco-2 cells. Moreover, in the apical sodium bile acid transporter (ASBT) transfected MDCK, the insulin/DCK complex showed interaction with ASBT, and also demonstrated absorption through passive diffusion. We could not find that any evidence of endocytosis in relation to the uptake of insulin complex in vitro. In the rat intestine model, the highest absorption of insulin complex was observed in the jejunum at 1 h and then in the ileum at 2-4 h. In PK/PD study, the complex showed a similar PK profile to that of SC insulin. Overall, the study showed that the effect of DCK on enhancing the absorption of insulin resulted from transcellular processes as well as bile acid transporter activity.

  11. Laforin, a dual specificity phosphatase involved in Lafora disease, is present mainly as monomeric form with full phosphatase activity.

    Directory of Open Access Journals (Sweden)

    Vikas V Dukhande

    Full Text Available Lafora Disease (LD is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs. LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both in vitro and in vivo. Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.

  12. Purification, characterization and regulation of a monomeric L-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

    NARCIS (Netherlands)

    Boer, L. de; Rijssel, M. van; Euverink, G.J.; Dijkhuizen, L.

    1989-01-01

    In Nocardia sp. 239 D-phenylalanine is converted into L-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent L-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on L- or D-phenylalanine and in batch cultures hi

  13. Symmetry breaking in navigating cells : The role of heterotrimeric and monomeric G-proteins in Dictyostelium chemotaxis

    NARCIS (Netherlands)

    Kataria, Rama

    2015-01-01

    Chemotaxis is the property of cells to migrate towards gradients of chemicals. It is a highly dynamic process that involves directional sensing, cellular polarity and cell motility. Chemotaxis is important for all organisms for many processes including, immune reponse, embryo organization and it’s a

  14. Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yang [Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala (Sweden); Näsvall, Joakim [Uppsala University, Biomedical Center, Box 582, SE-751 23 Uppsala (Sweden); Wu, Shiying [Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala (Sweden); Andersson, Dan I. [Uppsala University, Biomedical Center, Box 582, SE-751 23 Uppsala (Sweden); Selmer, Maria, E-mail: maria.selmer@icm.uu.se [Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala (Sweden)

    2015-10-31

    The crystal structure of the aminoglycoside-adenylating enzyme AadA is reported together with functional experiments providing insights into its oligomeric state, ligand binding and catalysis. Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

  15. Field Evaluation of Solvent-Free Sampling with Di-n-butylamine for the Determination of Airborne Monomeric and Oligomeric 1,6-Hexamethylene Diisocyanate

    Science.gov (United States)

    2014-03-27

    in other organ systems . Acute exposure to high levels of HDI vapor has been linked to central nervous system effects including lightheadedness...Species Exposure Duration NOAEL LOAEL LD50 Acute Exposures Rats 6 hr 4-8 hr - 5 ppm 3 ppm 11 ppm 44 ppm - Guinea Pig 2-6 hr 0.5 ppm 1.8 ppm 4...sensitization in guinea pigs , rabbits, and mice. Although dermal absorption is thought to be slow, HDI reacts with the skin proteins to produce a

  16. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

    NARCIS (Netherlands)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

  17. Molecular structure (monomeric and dimeric) and hydrogen bonds in 5-benzyl 2-thiohydantoin studied by FT-IR and FT-Raman spectroscopy and DFT calculations.

    Science.gov (United States)

    Deval, Vipin; Kumar, Amit; Gupta, Vineet; Sharma, Anamika; Gupta, Archana; Tandon, Poonam; Kunimoto, Ko-Ki

    2014-11-11

    In the present work the structural and spectral characteristics of 5-benzyl-2-thiohydantoin (5-BTH) have been studied by methods of infrared, Raman spectroscopy and quantum chemistry. Electrostatic potential surface, optimized geometry, harmonic vibrational frequencies, infrared intensities and activities of Raman scattering were calculated by density functional theory (DFT) employing B3LYP with complete relaxation in the potential energy surface using 6-311G++(d,p) basis set. Our results support the hydrogen bonding pattern proposed by reported crystalline structure. Stability of the molecule arising from hyperconjugative interactions, charge delocalization have been analyzed using natural bond orbital (NBO) analysis. The 13C nuclear magnetic resonance (NMR) chemical shifts of the molecule are calculated by the gauge independent atomic orbital (GIAO) method and compared with experimental results. UV-vis spectrum of the compound was recorded in methanol solvent. The TD-DFT calculations have been performed to explore the influence of electronic absorption spectra in the gas phase, as well as in solution environment using PCM and 6-311++G(d,p) basis set. In addition, the thermodynamic properties of the compound were calculated at different temperatures and corresponding relations between the properties and temperature were also studied.

  18. The Caenorhabditis elegans pericentriolar material components SPD-2 and SPD-5 are monomeric in the cytoplasm before incorporation into the PCM matrix

    DEFF Research Database (Denmark)

    Wueseke, Oliver; Bunkenborg, Jakob; Hein, Marco Y;

    2014-01-01

    Centrosomes are the main microtubule-organizing centers in animal cells. Centrosomes consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that assembles from cytoplasmic components. In Caenorhabditis elegans embryos, interactions between the coiled-coil proteins...

  19. 单体香料的合成技术研究进展%Research progress in synthesis techniques of monomeric spice

    Institute of Scientific and Technical Information of China (English)

    赵晓娟; 白卫东

    2011-01-01

    文章介绍了近年来单体香料合成技术的研究进展,对典型的几种合成技术,如生物技术、新型催化技术、手性合成技术、区域和立体选择性合成技术,分别进行重点论述和分析,同时展望了香料合成技术的发展趋势.

  20. Assessment of the dietary intake of total flavan-3-ols, monomeric flavan-3-ols, proanthocyanidins and theaflavins in the European Union.

    Science.gov (United States)

    Vogiatzoglou, Anna; Mulligan, Angela A; Luben, Robert N; Lentjes, Marleen A H; Heiss, Christian; Kelm, Malte; Merx, Marc W; Spencer, Jeremy P E; Schroeter, Hagen; Kuhnle, Gunter G C

    2014-04-28

    Dietary interventions with flavan-3-ols have shown beneficial effects on vascular function. The translation of these findings into the context of the health of the general public requires detailed information on habitual dietary intake. However, only limited data are currently available for European populations. Therefore, in the present study, we assessed the habitual intake of flavan-3-ol monomers, proanthocyanidins (PA) and theaflavins in the European Union (EU) and determined their main food sources using the EFSA (European Food Safety Authority) Comprehensive European Food Consumption Database. Data for adults aged 18-64 years were available from fourteen European countries, and intake was determined using the FLAVIOLA Flavanol Food Composition Database, developed for the present study and based on the latest US Department of Agriculture and Phenol-Explorer databases. The mean habitual intake of flavan-3-ol monomers, theaflavins and PA ranged from 181 mg/d (Czech Republic) to 793 mg/d (Ireland). The highest intakes of flavan-3-ol monomers and theaflavins were observed in Ireland (191/505 mg/d) and the lowest intakes in Spain (24/9 mg/d). In contrast, the daily intake of PA was highest in Spain (175 mg/d) and lowest in The Netherlands (96 mg/d). Main sources were tea (62%), pome fruits (11%), berries (3%) and cocoa products (3%). Tea was the major single contributor to monomer intake (75%), followed by pome fruits (6%). Pome fruits were also the main source of PA (28%). The present study provides important data on the population-based intake of flavanols in the EU and demonstrates that dietary intake amounts for flavan-3-ol monomers, PA and theaflavins vary significantly across European countries. The average habitual intake of flavan-3-ols is considerably below the amounts used in most dietary intervention studies.

  1. Ultrahigh and High Resolution Structures and Mutational Analysis of Monomeric Streptococcus pyogenes SpeB Reveal a Functional Role for the Glycine-rich C-terminal Loop

    Energy Technology Data Exchange (ETDEWEB)

    González-Páez, Gonzalo E.; Wolan, Dennis W. (Scripps)

    2012-09-05

    Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 {angstrom} resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC{sub 50} values for trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.

  2. Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits.

    Science.gov (United States)

    Yasmeen, Anila; Ringe, Rajesh; Derking, Ronald; Cupo, Albert; Julien, Jean-Philippe; Burton, Dennis R; Ward, Andrew B; Wilson, Ian A; Sanders, Rogier W; Moore, John P; Klasse, Per Johan

    2014-05-29

    The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus. The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs.

  3. Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties.

    Science.gov (United States)

    Don Paul, Craig; Traore, Daouda A K; Byres, Emma; Rossjohn, Jamie; Devenish, Rodney J; Kiss, Csaba; Bradbury, Andrew; Wilce, Matthew C J; Prescott, Mark

    2011-10-01

    Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V(M) = 2.26 Å(3) Da(-1)) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.

  4. Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors.

    Science.gov (United States)

    Sharma, Navneet; Fahr, Jochen; Renaux, Bernard; Saifeddine, Mahmoud; Kumar, Rajeev; Nishikawa, Sandra; Mihara, Koichiro; Ramachandran, Rithwik; Hollenberg, Morley D; Rancourt, Derrick E

    2013-12-01

    Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.

  5. Role of polymeric endosomolytic agents in gene transfection: a comparative study of poly(L-lysine) grafted with monomeric L-histidine analogue and poly(L-histidine).

    Science.gov (United States)

    Hwang, Hee Sook; Hu, Jun; Na, Kun; Bae, You Han

    2014-10-13

    Endosomal entrapment is one of the main barriers that must be overcome for efficient gene expression along with cell internalization, DNA release, and nuclear import. Introducing pH-sensitive ionizable groups into the polycationic polymers to increase gene transfer efficiency has proven to be a useful method; however, a comparative study of introducing equal numbers of ionizable groups in both polymer and monomer forms, has not been reported. In this study, we prepared two types of histidine-grafted poly(L-lysine) (PLL), a stacking form of poly(L-histidine) (PLL-g-PHis) and a mono-L-histidine (PLL-g-mHis) with the same number of imidazole groups. These two types of histidine-grafted PLL, PLL-g-PHis and PLL-g-mHis, showed profound differences in hemolytic activity, cellular uptake, internalization, and transfection efficiency. Cy3-labeled PLL-g-PHis showed strong fluorescence in the nucleus after internalization, and high hemolytic activity upon pH changes was also observed from PLL-g-PHis. The arrangement of imidazole groups from PHis also provided higher gene expression than mHis due to its ability to escape the endosome. mHis or PHis grafting reduced the cytotoxicity of PLL and changed the rate of cellular uptake by changing the quantity of free ε-amines available for gene condensation. The subcellular localization of PLL-g-PHis/pDNA measured by YOYO1-pDNA intensity was highest inside the nucleus, while the lysotracker, which stains the acidic compartments was lowest among these polymers. Thus, the polymeric histidine arrangement demonstrate the ability to escape the endosome and trigger rapid release of polyplexes into the cytosol, resulting in a greater amount of pDNA available for translocation to the nucleus and enhanced gene expression.

  6. The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules

    NARCIS (Netherlands)

    Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

    2006-01-01

    Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including

  7. SAXS Studies of the Endoglucanase Cel12A from Gloeophyllum trabeum Show Its Monomeric Structure and Reveal the Influence of Temperature on the Structural Stability of the Enzyme

    Directory of Open Access Journals (Sweden)

    Lis S. Miotto

    2014-07-01

    Full Text Available Endoglucanases are key enzymes applied to the conversion of biomass aiming for second generation biofuel production. In the present study we obtained the small angle X-ray scattering (SAXS structure of the G. trabeum endo-1,4-β-glucanase Cel12A and investigated the influence of an important parameter, temperature, on both secondary and tertiary structure of the enzyme and its activity. The CD analysis for GtCel12A revealed that changes in the CD spectra starts at 55 °C and the Tm calculated from the experimental CD sigmoid curve using the Boltzmann function was 60.2 ± 0.6 °C. SAXS data showed that GtCel12A forms monomers in solution and has an elongated form with a maximum diameter of 60 ± 5 Å and a gyration radius of 19.4 ± 0.1 Å as calculated from the distance distribution function. Kratky analysis revealed that 60 °C is the critical temperature above which we observed clear indications of denaturation. Our results showed the influence of temperature on the stability and activity of enzymes and revealed novel structural features of GtCel12A.

  8. “Covalent Hydration” Reactions in Model Monomeric Ru 2,2'-Bipyridine Complexes: Thermodynamic Favorability as a Function of Metal Oxidation and Overall Spin States

    Energy Technology Data Exchange (ETDEWEB)

    Ozkanlar, Abdullah; Cape, Jonathan L.; Hurst, James K.; Clark, Aurora E.

    2011-09-05

    Density functional theory (DFT) has been used to investigate the plausibility of water addition to the simple mononuclear ruthenium complexes, [(NH{sub 3}){sub 3}(bpy)Ru=O]{sup 2+}/{sup 3+} and [(NH{sub 3}){sub 3}(bpy)RuOH]{sup 3+}, in which the OH fragment adds to the 2,2{prime}-bipyridine (bpy) ligand. Activation of bpy toward water addition has frequently been postulated within the literature, although there exists little definitive experimental evidence for this type of 'covalent hydration'. In this study, we examine the energetic dependence of the reaction upon metal oxidation state, overall spin state of the complex, as well as selectivity for various positions on the bipyridine ring. The thermodynamic favorability is found to be highly dependent upon all three parameters, with free energies of reaction that span favorable and unfavorable regimes. Aqueous addition to [(NH{sub 3}){sub 3}(bpy)Ru=O]{sup 3+} was found to be highly favorable for the S = 1/2 state, while reduction of the formal oxidation state on the metal center makes the reaction highly unfavorable. Examination of both facial and meridional isomers reveals that when bipyridine occupies the position trans to the ruthenyl oxo atom, reactivity toward OH addition decreases and the site preferences are altered. The electronic structure and spectroscopic signatures (EPR parameters and simulated spectra) have been determined to aid in recognition of 'covalent hydration' in experimental systems. EPR parameters are found to uniquely characterize the position of the OH addition to the bpy as well as the overall spin state of the system.

  9. Experimental and time-dependent density functional theory characterization of the UV-visible spectra of monomeric and μ-oxo dimeric ferriprotoporphyrin IX.

    Science.gov (United States)

    Kuter, David; Venter, Gerhard A; Naidoo, Kevin J; Egan, Timothy J

    2012-10-01

    Speciation of ferriprotoporphyrin IX, Fe(III)PPIX, in aqueous solution is complex. Despite the use of its characteristic spectroscopic features for identification, the theoretical basis of the unique UV-visible absorbance spectrum of μ-[Fe(III)PPIX](2)O has not been explored. To investigate this and to establish a structural and spectroscopic model for Fe(III)PPIX species, density functional theory (DFT) calculations were undertaken for H(2)O-Fe(III)PPIX and μ-[Fe(III)PPIX](2)O. The models agreed with related Fe(III)porphyrin crystal structures and reproduced vibrational spectra well. The UV-visible absorbance spectra of H(2)O-Fe(III)PPIX and μ-[Fe(III)PPIX](2)O were calculated using time-dependent DFT and reproduced major features of the experimental spectra of both. Transitions contributing to calculated excitations have been identified. The features of the electronic spectrum calculated for μ-[Fe(III)PPIX](2)O were attributed to delocalization of electron density between the two porphyrin rings of the dimer, the weaker ligand field of the axial ligand, and antiferromagnetic coupling of the Fe(III) centers. Room temperature magnetic circular dichroism (MCD) spectra have been recorded and are shown to be useful in distinguishing between these two Fe(III)PPIX species. Bands underlying major spectroscopic features were identified through simultaneous deconvolution of UV-visible and MCD spectra. Computed UV-visible spectra were compared to deconvoluted spectra. Interpretation of the prominent bands of H(2)O-Fe(III)PPIX largely conforms to previous literature. Owing to the weak paramagnetism of μ-[Fe(III)PPIX](2)O at room temperature and the larger number of underlying excitations, interpretation of its experimental UV-visible spectrum was necessarily tentative. Nonetheless, comparison with the calculated spectra of antiferromagnetically coupled and paramagnetic forms of the μ-oxo dimer of Fe(III)porphine suggested that the composition of the Soret band involves a mixture of π→π* and π→d(π) charge transfer transitions. The Q-band and charge transfer bands appear to amalgamate into a mixed low energy envelope consisting of excitations with heavily admixed π→π* and charge transfer transitions.

  10. Heterologous protection elicited by candidate monomeric recombinant HIV-1 gp120 vaccine in the absence of cross neutralising antibodies in a macaque model

    Directory of Open Access Journals (Sweden)

    Page Mark

    2012-07-01

    Full Text Available Abstract Background Current data suggest that an efficacious human immunodeficiency virus type 1 (HIV-1 vaccine should elicit both adaptive humoral and cell mediated immune responses. Such a vaccine will also need to protect against infection from a range of heterologous viral variants. Here we have developed a simian-human immunodeficiency virus (SHIV based model in cynomolgus macaques to investigate the breadth of protection conferred by HIV-1W61D recombinant gp120 vaccination against SHIVsbg and SHIVSF33 challenge, and to identify correlates of protection. Results High titres of anti-envelope antibodies were detected in all vaccinees. The antibodies reacted with both the homologous HIV-1W61D and heterologous HIV-1IIIB envelope rgp120 which has an identical sequence to the SHIVsbg challenge virus. Significant titres of virus neutralising antibodies were detected against SHIVW61D expressing an envelope homologous with the vaccine, but only limited cross neutralisation against SHIVsbg, SHIV-4 and SHIVSF33 was observed. Protection against SHIVsbg infection was observed in vaccinated animals but none was observed against SHIVSF33 challenge. Transfer of immune sera from vaccinated macaques to naive recipients did not confer protection against SHIVsbg challenge. In a follow-up study, T cell proliferative responses detected after immunisation with the same vaccine against a single peptide present in the second conserved region 2 of HIV-1 W61D and HIV-1 IIIB gp120, but not SF33 gp120. Conclusions Following extended vaccination with a HIV-1 rgp120 vaccine, protection was observed against heterologous virus challenge with SHIVsbg, but not SHIVSF33. Protection did not correlate with serological responses generated by vaccination, but might be associated with T cell proliferative responses against an epitope in the second constant region of HIV-1 gp120. Broader protection may be obtained with recombinant HIV-1 envelope based vaccines formulated with adjuvants that generate proliferative T cell responses in addition to broadly neutralising antibodies.

  11. A hydrophobic gold surface triggers misfolding and aggregation of the amyloidogenic Josephin domain in monomeric form, while leaving the oligomers unaffected.

    Directory of Open Access Journals (Sweden)

    Alessandra Apicella

    Full Text Available Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD, a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity.

  12. Comparison between the efficacy of dimeric and monomeric non-ionic contrast media (iodixanol vs iopromide) in urography in patients with macroscopic haematuria

    Energy Technology Data Exchange (ETDEWEB)

    Stacul, F.; Cova, M.; Pravato, M. [Department of Radiology, University of Trieste, Cattinara Hospital, Strada di Fiume, 447, 34149 Trieste (Italy); Floriani, I. [Istituto di Ricerche Farmacologiche Mario Negri, Milan (Italy)

    2003-04-01

    Non-ionic dimers induce less diuresis than non-ionic monomers, resulting in increased opacification of the urinary tract in intravenous urography. This trial compared the diagnostic efficacy of iodixanol and iopromide in patients with macroscopic haematuria. One hundred consecutive patients with normal renal function and macroscopic haematuria entered a double blind, comparative, randomised, parallel trial. Contrast media were given with bolus injection in doses of 300 mgI/kg b.w. Radiographs were blindly evaluated by three radiologists who analysed different parameters (calyceal density and filling, papillary blush detection, delineation of collecting ducts, renal pelvis opacification, visualisation of ureters, bladder density, bladder distention) and estimated the diagnostic confidence (whether abnormal findings were definitely absent, probably absent, doubtful, probably present or certainly present). Radiological diagnoses were compared with final diagnoses. Results were summarised as the ratio of the odds of having a worse performance of iopromide vs iodixanol. Iodixanol showed a significantly better calyceal density and filling [odds ratio (OR): 1.96; 95% confidence interval (CI): 1.60-2.41], a significantly better pelvis opacification (OR 2.91; CI 2.02-4.18) and a significantly more frequent papillary blush detection (OR 1.95; CI 1.29-2.95). Iopromide showed a significantly better ureteral visualisation (OR 0.67; CI 0.48-0.92) and a significantly higher bladder distention (OR 0.59; CI 0.36-0.99). Iodixanol allowed a significantly higher diagnostic confidence as to calyceal evaluation (OR 1.35; CI 1.01-1.79). No significant differences were found with regard to other parameters. The results confirmed theoretical expectations. The higher opacification provided by iodixanol allowed better results and a higher diagnostic confidence in the upper excretory pathway. (orig.)

  13. Monomeric 55-kDa guanidinobenzoatase switches to a serine proteinase activity upon tetramerization. Tetrameric proteinase SP 220 K appears as the native form.

    Science.gov (United States)

    Poustis-Delpont, C; Thaon, S; Auberger, P; Gerardi-Laffin, C; Sudaka, P; Rossi, B

    1994-05-20

    Guanidinobenzoatases are cell surface enzymes present in cells capable of migration or remodeling. The guanidinobenzoatase purified to homogeneity from human renal carcinoma did not display gelatinase activity under the 55-kDa form (Poustis-Delpont, C., Descomps, R., Auberger, P., Delque-Bayer, P., Sudaka, P., and Rossi, B. (1992) Cancer Res. 52, 3622-3628). We bring new insights into the structure-activity relationships of this enzyme using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]diisopropyl fluorophosphate labeling, gelatin zymography, and immunodetection using a polyclonal antibody raised against the 55-kDa entity. Upon aggregation into a 220-kDa form, the enzyme exhibited [3H]diisopropyl fluorophosphate labeling and diisopropyl fluorophosphate-inhibitable gelatinase activity whereas its capability to cleave p-nitrophenyl p'-guanidinobenzoate as a substrate was abolished. Thus, the guanidinobenzoatase property appears as a feature of a 55-kDa inactive form of a serine proteinase subunit. After boiling in the presence of sodium dodecyl sulfate (3% w/v), the 220-kDa entity subjected to SDS-polyacrylamide gel electrophoresis could be dissociated into a 55-kDa protein as shown by silver staining. The resulting 55-kDa band remained [3H]diisopropyl fluorophosphate-labeled and reacted with anti-55-kDa guanidinobenzoatase antibodies, strongly suggesting that the 220-kDa proteinase was a noncovalently associated tetramer. Interestingly, Triton X-100 extracts of renal carcinoma plasma membranes exhibited a 220-kDa serine proteinase activity, as expressed in gelatin zymography, which was barely detectable in the non-tumoral counterpart. It is noteworthy that an anti-55-kDa guanidinobenzoatase reactive 220-kDa species was also observed in renal carcinoma plasma membranes extracts as assessed by Western blot, whereas it was hardly visible in the non-tumoral counterpart. No signal was immunodetected at M(r) 55,000 in renal carcinoma and kidney cortex membranes in Western blot experiments. Taken together, our data support the idea that the enzyme is expressed under its tetrameric form in the membrane. The purified enzyme is able to exhibit a serine proteinase activity when it recovers its native tetrameric form. This high molecular weight tetrameric proteinase SP 220 K appears as a new member of the cell surface serine protease family.

  14. Identification of the Phenol Functionality in Deprotonated Monomeric and Dimeric Lignin Degradation Products via Tandem Mass Spectrometry Based on Ion-Molecule Reactions with Diethylmethoxyborane

    Science.gov (United States)

    Zhu, Hanyu; Max, Joann P.; Marcum, Christopher L.; Luo, Hao; Abu-Omar, Mahdi M.; Kenttämaa, Hilkka I.

    2016-08-01

    Conversion of lignin into smaller molecules provides a promising alternate and sustainable source for the valuable chemicals currently derived from crude oil. Better understanding of the chemical composition of the resulting product mixtures is essential for the optimization of such conversion processes. However, these mixtures are complex and contain isomeric molecules with a wide variety of functionalities, which makes their characterization challenging. Tandem mass spectrometry based on ion-molecule reactions has proven to be a powerful tool in functional group identification and isomer differentiation for previously unknown compounds. This study demonstrates that the identification of the phenol functionality, the most commonly observed functionality in lignin degradation products, can be achieved via ion-molecule reactions between diethylmethoxyborane (DEMB) and the deprotonated analyte in the absence of strongly electron-withdrawing substituents in the ortho- and para-positions. Either a stable DEMB adduct or an adduct that has lost a methanol molecule (DEMB adduct-MeOH) is formed for these ions. Deprotonated phenols with an adjacent phenol or hydroxymethyl functionality or a conjugated carboxylic acid functionality can be identified based on the formation of DEMB adduct-MeOH. Deprotonated compounds not containing the phenol functionality and phenols containing an electron-withdrawing ortho- or para-substituent were found to be unreactive toward diethylmethoxyborane. Hence, certain deprotonated isomeric compounds with phenol and carboxylic acid, aldehyde, carboxylic acid ester, or nitro functionalities can be differentiated via these reactions. The above mass spectrometry method was successfully coupled with high-performance liquid chromatography for the analysis of a complex biomass degradation mixture.

  15. A monomeric complex of ammonia and cuprous chloride: H{sub 3}N⋯CuCl isolated and characterised by rotational spectroscopy and ab initio calculations

    Energy Technology Data Exchange (ETDEWEB)

    Bittner, Dror M.; Zaleski, Daniel P.; Stephens, Susanna L.; Walker, Nicholas R., E-mail: nick.walker@newcastle.ac.uk, E-mail: a.c.legon@bristol.ac.uk [School of Chemistry, Newcastle University, Bedson Building, Newcastle upon Tyne, Tyne and Wear NE1 7RU (United Kingdom); Tew, David P.; Legon, Anthony C., E-mail: nick.walker@newcastle.ac.uk, E-mail: a.c.legon@bristol.ac.uk [School of Chemistry, University of Bristol, Bristol BS8 1TS (United Kingdom)

    2015-04-14

    The H{sub 3}N⋯CuCl monomer has been generated and isolated in the gas phase through laser vaporisation of a copper sample in the presence of low concentrations of NH{sub 3} and CCl{sub 4} in argon. The resulting complex cools to a rotational temperature approaching 2 K during supersonic expansion of the gas sample and is characterised by broadband rotational spectroscopy between 7 and 18.5 GHz. The spectra of six isotopologues are measured and analysed to determine rotational, B{sub 0}; centrifugal distortion, D{sub J}, D{sub JK}; and nuclear quadrupole coupling constants of Cu, Cl, and {sup 14}N nuclei, χ{sub aa} (X). The geometry of the complex is C{sub 3v} with the N, Cu, and Cl atoms located on the a inertial axis. Bond distances and the ∠(H —N⋯Cu) bond angle within the complex are precisely evaluated through fitting of geometrical parameters to the experimentally determined moments of inertia and through ab initio calculations at the CCSD(T)(F12*)/AVQZ level. The r(Cu —Cl), r(Cu —N), and ∠(H —N⋯Cu) parameters are, respectively, evaluated to be 2.0614(7) Å, 1.9182(13) Å, and 111.40(6)° in the r{sub 0} geometry, in good agreement with the ab initio calculations. Geometrical parameters evaluated for the isolated complex are compared with those established crystallographically for a solid-state sample of [Cu(NH{sub 3})Cl].

  16. Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Reynolds K Brobey

    Full Text Available The reactive oxygen species (ROS-sensitive apoptosis signal-regulating kinase 1 (ASK1 signaling complex is a key regulator of p38 MAPK activity, a major modulator of stress-associated with aging disorders. We recently reported that the ratio of free ASK1 to the complex-bound ASK1 is significantly decreased in Klotho-responsive manner and that Klotho-deficient tissues have elevated levels of free ASK1 which coincides with increased oxidative stress. Here, we tested the hypothesis that: 1 covalent interactions exist among three identified proteins constituting the ASK1 signaling complex; 2 in normal unstressed cells the ASK1, 14-3-3ζ and thioredoxin (Trx proteins simultaneously engage in a tripartite complex formation; 3 Klotho's stabilizing effect on the complex relied solely on 14-3-3ζ expression and its apparent phosphorylation and dimerization changes. To verify the hypothesis, we performed 14-3-3ζ siRNA knock-down experiments in conjunction with cell-based assays to measure ASK1-client protein interactions in the presence and absence of Klotho, and with or without an oxidant such as rotenone. Our results show that Klotho activity induces posttranslational modifications in the complex targeting 14-3-3ζ monomer/dimer changes to effectively protect against ASK1 oxidation and dissociation. This is the first observation implicating all three proteins constituting the ASK1 signaling complex in close proximity.

  17. Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme.

    Science.gov (United States)

    Li, N; Mattoo, A K

    1994-03-04

    1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene. The expression of an enzymatically active, wound-inducible tomato (Lycopersicon esculentum L. cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation. We constructed several deletion mutants of the gene, expressed these in E. coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC syntheses. Deletion of the COOH terminus through Arg429 results in complete inactivation of the enzyme. Deletion of 46-52 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme. The highly efficient, truncated ACC synthase was found to be a monomer of 52 +/- 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer. These results demonstrate that the non-conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.

  18. Synthesis of novel monomeric graphene quantum dots and corresponding nanocomposite with molecularly imprinted polymer for electrochemical detection of an anticancerous ifosfamide drug.

    Science.gov (United States)

    Bali Prasad, Bhim; Kumar, Anil; Singh, Ragini

    2017-02-20

    This paper reports a typical synthesis of a nanocomposite of functionalized graphene quantum dots and imprinted polymer at the surface of screen-printed carbon electrode using N-acryloyl-4-aminobenzamide, as a functional monomer, and an anticancerous drug, ifosfamide, as a print molecule (test analyte). Herein, graphene quantum dots in nanocomposite practically induced the electrocatalytic activity by lowering the oxidation overpotential of test analyte and thereby amplifying electronic transmission, without any interfacial barrier in between the film and the electrode surface. The differential pulse anodic stripping signal at functionalized graphene quantum dots based imprinted sensor was realized to be about 3- and 7-fold higher as compared to the traditionally made imprinted polymers prepared in the presence and the absence of graphene quantum dots (un-functionalized), respectively. This may be attributed to a pertinent synergism in between the positively charged functionalized graphene quantum dots in the film and the target analyte toward the enhancement of electro-conductivity of the film and thereby the electrode kinetics. In fact, the covalent attachment of graphene quantum dots with N-acryloyl-4-aminobenzamide molecules might exert an extended conjugation at their interface facilitating electro conducting to render the channelized pathways for the electron transport. The proposed sensor is practically applicable to the ultratrace evaluation of ifosfamide in real (biological/pharmaceutical) samples with detection limit as low as 0.11ngmL(-1) (S/N=3), without any matrix effect, cross-reactivity, and false-positives.

  19. The influence of monomeric resin and filler characteristics on the performance of experimental resin-based composites (RBCs) derived from a commercial formulation.

    LENUS (Irish Health Repository)

    Hahnel, Sebastian

    2012-04-01

    To explore experimental RBCs derived from a successful commercially available RBC (Grandio) to investigate resin monomer blend and filler parameters (volume fraction, density and diameter) on RBC performance.

  20. Rotational Spectroscopy of ClZnCH3 (tilde{X}1A1): Characterization of a Monomeric Grignard-Type Reagent

    Science.gov (United States)

    Kilchenstein, K. M.; Min, Jie; Bucchino, Matthew; Ziurys, Lucy M.

    2016-06-01

    The pure rotational spectrum of the organozinc halide, ClZnCH3 (tilde{X}1A1), has been measured using Fourier-transform microwave (FTMW) and millimeter-wave direct-absorption methods in the frequency range 10-296 GHz. This work is the first study of ClZnCH3 by gas-phase spectroscopy. The molecule was created in a DC discharge from the reaction of zinc vapor, produced either by a Broida-type oven or by laser ablation, with chloromethane in what appears to be a metal insertion process. Rotational and chlorine quadrupole constants were determined for three zinc isotopologues. The Zn - Cl bond was found to be partly ionic and significantly shorter than in EtZnCl.

  1. Binary mixtures with novel monomeric and dimeric surfactants: influence of the head group nature and number of hydrophobic chains on non-ideality.

    Science.gov (United States)

    Martín, Victoria Isabel; Rodríguez, Amalia; del Mar Graciani, María; Moyá, María Luisa

    2012-02-15

    The micellization and micellar growth in the mixtures of N,N-dimethyl, N-phenyl,N-dodecylammonium bromide, PH12, N,N-dimethyl,N-ciclohexylmethyl,N-dodecylammonium bromide, CH12, and their two dimeric counterparts m-dimethylphenyl-α-ω-bis(dodecyldimethylammonium) bromide, 12PH12, and m-dimethylciclohexyl-α-ω-bis(dodecyldimethylammonium) bromide, 12CH12, with dodecyltrimethylammoniumbromide, DTAB, and with N-decanoyl N-methylglucamide, MEGA10, were investigated at 303 K. Circular dichroism, CD, experiments showed the formation of mixed micelles. Two-dimensional, 2D, rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments indicated that the arrangement of the rings in the pure and mixed micelles is similar, with the rings bent into the micelle interior avoiding contact with water. Application of different theoretical approaches shows that PH12 and CH12 mixtures with DTAB and with MEGA10 behave almost ideally. The binary systems of 12PH12 and 12CH12 with DTAB and with MEGA10 show a non-ideal behavior. An increment in the solution mole fraction of MEGA10 and DTAB diminishes the tendency of the micellar aggregates to grow. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Monomeric 14-3-3ζ Has a Chaperone-Like Activity and Is Stabilized by Phosphorylated HspB6

    OpenAIRE

    Sluchanko, Nikolai N.; Artemova, Natalya V.; Sudnitsyna, Maria V.; Safenkova, Irina V.; Antson, Alfred A.; Levitsky, Dmitrii I.; Gusev, Nikolai B.

    2012-01-01

    Members of the 14-3-3 eukaryotic protein family predominantly function as dimers. The dimeric form can be converted into monomers upon phosphorylation of Ser58 located at the subunit interface. Monomers are less stable than dimers and have been considered to be either less active or even inactive during binding and regulation of phosphorylated client proteins. However, like dimers, monomers contain the phosphoserine-binding site and therefore can retain some functions of the dimeric 14-3-3. F...

  3. Identification of the Phenol Functionality in Deprotonated Monomeric and Dimeric Lignin Degradation Products via Tandem Mass Spectrometry Based on Ion-Molecule Reactions with Diethylmethoxyborane

    Science.gov (United States)

    Zhu, Hanyu; Max, Joann P.; Marcum, Christopher L.; Luo, Hao; Abu-Omar, Mahdi M.; Kenttämaa, Hilkka I.

    2016-11-01

    Conversion of lignin into smaller molecules provides a promising alternate and sustainable source for the valuable chemicals currently derived from crude oil. Better understanding of the chemical composition of the resulting product mixtures is essential for the optimization of such conversion processes. However, these mixtures are complex and contain isomeric molecules with a wide variety of functionalities, which makes their characterization challenging. Tandem mass spectrometry based on ion-molecule reactions has proven to be a powerful tool in functional group identification and isomer differentiation for previously unknown compounds. This study demonstrates that the identification of the phenol functionality, the most commonly observed functionality in lignin degradation products, can be achieved via ion-molecule reactions between diethylmethoxyborane (DEMB) and the deprotonated analyte in the absence of strongly electron-withdrawing substituents in the ortho- and para-positions. Either a stable DEMB adduct or an adduct that has lost a methanol molecule (DEMB adduct-MeOH) is formed for these ions. Deprotonated phenols with an adjacent phenol or hydroxymethyl functionality or a conjugated carboxylic acid functionality can be identified based on the formation of DEMB adduct-MeOH. Deprotonated compounds not containing the phenol functionality and phenols containing an electron-withdrawing ortho- or para-substituent were found to be unreactive toward diethylmethoxyborane. Hence, certain deprotonated isomeric compounds with phenol and carboxylic acid, aldehyde, carboxylic acid ester, or nitro functionalities can be differentiated via these reactions. The above mass spectrometry method was successfully coupled with high-performance liquid chromatography for the analysis of a complex biomass degradation mixture.

  4. A Hydrophobic Gold Surface Triggers Misfolding and Aggregation of the Amyloidogenic Josephin Domain in Monomeric Form, While Leaving the Oligomers Unaffected

    Science.gov (United States)

    Apicella, Alessandra; Soncini, Monica; Deriu, Marco Agostino; Natalello, Antonino; Bonanomi, Marcella; Dellasega, David; Tortora, Paolo; Regonesi, Maria Elena; Casari, Carlo Spartaco

    2013-01-01

    Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity. PMID:23527026

  5. On the Difference between Self-Assembling Process of Monomeric and Dimeric Surfactants with the Same Head to Tail Ratio: A Lattice Monte Carlo Simulation

    Directory of Open Access Journals (Sweden)

    Reza Behjatmanesh-Ardakani

    2013-01-01

    Full Text Available Experimental data show that gemini surfactants have critical micelle concentrations that are almost tenfold lower than the CMCs of single chain ones. It is believed that the spacer groups play an important role in this subject. Short hydrophilic or long hydrophobic spacers can reduce CMC dramatically. In this paper, self-assembling processes of double-chain and one-chain surfactants with the same head to tail ratio are compared. Dimeric chain structure is exactly double of single chain. In other words, hydrophilic-lyophilic balances of two chain models are the same. Two single chains are connected head-to-head to form a dimeric chain, without introducing extra head or tail beads as a spacer group. Premicellar, micellar, and shape/phase transition ranges of both models are investigated. To do this, lattice Monte Carlo simulation in canonical ensemble has been used. Results show that without introducing extra beads as spacer group, the CMC of (H3T32 as a dimeric surfactant is much lower than the CMC of its similar single chain, H3T3. For dimeric case of study, it is shown that bolaform aggregates are formed.

  6. A chromatographic estimate of the degree of surface heterogeneity of reversed-phase liquid chromatography packing materials II-Endcapped monomeric C18-bonded stationary phase

    Energy Technology Data Exchange (ETDEWEB)

    Gritti, Fabrice [University of Tennessee, Knoxville (UTK); Guiochon, Georges A [ORNL

    2006-01-01

    In a previous report, the heterogeneity of a non-endcapped C{sub 30}-bonded stationary phase was investigated, based on the results of the measurements of the adsorption isotherms of two neutral compounds (phenol and caffeine) and two ionizable compounds (sodium naphthalene sulfonate and propranololium chloride) by frontal analysis (FA). The same method is applied here for the characterization of the surface heterogeneity of two new brands of endcapped C{sub 18}-bonded stationary phases (Gemini and Sunfire). The adsorption isotherms of the same four chemicals were measured by FA and the results confirmed by the independent calculation of the adsorption energy distribution (AED), using the expectation-maximization (EM) method. The effect of the length of the bonded alkyl chain was investigated. Shorter alkyl-bonded-chains (C{sub 18} versus C{sub 30}) and the end-capping of the silica surface contribute to decrease the surface heterogeneity under the same experimental conditions (30% methanol, 25 mM NaCl). The AEDs of phenol and caffeine are bimodal with the C{sub 18}-bonded columns while they are trimodal and quadrimodal, respectively, with a non-endcapped C{sub 30}-bonded column. The 'supersites' (adsorption energy >20 kJ/mol) found on the C{sub 30}-Prontosil column and attributed to a cation exchange mechanism completely disappear on the C{sub 18}-Gemini and C{sub 18}-Sunfire, probably because the end-capping of the silica surface eliminates most if not all the ionic interactions.

  7. Adsorption mechanism in reversed-phase liquid chromatography. Effect of the surface coverage of a monomeric C18-silica stationary phase

    Energy Technology Data Exchange (ETDEWEB)

    Gritti, Fabrice [University of Tennessee, Knoxville (UTK); Guiochon, Georges A [ORNL

    2006-04-01

    The effect of the bonding density of the octadecyl chains onto the same silica on the adsorption and retention properties of low molecular weight compounds (phenol, caffeine, and sodium 2-naphthalene sulfonate) was investigated. The same mobile phase (methanol:water, 20:80, v/v) and temperature (T = 298 K) were applied and two duplicate columns (A and B) from each batch of packing material (neat silica, simply endcapped or C{sub 1} phase, 0.42, 1.01, 2.03, and 3.15 {micro}mol/m{sup 2} of C{sub 18} alkyl chains) were tested. Adsorption data of the three compounds were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) were calculated using the expectation-maximization method. Results confirmed earlier findings in linear chromatography of a retention maximum at an intermediate bonding density. From a general point of view, the saturation capacity of the adsorbent tends to decrease with increasing bonding density, due to the vanishing space intercalated between the C{sub 18} bonded chains and to the decrease of the specific surface area of the stationary phase. The equilibrium constants are maximum for an intermediary bonding density ({approx}2 {micro}mol/m{sup 2}). An enthalpy-entropy compensation was found for the thermodynamic parameters of the isotherm data. Weak equilibrium constants (small {Delta}H) and high saturation capacities (large {Delta}S) were observed at low bonding densities, higher equilibrium constants and lower saturation capacities at high bonding densities, the combinations leading to similar apparent retention in RPLC. The use of a low surface coverage column is recommended for preparative purposes.

  8. The ratio of monomeric to aggregated forms of Abeta40 and Abeta42 is an important determinant of amyloid-beta aggregation, fibrillogenesis, and toxicity

    OpenAIRE

    Jan, Asad; Gokce, Ozgun; Luthi-Carter, Ruth; Lashuel, Hilal A.

    2008-01-01

    Aggregation and fibril formation of amyloid-beta (Abeta) peptides Abeta40 and Abeta42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of Abeta40 to Abeta42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of Abeta. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Abeta40 to Abeta42, we probed the concentration- an...

  9. Copper-free monomeric and dendritic palladium catalysts for the Sonogashira reaction: substituent effects, synthetic applications, and the recovery and re-use of the catalysts.

    Science.gov (United States)

    Heuzé, Karine; Méry, Denise; Gauss, Dominique; Blais, Jean-Claude; Astruc, Didier

    2004-08-20

    A series of bis(tert-butylphosphine)- and bis(cyclohexylphosphine)-functionalized Pd(II) monomers and polyamino (DAB) dendritic catalysts were synthesized and investigated for Sonogashira carbon-carbon coupling reactions in a copper-free procedure. The influence of phosphine substituents (tBu versus Cy) on the reaction kinetics was investigated by a GPC technique to monitor the reactions. The dendritic catalysts containing the cyclohexylphosphine ligands could be recovered and reused without loss of efficiency until the fifth cycle. The dendritic Pd(II) catalysts show a negative dendritic effect, that is, the catalyst efficiency decreases as the dendrimer generation increases.

  10. Monomeric immunoglobulin E stabilizes FcepsilonRIalpha from the human basophil cell line KU812 by protecting it from natural turnover

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Hansen, Jens Bo; Dissing, S;

    2003-01-01

    The high affinity IgE receptor (FcepsilonRI) on mast cells and basophils is up-regulated by its own ligand IgE; however, the mechanism is unknown.......The high affinity IgE receptor (FcepsilonRI) on mast cells and basophils is up-regulated by its own ligand IgE; however, the mechanism is unknown....

  11. A New Silarylene-Siloxane Monomeric Resin for Structural Composites: Cure-Chemistry Insight and Thermal Properties of the Cured Matrix

    Science.gov (United States)

    2013-03-01

    scan (note the broad endotherm from 30 to 130°C). The second heating scan clearly shows the crosslinking exotherm between 300 and 400°C. The...evaporation of 2PEPPSi2 between 300 and 350°C would have appeared as an endotherm , but the large crosslinking exotherm completely masked the process. A...The definitions used in this report are as follows. A resin is a formulation of monomers and additives. A monomer is a relatively low-molar

  12. MULTIPLE PROJECTIONS SYSTEM (MPS): USER'S MANUAL VERSION 2.0

    Science.gov (United States)

    The document is a user's manual for Multiple Projections System (MPS) Version 2.0, based on the 3% reasonable further progress (RFP) tracking system that was developed in FY92/FY93. The 3% RFP tracking system is a Windows application, and enhancements to convert the 3% RFP track...

  13. GK4, a G-protein-coupled receptor with a phosphatidylinositol phosphate kinase domain in Phytophthora infestans, is involved in sporangia development and virulence.

    Science.gov (United States)

    Hua, Chenlei; Meijer, Harold J G; de Keijzer, Jeroen; Zhao, Wei; Wang, Yuanchao; Govers, Francine

    2013-04-01

    For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G-protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a seven-transmembrane spanning (7-TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7-TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full-length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.

  14. Automated image analysis to quantify the subnuclear organization of transcriptional coregulatory protein complexes in living cell populations

    Science.gov (United States)

    Voss, Ty C.; Demarco, Ignacio A.; Booker, Cynthia F.; Day, Richard N.

    2004-06-01

    Regulated gene transcription is dependent on the steady-state concentration of DNA-binding and coregulatory proteins assembled in distinct regions of the cell nucleus. For example, several different transcriptional coactivator proteins, such as the Glucocorticoid Receptor Interacting Protein (GRIP), localize to distinct spherical intranuclear bodies that vary from approximately 0.2-1 micron in diameter. We are using multi-spectral wide-field microscopy of cells expressing coregulatory proteins labeled with the fluorescent proteins (FP) to study the mechanisms that control the assembly and distribution of these structures in living cells. However, variability between cells in the population makes an unbiased and consistent approach to this image analysis absolutely critical. To address this challenge, we developed a protocol for rigorous quantification of subnuclear organization in cell populations. Cells transiently co-expressing a green FP (GFP)-GRIP and the monomeric red FP (mRFP) are selected for imaging based only on the signal in the red channel, eliminating bias due to knowledge of coregulator organization. The impartially selected images of the GFP-coregulatory protein are then analyzed using an automated algorithm to objectively identify and measure the intranuclear bodies. By integrating all these features, this combination of unbiased image acquisition and automated analysis facilitates the precise and consistent measurement of thousands of protein bodies from hundreds of individual living cells that represent the population.

  15. Enhanced rifampicin delivery to alveolar macrophages by solid lipid nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Chuan Junlan [West China School of Pharmacy, Sichuan University, Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education (China); Li Yanzhen [Tianjin Institute of Pharmaceutical Research, State Key Laboratory of Drug Delivery Technology and Pharmacokinetics (China); Yang Likai; Sun Xun [West China School of Pharmacy, Sichuan University, Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education (China); Zhang Qiang [Peking University, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences (China); Gong Tao, E-mail: gongtaoy@126.com; Zhang Zhirong, E-mail: zrzzl@vip.sina.com [West China School of Pharmacy, Sichuan University, Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education (China)

    2013-05-15

    The present study aimed at developing a drug delivery system targeting the densest site of tuberculosis infection, the alveolar macrophages (AMs). Rifampicin (RFP)-loaded solid lipid nanoparticles (RFP-SLNs) with an average size of 829.6 {+-} 16.1 nm were prepared by a modified lipid film hydration method. The cytotoxicity of RFP-SLNs to AMs and alveolar epithelial type II cells (AECs) was examined using MTT assays. The viability of AMs and AECs was above 80 % after treatment with RFP-SLNs, which showed low toxicity to both AMs and AECs. Confocal Laser Scanning Microscopy was employed to observe the interaction between RFP-SLNs and both AMs and AECs. After incubating the cells with RFP-SLNs for 2 h, the fluorescent intensity in AMs was more and remained longer (from 0.5 to 12 h) when compared with that in AECs (from 0.5 to 8 h). In vitro uptake characteristics of RFP-SLNs in AMs and AECs were also investigated by detection of intracellular RFP by High performance liquid chromatography. Results showed that RFP-SLNs delivered markedly higher RFP into AMs (691.7 ng/mg in cultured AMs, 662.6 ng/mg in primary AMs) than that into AECs (319.2 ng/mg in cultured AECs, 287.2 ng/mg in primary AECs). Subsequently, in vivo delivery efficiency and the selectivity of RFP-SLNs were further verified in Sprague-Dawley rats. Under pulmonary administration of RFP-SLNs, the amount of RFP in AMs was significantly higher than that in AECs at each time point. Our results demonstrated that solid lipid nanoparticles are a promising strategy for the delivery of rifampicin to alveolar macrophages selectively.

  16. [A CLINICAL EXPERIENCE OF RIFAMPICIN SUPPOSITORY FOR THE TREATMENT OF PULMONARY TUBERCULOSIS].

    Science.gov (United States)

    Tsubota, Noriyuki; Tanimukai, Shigeatsu

    2015-06-01

    The usefulness of a rifampicin (RFP) suppository for treatment of pulmonary tuberculosis was examined in patients who had difficulty with oral consumption of medication. Among inpatients receiving first-time treatment for pulmonary tuberculosis susceptible to both isoniazid (INH) and RFP, and who underwent standard 3- or 4-drug treatments including INH and RFP, we compared the number of days required for obtaining two and three consecutive negative sputum smears and cultures, respectively, in patients who received hospital-made suppositories or standard oral RFP administration. There was no significant difference between groups in the number of days required for negative cultures and smears; although the times were equivalent, there were more number of elderly patients and those in generally poor condition in the RFP suppository group than the oral intake group. RFP suppositories may be one method for administration of standard tuberculosis treatment in patients with difficulty in oral consumption of medication.

  17. Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.

    Directory of Open Access Journals (Sweden)

    Adil Bakayan

    Full Text Available Bioluminescence recording of Ca(2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET to the green fluorescent protein (GFP. This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+ in various cell compartments. In addition, they would also serve to monitor Ca(2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA showed the highest BRET efficiency (largest energy transfer critical distance R(0 and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+ oscillations in single HeLa cells expressing tdTA. Ca(2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+ activity of HeLa cells injected subcutaneously into mice, and Ca(2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+ sensor reported to date and is, therefore, a promising probe to study Ca(2+ dynamics in whole organisms or tissues expressing the transgene.

  18. Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples.

    Directory of Open Access Journals (Sweden)

    Joachim Goedhart

    Full Text Available BACKGROUND: Fluorescence Resonance Energy Transfer (FRET between the green fluorescent protein (GFP variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells.

  19. Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants.

    Science.gov (United States)

    Palencia, Edwin Rene; Glenn, Anthony Elbie; Hinton, Dorothy Mae; Bacon, Charles Wilson

    2013-09-01

    Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli-maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP1) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri. © 2013.

  20. Adsorption mechanism and collapse propensities of the full-length, monomeric Aβ(1-42) on the surface of a single-walled carbon nanotube: a molecular dynamics simulation study

    National Research Council Canada - National Science Library

    Jana, Asis K; Sengupta, Neelanjana

    2012-01-01

    Though nanomaterials such as carbon nanotubes have gained recent attention in biology and medicine, there are few studies at the single-molecule level that explore their interactions with disease-causing proteins...

  1. Reactions of Monomeric [1,2,4-(Me3C)3C5H2]2CeH and CO with orwithout H2:An Experimental and Computational Study

    Energy Technology Data Exchange (ETDEWEB)

    Werkema, Evan L.; Maron, Laurent; Eisenstein, Odile; Andersen,Richard A.

    2006-09-07

    Addition of CO to [1,2,4-(Me3C)3C5H2]2CeH, Cp'2CeH, intoluene yields the cis (Cp'2Ce)2(mu-OCHCHO), in which the cis enediolategroup bridges the two metallocene fragments. The cis enediolatequantitatively isomerizes intramolecularly to the trans-enediolate inC6D6 at 100oC over seven months. When the solvent is pentane,Cp'2Ce(OCH2)CeCp'2 forms, in which the oxomethylene group or theformaldehyde dianion bridges the two metallocene fragments. The cisenediolate is suggested to form by insertion of CO into the Ce-C bond ofCp'2Ce(OCH2)CeCp'2 generating Cp'2CeOCH2COCeCp'2. The stereochemistry ofthe cis-enediolate is determined by a 1,2-hydrogen shift in the OCH2COfragment that has the OC(H2) bond anti periplanar relative to the carbenelone pair. The bridging oxomethylene complex reacts with H2, but not withCH4, to give Cp'2CeOMe, which is also the product of the reaction betweenCp'2CeH and a mixture of CO and H2. The oxomethylene complex reacts withCO to give the cis enediolate complex. DFT calculations on C5H5 modelmetallocenes show that the reaction of Cp2CeH with CO and H2 to giveCp2CeOMe is exoergic by 50 kcal mol-1. The net reaction proceeds by aseries of elementary reactions that occur after the formyl complex,Cp2Ce(eta-2 CHO), is formed by further reaction with H2. The key pointthat emerges from the calculated potential energy surface is thebifunctional nature of the metal formyl in which the carbon atom behavesas a donor and acceptor. Replacing H2 by CH4 increases the activationenergy barrier by 17 kcal mol-1.

  2. NBO, NMR, UV, FT-IR, FT-Raman spectra and molecular structure (monomeric and dimeric structures) investigation of 4-Chloro-3,5-Xylenol: A combined experimental and theoretical study

    Science.gov (United States)

    Arivazhagan, M.; Gayathri, R.

    2013-12-01

    In this work, a joint experimental (FTIR and FT-Raman) and theoretical (DFT and ab initio) study on the structure and the vibrations of 4-Chloro-3,5-Xylenol (CXL) are compared and analyzed. CXL is a chlorinated phenolic antiseptic which is a bactericide against most gram-positive bacteria. The first hyperpolarizability (β0) of this novel molecular system and related non-linear properties of CXL are calculated using HF/6-311++G(d,p) method on the finite-field approach. The energy and oscillator strength calculated using absorption spectra (UV-Vis spectrum), this spectral analysis confirms the charge transfer of the molecule. The theoretical 13C nuclear magnetic resonance (NMR) chemical shifts of the molecule were calculated by Gauge Including Atomic Orbital (GIAO) method, to analyze the molecular environment as well as the delocalization activities of electron clouds. The directly calculated ionization potential (IP), electron affinity (EA), electronegativity (χ), chemical hardness (η), first electron excitation energy (τ) and electrophilicity index (ω) as well as local reactivity (S) analyzed using HOMO and LUMO energies; the energy band gap are also determined. NBO analysis shows that charge in electron density(ED) in the σ* and π* antibonding orbitals and E(2) energies confirms the occurrence of ICT (Intramolecular Charge Transfer) within the molecule. Inter molecular hydrogen bonds exist between -OH group, give the evidence for the formation of dimer entities in the title molecule. The influences of chlorine atom, hydroxyl group and methyl group on the geometry of benzene and its normal modes of vibrations (monomer and dimer of CXL) have also been discussed. Finally the calculated results were applied to simulate Infrared and Raman spectra of the title molecule which show good agreement with observed spectra.

  3. Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in Vivo

    Science.gov (United States)

    Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Steven W. Newell; richard W. Hemingway; Vittorio Bottari; Jean-Pierre Perchellet

    1995-01-01

    m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce ODC activity at least as much as 3 ug of 12-O-tetradecanoylphorbol-13-acetate (TPA). ODC induction peaks...

  4. NBO, NMR, UV, FT-IR, FT-Raman spectra and molecular structure (monomeric and dimeric structures) investigation of 4-Chloro-3,5-Xylenol: a combined experimental and theoretical study.

    Science.gov (United States)

    Arivazhagan, M; Gayathri, R

    2013-12-01

    In this work, a joint experimental (FTIR and FT-Raman) and theoretical (DFT and ab initio) study on the structure and the vibrations of 4-Chloro-3,5-Xylenol (CXL) are compared and analyzed. CXL is a chlorinated phenolic antiseptic which is a bactericide against most gram-positive bacteria. The first hyperpolarizability (β0) of this novel molecular system and related non-linear properties of CXL are calculated using HF/6-311++G(d,p) method on the finite-field approach. The energy and oscillator strength calculated using absorption spectra (UV-Vis spectrum), this spectral analysis confirms the charge transfer of the molecule. The theoretical (13)C nuclear magnetic resonance (NMR) chemical shifts of the molecule were calculated by Gauge Including Atomic Orbital (GIAO) method, to analyze the molecular environment as well as the delocalization activities of electron clouds. The directly calculated ionization potential (IP), electron affinity (EA), electronegativity (χ), chemical hardness (η), first electron excitation energy (τ) and electrophilicity index (ω) as well as local reactivity (S) analyzed using HOMO and LUMO energies; the energy band gap are also determined. NBO analysis shows that charge in electron density(ED) in the σ(*) and π(*) antibonding orbitals and E((2)) energies confirms the occurrence of ICT (Intramolecular Charge Transfer) within the molecule. Inter molecular hydrogen bonds exist between -OH group, give the evidence for the formation of dimer entities in the title molecule. The influences of chlorine atom, hydroxyl group and methyl group on the geometry of benzene and its normal modes of vibrations (monomer and dimer of CXL) have also been discussed. Finally the calculated results were applied to simulate Infrared and Raman spectra of the title molecule which show good agreement with observed spectra.

  5. Studies on 5 Monomeric Flavan-3-ol of Wines by High Performance Liquid Chromatography%HPLC法同时测定葡萄酒中5种黄烷-3-醇单体的研究

    Institute of Scientific and Technical Information of China (English)

    温鹏飞; 陈建业; 黄卫东; 梁学军

    2006-01-01

    以中国产干红和干白葡萄酒为试材,采用LiChrospher 100 RP-18e色谱柱(250 mm×4.0 mm I.D.,5μm),冰乙酸水溶液为流动相,梯度洗脱,流速1.0 mL/min,柱温30℃,检测波长280 nm,直接进样,成功地同时分离了葡萄酒中的C、EgC、EgCg、EC和EcG 5种黄烷-3-醇单体并测定了其含量,建立了一种简单、经济、快速、准确的同时测定葡萄酒中5种黄-3-醇单体的高效液相色谱法.除张裕干红葡萄酒中未检出EcG外,不同厂家生产的葡萄酒中均检出5种黄烷-3-醇单体.

  6. Determination of Monomeric and Polymeric Flavan-3-ols in Grape Berry by High Performance Liquid Chromatography%葡萄果实中黄烷-3-醇及其聚合体的HPLC检测

    Institute of Scientific and Technical Information of China (English)

    温鹏飞; 陈建业; 李景明; 万嗣宝; 孔维府; 黄卫东

    2006-01-01

    以'赤霞珠'葡萄果实(Vitis vinifera L.'Cabernet Sauvignon')为试材,采用LiChrospher 100 RP-18e色谱柱(250 mm×4.0 mm I.D.,5 μm),冰乙酸∶水溶液为流动相,梯度洗脱流速为1.0 mL/min,柱温30℃,检测波长280 nm,成功地分离了(+)-儿茶素[(+)-catechin,CAT]、(-)-表棓儿茶素[(-)-epigallocatechin,EGC]、(-)-表棓儿茶素没食子酸酯[(-)-epigallocatechin gallate,EGCG]、(-)-表儿茶素[(-)-epicatechin,EC]和(-)-表儿茶素没食子酸酯[(-)-epicatechin gallate,ECG]等5种葡萄果实中最为重要的黄烷-3-醇及其衍生物,建立了一种快速、准确并可以同时测定葡萄果实中5种黄烷-3-醇及其衍生物的高效液相色谱法;此外,经TSK HW-50(F)柱分离纯化,间苯三酚存在下酸解后进行HPLC分析,建立了一种快速、准确测定葡萄果实中多聚黄烷-3-醇平均聚合度、平均分子量及其组成的方法.果实样品检测结果表明:黄烷-3-醇及其多聚体主要积累于果皮和种子中.

  7. Influence of Exciplex formation on the electroluminescent properties of dimeric Zn (II) bis-2-(2'-hydroxyphenyl) benzoxazole complex and monomeric Zn (II) 2-(1'-hydroxynaphthyl) benzothiazole complex

    Science.gov (United States)

    Prakash, Sattey; Anand, R. S.; Manoharan, S. Sundar

    2011-10-01

    In this paper we present the factors affecting electroluminescent properties of Zinc complexes of oxazole & thiazole derivatives. Electroluminescent spectra of the Zinc (II) complex of bis-[2-(2'-hydroxyphenyl) benzoxazole], [Zn (HPBO)2]2 and 2-(1'-hydroxynaphthyl) benzothiazole [Zn (HNBT)2] show unusual broadening and shows structural and photophysical similarity with [Zn (HPBT)2]2, a dimeric complex. The [Zn (HPBO)2]2 complex as an emissive layer in the device structure ITO /PEDOT:PSS /TPD (30nm) /[Zn (HPBO)2]2 (60nm) /BCP (6nm) /Ca (3nm) /Al (200nm) shows a broad bluish green emission, with a full width at half maxima (FWHM1˜70nm). The EL spectra is much broader compared to the PL spectra because of exciplex formation at the interfacial region between the emissive layer (EML) & hole transport layer (HTL). We also show the device performance of Zinc 2-(1'-hydroxynaphthyl) benzothiazole [Zn (HNBT)2] complex as emissive layer. Distinctly this device shows a broad greenish yellow emission with a peak maxima at 535nm and 690nm, owing to the exciplex formation between electron transport layer (ETL) and emissive layer (EML), which is in sharp contrast to the exciplex formation across the HTL-EML interface observed for the [Zn (HPBO)2]2 complex.

  8. Comparison of Design-Build to Design-Bid-Build as a Project Delivery Method

    Science.gov (United States)

    2001-12-01

    Design Activities Develop RFP Issue RFP and Receive Proposals Evaluate Proposals and Award Contract Administer Contract Post Occupancy Evaluation...Award Contract Administer Contract Post Occupancy Evaluation and Lessons Learned Fragmented Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6

  9. Comparison of Design-Build to Design-Bid Build as a Project Delivery Method

    Science.gov (United States)

    2001-12-01

    RFP Issue RFP and Receive Proposals Evaluate Proposals and Award Contract Administer Contract Post Occupancy Evaluation and Lessons...Contract Post Occupancy Evaluation and Lessons Learned Fragmented Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6 Phase 3 Phase 4 Phase 5

  10. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET

    NARCIS (Netherlands)

    Subach, F.V.; Zhang, L.; Gadella, T.W.J.; Gurskaya, N.G.; Lukyanov, K.A.; Verkhusha, V.V.

    2010-01-01

    We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF stat

  11. The Consulting Challenge: A Case Competition

    Science.gov (United States)

    Sachau, Daniel A.; Naas, Patricia A.

    2010-01-01

    The Consulting Challenge is a yearly case competition in which teams of graduate students respond to a request for proposals (RFP) for consulting services. The case and RFP are based on a problem that a host organization has experienced. Over 3 days, students meet with representatives of the host organization, analyze data, prepare a proposal for…

  12. Improving Evaluation in Nonprofit Organizations: A Study of How Evaluation Requests for Proposals Are Developed, Responded to, and Fulfilled

    Science.gov (United States)

    Matthews, Nakia S.

    2015-01-01

    It is often necessary for nonprofit organizations (NPOs) to formulate a Request for Proposal (RFP) to procure essential contracted services. This is most common when seeking the services of an external evaluator. Since most NPOs do not have internal evaluation staff, developing an appropriate RFP can be quite challenging. With limited literature…

  13. Dynamics of red fluorescent dental plaque during experimental gingivitis-A cohort study

    NARCIS (Netherlands)

    van der Veen, M.H.; Volgenant, C.M.C.; Keijser, B.; ten Cate, J.M.; Crielaard, W.

    2016-01-01

    Objectives: The dynamics of red fluorescent plaque (RFP) in comparison to clinical plaque and bleeding scores were studied during an experimental gingivitis protocol in a cohort of healthy participants. Methods: Forty-one participants were monitored for RFP before (24 h plaque), during 14 days

  14. 23 CFR 636.201 - What selection procedures and award criteria may be used?

    Science.gov (United States)

    2010-04-01

    ... (RFQ followed by RFP) § 636.202 Lowest price, Adjusted low-bid (price per quality point), meets criteria/low bid, weighted criteria process, fixed price/best design, best value. (b) Single Phase (RFP... appropriate for your project (based on the criteria in § 636.202), you may use a single phase...

  15. Pharmaceutical study of suppository formulations for improved in vivo kinetics of rifampicin.

    Science.gov (United States)

    Taki, Hisashi; Ogawa, Kenji; Nikai, Toshiaki

    2008-06-01

    To study rifampicin (RFP) suppository formulations that were prepared by adding an absorption promoter and a holding agent to conventional rifampicin suppositories in order to achieve higher blood drug levels. The subjects were 3 healthy volunteers who gave consent to participate in this study. Suppository formulations were prepared by adding sodium caprinate (the absorption promoter) to 600 mg, 750 mg, or 900 mg of RFP at about 3% of the suppository weight and sodium alginate (the holding agent) at 25% of the RFP content. Serum samples collected at 2, 6, and 10 hours after insertion of a suppository were subjected to RFP concentration analysis. In subject no. 1, the maximum concentration was 0.807 Lg/ml, 1.093 microg/ml, and 1.291 microg/ml after administration of a suppository containing 600 mg, 750 mg, or 900 mg of RFP, respectively. Since an injectable RFP formulation has not been approved in Japan, formulation studies of RFP suppositories are important to achieve a better clinical response and to prevent the development of resistance. The present formulation that delivered a blood concentration of RFP considerably higher than 1 microg/ml was considered to have therapeutic potential. Its clinical utility will be examined in further formulation studies.

  16. A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

    Science.gov (United States)

    Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

    2011-10-01

    To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

  17. Application of Modal Parameter Estimation Methods for Continuous Wavelet Transform-Based Damage Detection for Beam-Like Structures

    Directory of Open Access Journals (Sweden)

    Zhi Qiu

    2015-02-01

    Full Text Available This paper presents a hybrid damage detection method based on continuous wavelet transform (CWT and modal parameter identification techniques for beam-like structures. First, two kinds of mode shape estimation methods, herein referred to as the quadrature peaks picking (QPP and rational fraction polynomial (RFP methods, are used to identify the first four mode shapes of an intact beam-like structure based on the hammer/accelerometer modal experiment. The results are compared and validated using a numerical simulation with ABAQUS software. In order to determine the damage detection effectiveness between the QPP-based method and the RFP-based method when applying the CWT technique, the first two mode shapes calculated by the QPP and RFP methods are analyzed using CWT. The experiment, performed on different damage scenarios involving beam-like structures, shows that, due to the outstanding advantage of the denoising characteristic of the RFP-based (RFP-CWT technique, the RFP-CWT method gives a clearer indication of the damage location than the conventionally used QPP-based (QPP-CWT method. Finally, an overall evaluation of the damage detection is outlined, as the identification results suggest that the newly proposed RFP-CWT method is accurate and reliable in terms of detection of damage locations on beam-like structures.

  18. Generation of red fluorescent protein transgenic dogs.

    Science.gov (United States)

    Hong, So Gun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Teoan; Kwon, Mo Sun; Koo, Bon Chul; Ra, Jeong Chan; Kim, Dae Yong; Ko, CheMyong; Lee, Byeong Chun

    2009-05-01

    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP.

  19. 48 CFR 415.207 - Handling proposals and information.

    Science.gov (United States)

    2010-10-01

    ... Proposals RFP Offeror 1. To the best of my knowledge and belief, no conflict of interest exists that may... purposes of the Freedom of Information Act (5 U.S.C. 552). (d) The contracting officer shall attach a cover...

  20. Tank Bladders for Advanced Monopropellants Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In response to RFP S8.04 Spacecraft Propulsion, innovations in propulsion technologies are needed to increase the capabilities of the Science Mission Directorate...

  1. Three Dimensional Situational Awareness Sensor to Assist Descent and Landing of the Mars Lander Spacecraft Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In order to address NASA's needs identified in the RFP, TetraVue proposes the use of a unique, non GPS and non-observer position dependent 3D sensor system to...

  2. The TITAN reversed-field-pinch fusion reactor study

    Energy Technology Data Exchange (ETDEWEB)

    1990-01-01

    This paper on titan plasma engineering contains papers on the following topics: reversed-field pinch as a fusion reactor; parametric systems studies; magnetics; burning-plasma simulations; plasma transient operations; current drive; and physics issues for compact RFP reactors.

  3. LIDAR: Malheur NWR

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This project was funded through the Region 1 Inventory and Monitoring Initiative RFP in 2011. LiDAR has been identified by the Malheur Lake Work Group as critical...

  4. Sleeping and Driving Don't Mix: Sleep Quiz

    Science.gov (United States)

    ... to rest, the less opportunity there is for crime. Always lock your doors and roll up the ... are asleep? back to results Navigation Research Current Projects Completed Projects RFP About Overview Mission Staff Board ...

  5. RANT Building Upgrade Pre-Proposal Meeting

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Melinda Ann [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Meadows, Darren W. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Rodriguez, Roger R. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); MacFarlane, Eric Robert [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Ortega, Adan Eduardo [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Lopez Escobedo, Gabriela Maria [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2017-07-31

    This presentation is designed to provide an overview of the project scope and RFP; discuss interfaces for the construction project; highlight key expectations and deliverables from the subcontractor; and address questions and provide clarification of the project scope.

  6. Renewable Energy Contract Development Best Practices

    Science.gov (United States)

    This page provides information on how available resources can help organizations effectively navigate the solar contract development process with the goal of RFP and contract transparency, completeness, and accountability of all involved parties.

  7. 7 CFR 1783.9 - What are the criteria for scoring applications?

    Science.gov (United States)

    2010-01-01

    ... service area to be covered by the RFP loans, and appears likely to be sustainable. Up to 40 points. (3.... (7) Administrator's discretion, considering such factors as creative outreach ideas for marketing...

  8. 77 FR 31268 - Determination of Attainment for the Paul Spur/Douglas PM10

    Science.gov (United States)

    2012-05-25

    ... schedule. In January 2012, ADEQ replaced one of the partisol samplers with a continuous tapering element... the definition of RFP in section 171(1), and incorporated in section 189(c)(1), to be a...

  9. Fossil AGN jets as ultra high energy particle accelerators

    OpenAIRE

    2007-01-01

    Remnants of AGN jets and their surrounding cocoons leave colossal magnetohydrodynamic (MHD) fossil structures storing total energies ~10^{60} erg. The original active galacic nucleus (AGN) may be dead but the fossil will retain its stable configuration resembling the reversed-field pinch (RFP) encountered in laboratory MHD experiments. In an RFP the longitudinal magnetic field changes direction at a critical distance from the axis, leading to magnetic re-connection there, and to slow decay of...

  10. Air Force Air Refueling: The KC-X Aircraft Acquisition Program

    Science.gov (United States)

    2008-08-04

    in demand when airlift assets are stretched most thin during the early deployment phase of a conflict.”65 This may be considered significant to some...for proposals (RFP) in early 2007.73 The Boeing Company responded to the RFP with the KC-767, a variant of the commercial 767-200, while Northrop...Industries United States outer rear spar, main landing gearsupport, ribs (for BAE) Ciba-Geigy Corp. Federal Republic of Germany HTA /6376 prepreg on

  11. Use of Helical Fields to Allow a Long Pulse Reversed Field Pinch

    Energy Technology Data Exchange (ETDEWEB)

    A. Boozer and N. Pomphrey

    2008-11-20

    The maintenance of the magnetic configuration of a Reversed Field Pinch (RFP) is an unsolved problem. Even a toroidal loop voltage does not suffice to maintain the magnetic configuration in axisymmetry but could if the plasma had helical shaping. The theoretical tools for plasma optimization using helical shaping have advanced, so an RFP could be relatively easily designed for optimal performance with a spatially constant toroidal loop voltage. A demonstration that interesting solutions exist is given.

  12. Autonomous Star Tracker Algorithms

    DEFF Research Database (Denmark)

    Betto, Maurizio; Jørgensen, John Leif; Kilsgaard, Søren

    1998-01-01

    Proposal, in response to an ESA R.f.P., to design algorithms for autonomous star tracker operations.The proposal also included the development of a star tracker breadboard to test the algorithms performances.......Proposal, in response to an ESA R.f.P., to design algorithms for autonomous star tracker operations.The proposal also included the development of a star tracker breadboard to test the algorithms performances....

  13. Uncarboxylated Osteocalcin and Gprc6a Axis Produce Intratumoral Androgens in Castration-Resistant Prostate Cancer

    Science.gov (United States)

    2016-05-01

    None. Figure 5. Bone tumor growth of VCaP osteocalcin transfectants. A. Whole body mice luciferase imaging for bone tumors. B. Quantitation of...Figure 1). Conditioned media was collected from RFP, osteocalin and mutated Osteocalcin expressing cells. ELISA quatitiation show that...transfectants expressing RFP, Osteocalcin and 3E/Q osteocalcin cells were injected into tibae of mice. Mass spectrometric quantitation of Testosterone and

  14. Study of ITG modes in RFX-mod using TRB code

    CERN Document Server

    Sattin, F; Guo, S C

    2010-01-01

    We present here a study about the stability of Ion-Temperature-Gradient drift turbulence in the Quasi-Single-Helicity regime of RFX-mod Reversed Field Pinch (RFP) using the TRB fluid electrostatic turbulence code. Our results suggest that present-days RFP plasmas are marginally stable against this kind of turbulence. The onset of the instability may be envisaged for close future regimes, in the presence of hotter plasmas with sharper internal transport barriers.

  15. MODEL REQUEST FOR PROPOSALS TO PROVIDE ENERGY AND OTHER ATTRIBUTES FROM AN OFFSHORE WIND POWER PROJECT

    Energy Technology Data Exchange (ETDEWEB)

    Jeremy Firestone; Dawn Kurtz Crompton

    2011-10-22

    This document provides a model RFP for new generation. The 'base' RFP is for a single-source offshore wind RFP. Required modifications are noted should a state or utility seek multi-source bids (e.g., all renewables or all sources). The model is premised on proposals meeting threshold requirements (e.g., a MW range of generating capacity and a range in terms of years), RFP issuer preferences (e.g., likelihood of commercial operation by a date certain, price certainty, and reduction in congestion), and evaluation criteria, along with a series of plans (e.g., site, environmental effects, construction, community outreach, interconnection, etc.). The Model RFP places the most weight on project risk (45%), followed by project economics (35%), and environmental and social considerations (20%). However, if a multi-source RFP is put forward, the sponsor would need to either add per-MWh technology-specific, life-cycle climate (CO2), environmental and health impact costs to bid prices under the 'Project Economics' category or it should increase the weight given to the 'Environmental and Social Considerations' category.

  16. Validación de un método de cromatografía líquida para la determinación de rifampicina en plasma humano

    Directory of Open Access Journals (Sweden)

    Luis Moreno-Exebio

    Full Text Available Objetivos: Validar un método de cromatografía líquida de alta resolución (HPLC para la determinación de rifampicina (RFP en plasma humano. Materiales y métodos. Se desarrolló un método HPLC para la determinación de RFP en plasma. La separación fue realizada por cromatografía de fase reversa con una columna C18 y una fase móvil compuesta por una mezcla de acetonitrilo y solución amortiguadora de fosfato de potasio monobásico 0,05 M (38:62 v/v a 335 nm. En el cual se empleó como estándar interno rifampicina quinona (RFP-QN. Resultados. Los tiempos de retención de RFP y RFP-QN fueron 7,81 y 12,26 minutos, respectivamente. El ensayo fue lineal de 0,5 a 250 ug/mL Los parámetros evaluados de precisión, exactitud, selectividad, linealidad, recuperación cumplieron con lo establecido en las guías internacionales de validación de métodos bioanalíticos. Conclusiones. El método HPLC desarrollado es simple, específico, sensible, selectivo y lineal para un amplio rango de concentraciones de RFP en plasma

  17. MODEL REQUEST FOR PROPOSALS TO PROVIDE ENERGY AND OTHER ATTRIBUTES FROM AN OFFSHORE WIND POWER PROJECT

    Energy Technology Data Exchange (ETDEWEB)

    Jeremy Firestone; Dawn Kurtz Crompton

    2011-10-22

    This document provides a model RFP for new generation. The 'base' RFP is for a single-source offshore wind RFP. Required modifications are noted should a state or utility seek multi-source bids (e.g., all renewables or all sources). The model is premised on proposals meeting threshold requirements (e.g., a MW range of generating capacity and a range in terms of years), RFP issuer preferences (e.g., likelihood of commercial operation by a date certain, price certainty, and reduction in congestion), and evaluation criteria, along with a series of plans (e.g., site, environmental effects, construction, community outreach, interconnection, etc.). The Model RFP places the most weight on project risk (45%), followed by project economics (35%), and environmental and social considerations (20%). However, if a multi-source RFP is put forward, the sponsor would need to either add per-MWh technology-specific, life-cycle climate (CO2), environmental and health impact costs to bid prices under the 'Project Economics' category or it should increase the weight given to the 'Environmental and Social Considerations' category.

  18. Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins.

    Science.gov (United States)

    Pires, S F; DaRocha, W D; Freitas, J M; Oliveira, L A; Kitten, G T; Machado, C R; Pena, S D J; Chiari, E; Macedo, A M; Teixeira, S M R

    2008-03-01

    Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

  19. Electrical modeling of the Reversed Field Pinch configuration

    Science.gov (United States)

    Cavazzana, Roberto

    2016-10-01

    Starting from the Poynting theorem, a two port equivalent formulation for the Reversed Field Pinch (RFP) is obtained. At first a general formulation applicable to any sort of underlying MHD physics is derived. Then its specialization is discussed, showing that: i) the toroidal field reversal is guided from outside the plasma by the external imposed boundary conditions; ii) the classic textbook RFP derivation with the toroidal flux Φt conserved is only a particular choice among the many possible. Here a parametric force free MHD family of equilibria is used to derive the two port equation of a realistic RFP boundary condition. The key master parameter turns out to be the edge safety factor q (a) = a / R .Bt (a) /Bp (a) , whereas Φt becomes a free variable determined by the RFP self-organization processes. As a by product a correct expression for the resistive component of the toroidal loop voltage is given. The two port model obtained is finally closed by adding the poloidal and toroidal power supply networks and evolved by means of a SPICE simulator. The results enlighten some peculiarities found in the RFP transient operations: RFP startup and formation, pulsed poloidal current drive (PPCD) and oscillating field current drive (OFCD).

  20. Synthesis, structure and NMR characterization of a new monomeric aluminophosphate [ dl-Co(en) 3] 2[Al(HPO 4) 2(H 1.5PO 4) 2(H 2PO 4) 2](H 3PO 4) 4 containing four different types of monophosphates

    Science.gov (United States)

    Chen, Peng; Li, Jiyang; Xu, Jun; Duan, Fangzheng; Deng, Feng; Xu, Ruren

    2009-03-01

    A new zero-dimensional (0D) aluminophosphate monomer [ dl-Co(en) 3] 2[Al(HPO 4) 2(H 1.5PO 4) 2(H 2PO 4) 2](H 3PO 4) 4 (designated AlPO-CJ38) with Al/P ratio of 1/6 has been solvothermally prepared by using racemic cobalt complex dl-Co(en) 3Cl 3 as the template. The Al atom is octahedrally linked to six P atoms via bridging oxygen atoms, forming a unique [Al(HPO 4) 2(H 1.5PO 4) 2(H 2PO 4) 2] 6- monomer. Notably, there exists intramolecular symmetrical O⋯H⋯O bonds, which results in pseudo-4-rings stabilized by the strong H-bonding interactions. The structure is also featured by the existence of four different types of monophosphates that have been confirmed by 31P NMR and 1H NMR spectra. The crystal data are as follows: AlPO-CJ38, [ dl-Co(en) 3] 2[Al(HPO 4) 2(H 1.5PO 4) 2(H 2PO 4) 2](H 3PO 4) 4, M = 1476.33, monoclinic, C2/ c (No. 15), a = 36.028(7) Å, b = 8.9877(18) Å, c = 16.006(3) Å, β = 100.68(3)°, U = 5093.2(18) Å 3,Z = 4, R1 = 0.0509 ( I > 2 σ( I)) and wR2 = 0.1074 (all data). CCDC number 689491.

  1. Hydrogen for X-group exchange in CH3X, X = Cl, Br, I, OMe and NMe2 byMonomeric [1,2,4-(Me3C)3C5H2]2CeH: Experimental and Computational Support for a Carbenoid Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Werkema, Evan; Andersen, Richard; Yahia, Ahmed; Maron, Laurent; Eisenstein, Odile

    2009-05-15

    The reaction between [1,2,4-(Me3C)3C5H2]2CeH, referred to as Cp'2CeH, andCH3X where X is Cl, Br, I, OMe and NMe2, are described. The reactions fall intothree distinct classes. Class a, where X = Cl, Br and I rapidly form Cp'2CeX and CH4without formation of identifiable intermediates in the 1H NMR spectra. Class b, whereX = OMe proceeds rapidly to Cp'2Ce(eta2-CH2OMe) and H2 and then to Cp'2CeOMeand CH4. The methoxymethyl derivative is sufficiently stable to be isolated andcharacterized and it is rapidly converted to Cp'2CeOMe in presence of BPh3. Class c,where X = NMe2 does not result in formation of Cp'2CeNMe2, but deuterium labelingexperiments show that H for D exchange occurs in NMe3. Density functionalcalculations DFT(B3PW91) on the reaction of (C5H5)2CeH, referred to as Cp2CeH,and CH3X show that the barrier for alpha-CH activation, resulting in formation ofCp2Ce(eta2-CH2X), proceeds with a relatively low activation barrier (DeltaG++) but thesubsequent ejection of CH2 and trapping by H2 has a higher barrier; the height of thesecond barrier lies in the order F, Cl, Br, I< OMe<< NMe2, consistent with theexperimental studies. The DFT calculations also show that the two-step reaction,which proceeds through a carbenoid intermediate, has a lower barrier than a directone-step sigma bond metathesis mechanism. The reaction of Cp2CeCH2OMe and BPh3 is calculated to be a low barrier process and the ylide, CH2(+)BPh3(-), is a transition state and not an intermediate.

  2. Synthesis, Crystal Structure and Properties of Monomeric Copper(Ⅱ) Complexes of Pyrrole-Containing Tridentate Schiff-Base Ligands%含吡咯三齿席夫碱配体的单核铜(Ⅱ)配合物的合成、晶体结构和性质

    Institute of Scientific and Technical Information of China (English)

    李荣清; 赵朴素; 张宇; 张载超; 刘仲雨; 赵振

    2012-01-01

    Two copper(Ⅱ) complexes of the homologous tridentate Schiff-base ligands (L1)- and (L2)-,[CuL1I] (1)and [CuL2I] (2),have been synthesised and characterised by IR,ES-MS,UV-Vis,single crystal X-ray diffraction and thermogravimetric analysis ((L1)- and (L2)- are the deprotonated forms of 2-(2-pyridyl)-N-[1-(1H-pyrrol-2-yl)ethylidene]ethanamine (HL1) and N-[1-(1H-pyrrol-2-yl)ethylidene](2-pyridyl)methanamine (HL2),respectively).X-ray crystal structure determination carried out on 1 reveals that it is a distorted square planar copper(Ⅱ) monomer.The crystal structure indicates that the intermolecular interaction is also a factor which can influence the structural properties of copper(Ⅱ) complexes in addition to ligand flexibility and steric constraints.CCDC:862124.%合成和表征了两个类似的三齿席夫碱配体(L1)-和(L2)-的铜(Ⅱ)配合物[CuL1I] (1)和[CuL2I] (2)(HL1为2-(2- pyridyl)-N-[1 -(1H-pyrrol-2-yl)ethylidene]ethanamine;HL2为N-[1-(1H-pyrrol-2-yl)ethylidene](2-pyridyl)methanamine),并用红外光谱、电喷雾质谱、紫外-可见光谱、单晶X-射线衍射和热重分析等手段对配合物进行了表征.对配合物1的X-射线晶体结构测定表明它是一个畸变的平面正方形单核铜(Ⅱ)配合物.晶体结构还表明,除了配体的柔性和空间限制外,分子间的相互作用也是影响铜(Ⅱ)配合物结构的因素.

  3. Hydrogen for X-group exchange in CH3X, X = Cl, Br, I, OMe and NMe2 byMonomeric [1,2,4-(Me3C)3C5H2]2CeH: Experimental and Computational Support for a Carbenoid Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Werkema, Evan; Andersen, Richard; Yahia, Ahmed; Maron, Laurent; Eisenstein, Odile

    2009-05-15

    The reaction between [1,2,4-(Me3C)3C5H2]2CeH, referred to as Cp'2CeH, andCH3X where X is Cl, Br, I, OMe and NMe2, are described. The reactions fall intothree distinct classes. Class a, where X = Cl, Br and I rapidly form Cp'2CeX and CH4without formation of identifiable intermediates in the 1H NMR spectra. Class b, whereX = OMe proceeds rapidly to Cp'2Ce(eta2-CH2OMe) and H2 and then to Cp'2CeOMeand CH4. The methoxymethyl derivative is sufficiently stable to be isolated andcharacterized and it is rapidly converted to Cp'2CeOMe in presence of BPh3. Class c,where X = NMe2 does not result in formation of Cp'2CeNMe2, but deuterium labelingexperiments show that H for D exchange occurs in NMe3. Density functionalcalculations DFT(B3PW91) on the reaction of (C5H5)2CeH, referred to as Cp2CeH,and CH3X show that the barrier for alpha-CH activation, resulting in formation ofCp2Ce(eta2-CH2X), proceeds with a relatively low activation barrier (DeltaG++) but thesubsequent ejection of CH2 and trapping by H2 has a higher barrier; the height of thesecond barrier lies in the order F, Cl, Br, I< OMe<< NMe2, consistent with theexperimental studies. The DFT calculations also show that the two-step reaction,which proceeds through a carbenoid intermediate, has a lower barrier than a directone-step sigma bond metathesis mechanism. The reaction of Cp2CeCH2OMe and BPh3 is calculated to be a low barrier process and the ylide, CH2(+)BPh3(-), is a transition state and not an intermediate.

  4. Cry1AH蛋白活性片段在溶液中的单体和寡聚体杀虫活性分析%Toxicity analysis of activated Cry1Ah of monomeric and oligomeric forms in the solution

    Institute of Scientific and Technical Information of China (English)

    周子珊; 束长龙; 梁革梅; 苍晶; 宋福平; 张杰

    2011-01-01

    [目的]分析NaCl对寡聚化的影响,以及寡聚化对Cry1Ah杀虫活性的影响.[方法]利用Superdex-75分子筛,将Cry1Ah活性片段的寡聚体和单体进行分离,通过戊二醛交联试脸分析寡聚化程度,结合生物活性测定,分析NaCl对寡聚化的影响以及寡聚化对Cry1Ah毒力的影响.[结果]在纯化的组分峰Ⅰ中寡聚体占总蛋白的63.35%.NaCl时寡聚化有促进作用,而寡聚体又以多种形式存在,并且表现出不同的生物活性.寡聚体峰Ⅰ、寡聚体峰Ⅱ、单体峰Ⅲ对棉铃虫的LC50分别为47.192、3.329、3.977μg/mL.[结论]NaCl对寡聚化有促进作用,而且,寡聚体的毒力比Cry1Ah活性片段的毒力有不同程度的降低,最多下降49.6倍.推测这种体外形成的寡聚体不同于昆虫体内形成的寡聚体,引起插膜穿孔能力下降,导致毒力不同.%[Objective] Our aim is to analyze the effect of sodium chloride on oligomerization, and the toxicity of oligomeric units. [Method] To analyze the effect of sodium chloride on oligomerization, and toxicity of oligomeric units, toxin protein was purified by chromatography with Superdex-75 to separate the oligomeric units and monomer unit. Oligomerization reaction was detected by glutaraldehyde crosslinking, and bioassay of all the oligomeric units was performed against the larvae of Helicoverpa armigera. [Result] Oligomeric units accounted for 63.35% of total Cry1Ah toxin in Peak 1. Sodium chloride could promote oligomerization. The 50% lethal concentrations (LC50) of oligomeric units (Peak 1 and Peak 2) and monomer unit (Peak 3) against H. armigera were 47.192 μg/mL, 3. 329 μg/mL and 3. 977 μg/mL, respectively. [Conclasion] Sodium chloride could promote oligomerization; the toxicity of oligomeric units declined compared with Cry1Ah polypeptide: Peak1 was 49.6 fold lower than that of activated Cry1Ah polypeptide. We deduced that the oligomerization was different in structure between vivo and in vitro reactions, and thereby pore-forming ability and insecticidal activity were decreased, respectively.

  5. Preparation and characterization of rifampicin loaded poly (lactic-co-glycolic acid) copolymer nanoparticles%利福平聚乳酸-羟基乙酸共聚物纳米粒的成型工艺及制剂学性质分析

    Institute of Scientific and Technical Information of China (English)

    蔡鑫君; 徐颖颖; 李范珠; 叶晓莉

    2012-01-01

    目的 优化利福平聚乳酸-羟基乙酸共聚物纳米粒(RFP-PLGA-NPs)的制备工艺,并分析其制剂学性质.方法 以PLGA为载体,采用改良的自乳化溶剂蒸发法(M-SESD)制备RFP-PLGA-NPs.以粒径、包封率、载药量为指标,采用正交设计法优化处方和制备工艺.结果 制备RFP-PLGA-NPs的优化条件为PLGA 100mg,poloxamer 188质量分数1.0%,丙酮与乙醇体积比3∶1,有机相体积15 mL.按优化条件所制备的RFP-PLG-NPs的粒径为(128.73±4.07) nm,多分散系数(PDI)为0.046~0.105,包封率(65.84±0.69)%,载药量(3.78±0.14)%.结论 该工艺简单、稳定性好,为后续RFP-PLGA-NPs的体内研究奠定了基础.%AIM To prepare rifampicin poly(lactic-co-glycolic acid)copolymer(FLGA) nanoparticles ( RFP-PLGA-NPs)with optimization. METHODS RFP-PLGA-NPs were prepared by modified spontaneous emulsification solvent difiusion(M-SESD)with the PLGA as its carrier.The orthogonal design was carried out to optimize the preparing technology according to particle size,entrapment efficiency and drug loading of RFP-PLGA-NPs. RESULTS The optimal conditions for the preparation of RFP-PLGA-NPs were 100 mg PLGA, 1.0 % poloxamer 188( m/V),acetone/ethanol 3 = 1( V/ V) and 15 mL organic phase. The mean particle size of the resulted RFP-PLGA-NPs was (128.73 ± 4.07)nm and the polydispersity index(PDI) was 0.046 - 0.105. the average entrapment efficiency and drug loading was (65.84 ± 0.69)% and (3.78±0.14)% , respectively. CONCLUSION An optimized nanoparticle drug delivery system is obtained by M-SESD and provides foundation for further pharmacokinetic research.

  6. [An experimental study on a slow-release complex with rifampicin-polylactic-co-glycolic acid-calcium 
phosphate cement].

    Science.gov (United States)

    Wu, Jianhuang; Ding, Zhou; Lei, Qing; Li, Miao; Liang, Yan; Lu, Tao

    2016-09-28

    目的:制备利福平(rifampicin,RFP)-聚乳酸-羟基乙酸(polylactic-co-glycolic acid,PLGA)-磷酸钙骨水泥(calcium phosphate cement,CPC)缓释复合体(RFP-PLGA-CPC复合体),并研究其理化性质及体外释药性能。方法:采用乳化-溶剂挥发法制备RFP-PLGA缓释微球。实验分为CPC组、包埋了RFP的CPC组(RFP-CPC组)、载有RFP的PLGA缓释微球与自固化CPC复合体组(RFP-PLGA-CPC复合体组)。测定3组材料的凝固时间﹑孔隙率。通过体外药物释放实验观察释药前后的抗压强度、断面形态的变化以及体外释药情况。结果:CPC组的凝固时间最短,RFP-PLGA-CPC复合体组的凝固时间最长。CPC组的孔隙率同RFP-CPC组比较,差异有统计学意义(P<0.05);CPC组和RFP-CPC组的孔隙率与RFP-PLGA-CPC复合体组比较,差异均有统计学意义(均P<0.01)。RFP-PLGA-CPC复合体组的抗压强度与CPC组比较,差异有统计学意义(P<0.01);而RFP-CPC组和CPC组之间的抗压强度随着时间的变化逐渐表现出显著性差异(3 d: P<0.05;30和60 d:P<0.01)。CPC组在降解过程中的抗压强度的变化不大。PLGA微球的大小均一,粒径基本在100~150 μm之间,微球的形态呈现出球体或是类球体,微球的表面圆润光滑,无杂质附着; CPC组的断面空隙在浸泡3 d直至60 d都没有明显变化;而RFP-CPC组的微结构变化亦不大,其断面均是小的微粒形成的;RFP-PLGA-CPC复合体组断面的孔隙明显增多,一直到60 d时PLGA微球逐渐消失,剩下空洞。RFP-PLGA-CPC复合体组无明显短时间内药物大量释放现象,60 d累计释药率达到近95%,将该复合体释药行为进行线性拟合,发现药物以恒速进行局部释放,符合零级动力学方程F=0.168×t。结论:RFP-PLGA-CPC复合体孔隙率显著高于CPC,能够持续缓慢释放有效抗结核药物,并能较长时间维持一定的力学强度。.

  7. An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein✩

    Science.gov (United States)

    Rocha, Marcele N.; Corrêa, Célia M.; Melo, Maria N.; Beverley, Stephen M.; Martins-Filho, Olindo Assis; Madureira, Ana Paula; Soares, Rodrigo P.

    2013-01-01

    Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting. PMID:23312610

  8. Serum fibrosis markers can predict rapid fibrosis progression after liver transplantation for hepatitis C.

    Science.gov (United States)

    Pungpapong, Surakit; Nunes, David P; Krishna, Murli; Nakhleh, Raouf; Chambers, Kyle; Ghabril, Marwan; Dickson, Rolland C; Hughes, Christopher B; Steers, Jeffery; Nguyen, Justin H; Keaveny, Andrew P

    2008-09-01

    Although recurrent hepatitis C virus (HCV) after liver transplantation (LT) is universal, a minority of patients will develop cirrhosis within 5 years of surgery, which places them at risk for allograft failure. This retrospective study investigated whether 2 serum fibrosis markers, serum hyaluronic acid (HA) and YKL-40, could be used to predict rapid fibrosis progression (RFP) post-LT. These markers were compared with conventional laboratory tests, histological assessment, and hepatic stellate cell activity (HSCA), a key step in fibrogenesis, as assessed by immunohistochemical staining for alpha-smooth muscle actin. Serum and protocol liver biopsy samples were obtained from 46 LT recipients at means of 5 +/- 2 (biopsy 1) and 39 +/- 6 (biopsy 2) months post-LT, respectively. RFP was defined as an increase in the fibrosis score >or= 2 from biopsy 1 to biopsy 2 (a mean interval of 33 +/- 6 months). The ability of parameters at biopsy 1 to predict RFP was compared with the areas under receiver operating characteristic curves (AUROCs). Of the 46 subjects, 15 developed RFP. Serum HA and YKL-40 performed significantly better than conventional parameters and HSCA in predicting RFP post-LT for HCV at biopsy 1, with AUROCs of 0.89 and 0.92, respectively. The accuracy of serum HA >or= 90 microg/L and YKL-40 >or= 200 microg/L in predicting RFP at biopsy 1 was 80% and 96%, respectively. In conclusion, we found that elevated levels of serum HA and YKL-40 within the first 6 months after LT accurately predicted RFP. Larger studies evaluating the role of serum HA and YKL-40 in post-LT management are warranted.

  9. Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene%携带红色荧光蛋白报告基因的重组腺病毒载体的构建及其体外试验研究

    Institute of Scientific and Technical Information of China (English)

    卢建溪; 阳莉; 钱师宇; 王强

    2011-01-01

    目的:采用体外连接技术构建含红色荧光蛋白(RFP)报告基因的重组腺病毒载体,为基因治疗与基因疫苗研究提供重要工具.方法:将pTurboRFP-N上的含RFP基因片段,经酶切连接定向克隆至转移载体pShuttle上,构成重组质粒pShuttle-TurboRFP-N.用I-Ceu I/PI-Sce I双酶切重组质粒pShuttle-TurboRFP-N和骨架质粒pH5'040 pkGFP- II,回收目的片段,连接,获得重组腺病毒载体AdH5'.040.CMV.RFP-N.将其线性化后,经脂质体介导转染至AD293细胞内包装出重组腺病毒颗粒.通过荧光显微镜观察其在AD293细胞内的包装效率和RFP表达情况.扩增并再次感染AD293细胞检测病毒颗粒感染能力,并测定其生物活性及滴度.将所构建的重组腺病毒载体AdH5'.040.CMV.RFP-N与对照腺病毒载体AdH5.CMV.EGFP 在体外感染人肺癌细胞系A549 和乳腺癌细胞系MDA-MB-231.通过荧光蛋白RFP和EGFP的表达,比较它们对以上肿瘤细胞的感染效率.结果:经酶切鉴定,证明已正确构建重组腺病毒载体AdH5'.040.CMV.RFP-N;经AD293细胞包装成具有感染性的病毒颗粒,并能在真核细胞中高效表达目的基因;扩增纯化后获得重组腺病毒颗粒数为3.6×1015vp/L,滴度达1.8×1013pfu/L.重组腺病毒载体AdH5'.040.CMV.RFP-N感染人肺癌细胞系A549 和乳腺癌细胞系MDA-MB-231的感染效率,高于对照腺病毒载体AdH5.CMV.EGFP (P<0.05).结论:成功构建了携带RFP报告基因的重组腺病毒载体,并能够在AD293细胞中高效地表达,对肿瘤细胞的感染效率高.RFP对GFP是一种很好的代替和补充,为基因治疗与基因疫苗研究提供更多的荧光标签.%AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP - N was digested and ligated into pShuttle transfer vector to

  10. Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice

    Science.gov (United States)

    Hoffman, Robert M.

    2006-02-01

    We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

  11. Recommended plutonium release fractions from postulated fires. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Kogan, V.; Schumacher, P.M.

    1993-12-01

    This report was written at the request of EG&G Rocky Flats, Inc. in support of joint emergency planning for the Rocky Flats Plant (RFP) by EG&G and the State of Colorado. The intent of the report is to provide the State of Colorado with an independent assessment of any respirable plutonium releases that might occur in the event of a severe fire at the plant. Fire releases of plutonium are of interest because they have been used by EG&G to determine the RFP emergency planning zones. These zones are based on the maximum credible accident (MCA) described in the RFP Final Environmental Impact Statement (FEIS) of 1980, that MCA is assumed to be a large airplane crashing into a RFP plutonium building.The objective of this report was first, to perform a worldwide literature review of relevant release experiments from 1960 to the present and to summarize those findings, and second, to provide recommendations for application of the experimental data to fire release analyses at Rocky Flats. The latter step requires translation between experimental and expected RFP accident parameters, or ``scaling.`` The parameters of particular concern are: quantities of material, environmental parameters such as the intensity of a fire, and the physico-chemical forms of the plutonium. The latter include plutonium metal, bulk plutonium oxide powder, combustible and noncombustible wastes contaminated with plutonium oxide powder, and residues from plutonium extraction processes.

  12. Environmental Survey preliminary report, Rocky Flats Plant, Golden, Colorado

    Energy Technology Data Exchange (ETDEWEB)

    1987-06-01

    This report presents the preliminary findings of the Environmental Survey of the United States Department of Energy (DOE), Rocky Flats Plant (RFP), conducted August 11 through 22, 1986. The Survey is being conducted by an multidisciplinary team of environmental specialists, led and managed by the Office of Environment, Safety and Health's Office of Environmental Audit. Individual team members are outside experts supplied by a private contractor. The objective of the Survey is to identify environmental problems and areas of environmental risk associated with the RFP. The Survey covers all environmental media and all areas of environmental regulations. It is being performed in accordance with the DOE Environmental Survey Manual. The on-site phase of the Survey involves the review of existing site environmental data observations of the operations carried on at RFP, and interviews with site personnel. The Survey team developed a Sampling and Analysis Plan to assist in further assessing certain environmental problems identified during its on-site activates. The Sampling and Analysis Plan is being executed by DOE's Oak Ridge National Laboratory. When completed, the results will be incorporated into the RFP Environmental Survey Interim Report. The Interim Report will reflect the final determinations of the RFP Survey. 75 refs., 24 figs., 33 tabs.

  13. Molecular encapsulation of rifampicin as an inclusion complex of hydroxypropyl-β-cyclodextrin: design; characterization and in vitro dissolution.

    Science.gov (United States)

    He, Dan; Deng, Ping; Yang, Lin; Tan, Qunyou; Liu, Juan; Yang, Mei; Zhang, Jingqing

    2013-03-01

    A hydrophobic drug, rifampin (RFP), was molecularly encapsulated into hydroxylpropyl-β-cyclodextrin (HCD) to form a molecular inclusion complex (MRICD) with higher solubility and stability. A solid-state grinding method was applied to prepare MRICD for 0.5h. The inclusion ratio, binding constant and the change of Gibbs free energy estimated from the phase solubility diagram and/or by the ultraviolet-visible spectroscopic method were 1:1, ∼218 mol/L and -1.767 KJ/mol, respectively. Differential scanning calorimetry and Fourier transformed infrared spectra of MRICD confirmed the molecular interactions between RFP and HCD. Morphological differences between MICDH and RFP further confirmed the molecular encapsulation of RFP. The most probable configuration of MRICD was estimated via computer simulation. MRICD had a higher dissolution rate than free RFP. Weibull function fit well the dissolution data of MRICD. Broth macrodilution experiments indicated that MRICD had good antibacterial activity. MRICD might be a promising system for oral or parenteral drug delivery to treat bacterial infections.

  14. Women and family poultry production in rural Africa.

    Science.gov (United States)

    Gueye, E H

    2000-02-01

    Poultry production has existed for many generations in Africa, and almost every village household keeps chickens. The rural family poultry (RFP) are generally raised in free-range and/or backyard systems, which are traditional extensive husbandry systems. The development of an intensive poultry production has been the goal of the African government over the years. Despite efforts aiming for such goal, RFP is still very important in African countries that are both poor and net importers of food. It is a valuable asset because it can contribute significantly in alleviating poverty, securing food supply, and promoting gender equality. In view of this, interventions to improve RFP production systems should take into account the sociocultural issues, specifically gender-based aspects. It is noted that such interventions might, in addition to food security and poverty alleviation, also serve to promote gender equality. RFP development programs should be more women-friendly in order to facilitate women's participation, as RFP production in the region is generally a woman's business. Moreover, efforts to empower village women has to be envisaged cautiously as there is a serious risk of men taking over once the poultry sector becomes more profitable.

  15. Rifampicin-induced antineutrophil cytoplasmic antibody-positive vasculitis: a case report and review of the literature.

    Science.gov (United States)

    Ji, Guiyi; Zeng, Xuemei; Sandford, Andrew J; He, Jian-Qing

    2016-10-01

    Antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) is a pauci-immune necrotizing vasculitis that involves small vessels. Herein, we report an extremely rare case of rifampicin (RFP)-induced AAV. A 42-yearold female was transferred to the West China Hospital due to cough with phlegm for 3 months, fever for 1 month, and fatigue for 2 weeks. The patient was diagnosed with pulmonary tuberculosis (TB) and received anti-TB treatment with isoniazid, RFP, ethambutol, and pyrazinamide (PZA) at her local hospital. After 5 days of anti-TB treatment, her creatinine level rose to 420.2 μmol/L from a normal level prior to anti-TB treatment. Serum proteinase 3 (PR3)-ANCA was positive. After discontinuing the anti-TB drugs and administering protective renal treatment, her renal function improved, whereas PR3-ANCA remained positive. With RFP rechallenge after transfer to our hospital, the patient developed oliguria. Her urine volume increased gradually after RFP was discontinued 3 days later. Therefore, RFPinduced AAV was suspected. Eventually, the patient received prednisone and anti-TB therapy, including isoniazid, ethambutol, PZA, and moxifloxacin. After 2 months, PZA was discontinued. During 6 months of normal, and PR3-ANCA became negative at 4 months. This outcome is characteristic of RFP-induced AAV.

  16. Overrelaxation phenomena during the formation of reversed-field pinch plasmas

    Science.gov (United States)

    Mazur, S.; Nordlund, P.

    1995-10-01

    Experiments on the Extrap T1 reversed-field pinch (RFP) [Phys. Scr. 49, 224 (1994)] have shown that the formation of the RFP configuration is quite sensitive to the relative programming of the toroidal field and Ohmic heating circuits. In this paper, new measurements of the evolution of the current density profile and of the spectral structure of the fluctuations during the setup phase of RFP plasmas in the T1 experiment are presented. These measurements improve the understanding of the role of different spectral components in the dynamics of RFP formation. Under unfavorable (slow) setup conditions, comparatively high energy is accumulated in m=1 internal kinks prior to reversal of the edge toroidal field. At reversal, nonlinearly driven m=0 modes trigger a rapid broadening of the m=1 spectrum. This behavior is associated with a violent suppression of the current density in the core, leading to an overrelaxation of the discharge involving a hollowing of the parallel current density profile. The setup conditions are found to affect the volt-second consumption and plasma/wall interaction during RFP formation, as well as the flat-top discharge performance.

  17. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

    Directory of Open Access Journals (Sweden)

    Alexander S. Goryashchenko

    2015-07-01

    Full Text Available This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds, pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

  18. Characterization of metastasis formation and virotherapy in the human C33A cervical cancer model.

    Directory of Open Access Journals (Sweden)

    Ulrike Donat

    Full Text Available More than 90% of cancer mortalities are due to cancer that has metastasized. Therefore, it is crucial to intensify research on metastasis formation and therapy. Here, we describe for the first time the metastasizing ability of the human cervical cancer cell line C33A in athymic nude mice after subcutaneous implantation of tumor cells. In this model, we demonstrated a steady progression of lumbar and renal lymph node metastases during tumor development. Besides predominantly occurring lymphatic metastases, we visualized the formation of hematogenous metastases utilizing red fluorescent protein (RFP expressing C33A-RFP cells. RFP positive cancer cells were found migrating in blood vessels and forming micrometastases in lungs of tumor-bearing mice. Next, we set out to analyze the influence of oncolytic virotherapy in the C33A-RFP model and demonstrated an efficient virus-mediated reduction of tumor size and metastatic burden. These results suggest the C33A-RFP cervical cancer model as a new platform to analyze cancer metastases as well as to test novel treatment options to combat metastases.

  19. Magnetohydrodynamic simulations of noninductive helicity injection in the reversed-field pinch and tokamak

    Energy Technology Data Exchange (ETDEWEB)

    Sovinec, C.R.

    1995-12-31

    Numerical computation is used to investigate resistive magnetohydrodynamic (MHD) fluctuations in the reversed-field pinch (RFP) and in tokamak-like configurations driven solely by direct current (DC) helicity injection. A Lundquist number (S) scan of RFP turbulence without plasma pressure produces the weak scaling of S{sup -0.18} for the root-mean-square magnetic fluctuation level for 2.5x10{sup 3}{le}S{le}4x10{sup 4}. The temporal behavior of fluctuations and the reversal parameter becomes more regular as S is increased, acquiring a {open_quotes}sawtooth{close_quotes} shape at the largest value of S. Simulations with plasma pressure and anisotropic thermal conduction demonstrate energy transport resulting from parallel heat fluctuations. To investigate means of improving RFP energy confinement, three forms of current profile modification are tested. Radio frequency (RF) current drive is modeled with an auxiliary electron force, and linear stability calculations are used.

  20. Fluorochrome-based definition of naturally occurring Foxp3(+) regulatory T cells of intra- and extrathymic origin.

    Science.gov (United States)

    Petzold, Cathleen; Steinbronn, Nadine; Gereke, Marcus; Strasser, Ruth H; Sparwasser, Tim; Bruder, Dunja; Geffers, Robert; Schallenberg, Sonja; Kretschmer, Karsten

    2014-12-01

    Under physiological conditions, studies on the biology of naturally induced Foxp3(+) Treg cells of intra- and extrathymic origin have been hampered by the lack of unambiguous markers to discriminate the mature progeny of such developmental Treg-cell sublineages. Here, we report on experiments in double-transgenic mice, in which red fluorescent protein (RFP) is expressed in all Foxp3(+) Treg cells, whereas Foxp3-dependent GFP expression is exclusively confined to intrathymically induced Foxp3(+) Treg cells. This novel molecular genetic tool enabled us to faithfully track and characterize naturally induced Treg cells of intrathymic (RFP(+) GFP(+) ) and extrathymic (RFP(+) GFP(-) ) origin in otherwise unmanipulated mice. These experiments directly demonstrate that extrathymically induced Treg cells substantially contribute to the overall pool of mature Foxp3(+) Treg cells residing in peripheral lymphoid tissues of steady-state mice. Furthermore, we provide evidence that intra- and extrathymically induced Foxp3(+) Treg cells represent distinct phenotypic and functional sublineages.

  1. Magnetohydrodynamic simulations of noninductive helicity injection in the reversed-field pinch and tokamak

    Energy Technology Data Exchange (ETDEWEB)

    Sovinec, Carl R. [Univ. of Wisconsin, Madison, WI (United States)

    1995-11-01

    Numerical computation is used to investigate resistive magnetohydrodynamic (MHD) fluctuations in the reversed-field pinch (RFP) and in tokamak-like configurations driven solely by direct current (DC) helicity injection. A Lundquist number (S) scan of RFP turbulence without plasma pressure produces the weak scaling of S-0.18 for the root-mean-square magnetic fluctuation level for 2.5x103≤S≤4x104. The temporal behavior of fluctuations and the reversal parameter becomes more regular as S is increased, acquiring a "sawtooth" shape at the largest value of S. Simulations with plasma pressure and anisotropic thermal conduction demonstrate energy transport resulting from parallel heat fluctuations. To investigate means of improving RFP energy confinement, three forms of current profile modification are tested. Radio frequency (RF) current drive is modeled with an auxiliary electron force, and linear stability calculations are used.

  2. Improvement of the magnetic configuration in the reversed field pinch through successive bifurcationsa)

    Science.gov (United States)

    Lorenzini, R.; Agostini, M.; Alfier, A.; Antoni, V.; Apolloni, L.; Auriemma, F.; Barana, O.; Baruzzo, M.; Bettini, P.; Bonfiglio, D.; Bolzonella, T.; Bonomo, F.; Brombin, M.; Buffa, A.; Canton, A.; Cappello, S.; Carraro, L.; Cavazzana, R.; Chitarin, G.; Dal Bello, S.; De Lorenzi, A.; De Masi, G.; Escande, D. F.; Fassina, A.; Franz, P.; Gaio, E.; Gazza, E.; Giudicotti, L.; Gnesotto, F.; Gobbin, M.; Grando, L.; Guo, S. C.; Innocente, P.; Luchetta, A.; Manduchi, G.; Marchiori, G.; Marcuzzi, D.; Marrelli, L.; Martin, P.; Martini, S.; Martines, E.; Milani, F.; Moresco, M.; Novello, L.; Ortolani, S.; Paccagnella, R.; Pasqualotto, R.; Peruzzo, S.; Piovan, R.; Piovesan, P.; Piron, L.; Pizzimenti, A.; Pomaro, N.; Predebon, I.; Puiatti, M. E.; Rostagni, G.; Sattin, F.; Scarin, P.; Serianni, G.; Sonato, P.; Spada, E.; Soppelsa, A.; Spagnolo, S.; Spizzo, G.; Spolaore, M.; Taliercio, C.; Terranova, D.; Toigo, V.; Valisa, M.; Veltri, P.; Vianello, N.; Zaccaria, P.; Zaniol, B.; Zanotto, L.; Zilli, E.; Zuin, M.

    2009-05-01

    The reversed field pinch (RFP) is a magnetic configuration alternative to the tokamak that can be considered for a second generation of reactors. In this paper new remarkable results obtained in the RFP experiment RFX-mod are presented, showing that an internal transport barrier delimitates a large fraction of the plasma volume in a RFP when the current is raised to ˜1.5 MA. The formation of this transport barrier is related to a profound, spontaneous modification of the magnetic topology. Due to the occurrence of a saddle node bifurcation the plasma enters in the single helical axis state, which is theoretically known to be more resilient to chaos. This bifurcation is driven by the amplitude of the helical perturbation which dominates the mode spectrum.

  3. High-flux first-wall design for a small reversed-field pinch reactor

    Science.gov (United States)

    Cort, G. E.; Graham, A. L.; Christensen, K. E.

    To achieve the goal of a commercially economical fusion power reactor, small physical size and high power density should be combined with simplicity (minimized use of high technology systems). The Reversed-Field Pinch (RFP) is a magnetic confinement device that promises to meet these requirements with power densities comparable to those in existing fission power plants. To establish feasibility of such an RFP reactor, a practical design for a first wall capable of withstanding high levels of cyclic neutron wall loadings is needed. Associated with the neutron flux in the proposed RFP reactor is a time averaged heat flux of 4.5 MW/sq m with a conservatively estimated transient peak approximately twice the average value. The design for a modular first wall made from a high-strength copper alloy that will meet these requirements of cyclic thermal loading is presented. The heat removal from the wall is by subcooled water flowing in straight tubes at high linear velocities.

  4. The Controlled-releasing Drug Implant based on the Three Dimensional Printing Technology:Fabrication and Properties of Drug Releasing in vivo

    Institute of Scientific and Technical Information of China (English)

    WU Weigang; ZHENG Qixin; GUO Xiaodong; HUANG Weidong

    2009-01-01

    Three dimensional(3D)printing technology was utilized to fabricate a new type of drug implant with complicated architectures,employing levofloxacin(LVFX)and rifampicine(RFP) as model drugs.The prepared drug implant prototype consists of a double-layer structure,of which the upper region is a reservoir system containing RFP and the lower region is a matrix one containing LVFX.The release test in vivo revealed that LVFX was released in the early stage;no RFP was de-tected until 8th day;both of them continuously released more than 6 weeks.Therefore,3D printing technology provides a precise and feasible method to fabricate a controlled-releasing drug implant with complicated architectures and this drug implant may present a new strategy for the prophylaxis and treatment of bone diseases such as combined bone infections and bone tuberculosis in the near future.

  5. GPR41/FFAR3 and GPR43/FFAR2 as cosensors for short-chain fatty acids in enteroendocrine cells vs FFAR3 in enteric neurons and FFAR2 in enteric leukocytes

    DEFF Research Database (Denmark)

    Nøhr, Mark Klitgaard; Pedersen, Maria H; Gille, Andreas;

    2013-01-01

    in a subpopulation of enteroendocrine cells. Nevertheless, synthetic ligands specific for either FFAR3 or FFAR2 each released GLP-1 from colonic crypt cultures and the FFAR2 agonist mobilized intracellular Ca(2+) in FFAR2 positive enteroendocrine cells. It is concluded that FFAR3-mRFP serves as a useful marker....... Throughout the colon and rectum, FFAR3-mRFP was strongly expressed in the large population of peptide YY and GLP-1 cells and in the neurotensin cells of the proximal colon. A gradient of expression of FFAR3-mRFP was observed in the somatostatin cells from less than 5% in the stomach to more than 95...

  6. Influence of Via Stacking for High Density of Consumer Electronics Products

    Science.gov (United States)

    Nakanishi, Tohru; Hase, Tomohiro

    The stacked via technology becomes to be one of key technologies for the achievement of high density packaging as of today, however, it could not be said that the influence of via stacking has been understood sufficiently. The influence of one to five stacked VIA technologies are studied with the parameter of stress and strain on the view point of reliability, comparing the condition that these vias are located directly on the RFP (Resin Filled PTH (Pin Through Hole)), and the other condition that these are located left and right with some distance from RFP. The maximum is happened at the smallest neck of via, and it is recommended that the via stacking is designed with some distance from RFP. The guideline as to the optimized design of the substrate that has the stacked via is provided.

  7. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Yong Zhao

    Full Text Available Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0; thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100% were correctly detected through the assay. Of these isolates, 88/88 (100% were determined as RFP susceptible and 52/54 (96.3% were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  8. Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhao, Yong; Li, Guilian; Sun, Chongyun; Li, Chao; Wang, Xiaochen; Liu, Haican; Zhang, Pingping; Zhao, Xiuqin; Wang, Xinrui; Jiang, Yi; Yang, Ruifu; Wan, Kanglin; Zhou, Lei

    2015-01-01

    Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

  9. 应用双荧光报告系统检测斑马鱼microRNA的动态表达%Detection of dynamic expression of microRNAs in vivo using a dual-fluorescence reporter system/miRNA Tracer in zebrafish

    Institute of Scientific and Technical Information of China (English)

    杨红波; 梁巍; 刘新星; 朱作言; 林硕; 张博

    2012-01-01

    microRNAs (miRNAs) are short noncoding RNAs that have been found in a wide variety of organisms and many have been shown to play essential roles by regulating the stability and translation of target messenger RNAs (mRNAs) in animals and plants. Temporal and spatial expression is critical for the regulatory function of miRNAs. To analyze the dynamic expression of particular miRNA in vivo, we constructed a dual-fluorescence reporter system based on Tol2 trans-poson, in which two reporter genes, enhanced green fluorescent protein (eGFP) and monomeric red fluorescent protein 1 (mRFP1), were driven by the heat shock promoter (hsp) from zebrafish hsp70 gene in an opposite orientation. To sense the existence of a particular miRNA, the complementary DNA sequence of the corresponding miRNA was inserted into the 3'-UTR region of one of the two reporter genes. By injecting the corresponding plasmid DNA into zebrafish embryos, we were able to monitor the abundance and dynamics of miRNA miR-206 in live embryos. To further evaluate this method, we made a collection of transgenic zebrafish with stable integration of dual-fluorescence reporter plasmids targeting different miRNAs, including miR-206 and miR-219. Our results showed that this dual-fluorescence reporter system, which is also called miRNA Tracer, could faithfully monitor the appearance and disappearance of target miRNAs in defined cell lineages during zebrafish development in these fish lines. Our dual-fluorescence reporter/Tracer system provides an important tool for further in-depth studies on miRNAs in zebrafish.%microRNA(miRNA)是一类细胞内源表达的小分子非编码RNA,主要通过降解靶基因的mRNA或者抑制靶基因的翻译,在动植物的发育以及其他重要的生理过程中起调控作用.miRNA的功能跟它的表达位置与时间密切相关,但是目前尚缺乏一个能够在活体与个体水平稳定、持续地实时观察miRNA动态表达的方法.文章以斑马鱼为模式,建

  10. Changes in risk factor profile after ischemic stroke

    DEFF Research Database (Denmark)

    Hornnes, Nete

    up 1 year after stroke. We constructed a baseline risk factor profile (RFP) of 6 variables: smoking, excessive drinking, physical inactivity, untreated hypertension, no cholesterol-lowering, and no antithrombotic treatment/warfarin at discharge from hospital. Each item was rated 0 or 1 giving...... a maximum score of 6 points. Mean baseline RFP-score was 1.6 Results. After 1 year we found a reduction in current smoking (p=0.008) and in excessive drinking (p=0.0001). There was no change in physical activity and in untreated hypertension. There was an increase in the proportion of patients on lipid...

  11. Yeast Interacting Proteins Database: YPL125W, YDR502C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YPL125W KAP120 Karyopherin responsible for the nuclear import of ribosome maturation factor Rfp1p Row...s with this bait as bait (1) Rows with this bait as prey (0) YDR502C SAM2 S-adenosylmethio...o differentially regulated isozymes (Sam1p and Sam2p) Rows with this prey as prey (1) Row... gene name KAP120 Bait description Karyopherin responsible for the nuclear import of ribosome maturation factor Rfp1p Row...s with this bait as bait Rows with this bait as bait (1) Rows with this bait as prey Row

  12. Demonstration, testing and evaluation of nonintrusive characterization technologies at operable Unit 2 of Rocky Flats Plant. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1994-09-01

    A three-dimensional (3-D), high-resolution (HR) seismic reflection evaluation was conducted at the Rocky Flats Plant (RFP), near Golden, Colorado, to demonstrate the applicability of nonintrusive characterization techniques to detect buried objects, contamination, and geological/hydrological features at RFP. The evaluation was conducted as part of the U.S. Department of Energy`s (DOE) request for demonstration, testing and evaluation (DT&E) of nonintrusive techniques, under DOE Program Research and Development Announcement (PRDA) No. DE-RA05-09OR22000.

  13. ELECTROSTATIC MODE ASSOCIATED WITH PINCH VELOCITY IN RFPS

    Energy Technology Data Exchange (ETDEWEB)

    DELZANNO, GIAN LUCA [Los Alamos National Laboratory; FINN, JOHN M. [Los Alamos National Laboratory; CHACON, LUIS [Los Alamos National Laboratory

    2007-02-08

    The existence of a new electrostatic instability is shown for RFP (reversed field pinch) equilibria. This mode arises due to the non-zero equilibrium radial flow (pinch flow). In RFP simulations with no-stress boundary conditions on the tangential velocity at the radial wall, this electrostatic mode is unstable and dominates the nonlinear dynamics, even in the presence of the MHD modes typically responsible for the reversal of the axial magnetic field at edge. Nonlinearly, this mode leads to two beams moving azimuthally towards each other, which eventually collide. The electrostatic mode can be controlled by using Dirichlet (no-slip) boundary conditions on the azimuthal velocity at the radial wall.

  14. Post-mortem re-cloning of a transgenic red fluorescent protein dog.

    Science.gov (United States)

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

    2011-12-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

  15. A redox-dependent dimerization switch regulates activity and tolerance for reactive oxygen species of barley seed glutathione peroxidase

    DEFF Research Database (Denmark)

    Navrot, Nicolas; Skjoldager, Nicklas; Bunkenborg, Jakob

    2015-01-01

    Monomeric and dimeric forms of recombinant barley (Hordeum vulgare subsp. vulgare) glutathione peroxidase 2 (HvGpx2) are demonstrated to display distinctly different functional properties in vitro. Monomeric HvGpx2 thus has five fold higher catalytic efficiency than the dimer towards tert-butyl h...... active, but more oxidation-resistant dimer. ...

  16. Monomer formation and functioning of p hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide-water mixtures

    NARCIS (Netherlands)

    Kudryashova, E.V.; Visser, A.J.W.G.; Berkel, van W.J.H.

    2008-01-01

    It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric

  17. Chemistry of organogold compounds I. Syntheses and properties of dihalogold(III) N,N-dialkyldithiocarbamates and dialkylgold(III) N,N-dialkyldithiocarbamates

    NARCIS (Netherlands)

    Blaauw, H.J.A.; Nivard, R.J.F.; Kerk, G.J.M. van der

    Monomeric dialkylgold(III) N,N-dialkyldithiocarbamates (R2AuSSCNR'2) can be prepared in good yields from monomeric dibromogold(III) N,N-dialkyldithiocarbamates (Br2AuSSCNR'2) and alkylmagnesium halides or dialkylcadmium compounds. In addition N-ethylpiperidinium tetrachloroaurate can be converted

  18. Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus : Nucleotide-free and nucleotide-bound conformations

    NARCIS (Netherlands)

    Verdon, G.; Albers, S.V.; Dijkstra, B.W.; Driessen, A.J.M.; Thunnissen, A.M.W.H.

    2003-01-01

    The ABC-ATPase GlcV energizes a binding protein-dependent ABC transporter that mediates glucose uptake in Sulfolobus solfataricus. Here, we report high-resolution crystal structures of GlcV in different states along its catalytic cycle: distinct monomeric nucleotide-free states and monomeric

  19. Conversion of oligomeric starch, cellulose, hydrolysates or sugars to hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Silks, Louis A; Sutton, Andrew; Kim, Jin Kyung; Gordon, John Cameron; Wu, Ruilian; Kimball, David B.

    2017-09-05

    Embodiments of the present invention are directed to the conversion of a source material (e.g., a depolymerized oligosaccharide mixture, a monomeric sugar, a hydrolysate, or a mixture of monomeric sugars) to intermediate molecules containing 7 to 26 contiguous carbon atoms. These intermediates may also be converted to saturated hydrocarbons. Such saturated hydrocarbons are useful as, for example, fuels.

  20. Methods of forming boron nitride

    Science.gov (United States)

    Trowbridge, Tammy L; Wertsching, Alan K; Pinhero, Patrick J; Crandall, David L

    2015-03-03

    A method of forming a boron nitride. The method comprises contacting a metal article with a monomeric boron-nitrogen compound and converting the monomeric boron-nitrogen compound to a boron nitride. The boron nitride is formed on the same or a different metal article. The monomeric boron-nitrogen compound is borazine, cycloborazane, trimethylcycloborazane, polyborazylene, B-vinylborazine, poly(B-vinylborazine), or combinations thereof. The monomeric boron-nitrogen compound is polymerized to form the boron nitride by exposure to a temperature greater than approximately 100.degree. C. The boron nitride is amorphous boron nitride, hexagonal boron nitride, rhombohedral boron nitride, turbostratic boron nitride, wurzite boron nitride, combinations thereof, or boron nitride and carbon. A method of conditioning a ballistic weapon and a metal article coated with the monomeric boron-nitrogen compound are also disclosed.

  1. Methods of forming boron nitride

    Energy Technology Data Exchange (ETDEWEB)

    Trowbridge, Tammy L; Wertsching, Alan K; Pinhero, Patrick J; Crandall, David L

    2015-03-03

    A method of forming a boron nitride. The method comprises contacting a metal article with a monomeric boron-nitrogen compound and converting the monomeric boron-nitrogen compound to a boron nitride. The boron nitride is formed on the same or a different metal article. The monomeric boron-nitrogen compound is borazine, cycloborazane, trimethylcycloborazane, polyborazylene, B-vinylborazine, poly(B-vinylborazine), or combinations thereof. The monomeric boron-nitrogen compound is polymerized to form the boron nitride by exposure to a temperature greater than approximately 100.degree. C. The boron nitride is amorphous boron nitride, hexagonal boron nitride, rhombohedral boron nitride, turbostratic boron nitride, wurzite boron nitride, combinations thereof, or boron nitride and carbon. A method of conditioning a ballistic weapon and a metal article coated with the monomeric boron-nitrogen compound are also disclosed.

  2. 76 FR 57845 - Approval of Air Quality Implementation Plans; California; San Joaquin Valley; Attainment Plan for...

    Science.gov (United States)

    2011-09-16

    ... 16696 (March 25, 2011). S-COM-2 Boilers, Steam Generators and 4307 3rd Q--2008 October 2008 75 FR 1715... for boilers, reasonable further progress (RFP) and attainment demonstrations, transportation... Clean Fuels for Boilers H. Motor Vehicle Emissions Budgets for Transportation Conformity I. Other...

  3. SITE TECHNOLOGY CAPSULE: FILTER FLOW TECHNOLOGY, INC. - COLLOID POLISHING FILTER METHOD

    Science.gov (United States)

    The Filter Flow Technology, Inc. (FFT) Coloid Polishing Filter Method (CPFM) was demonstrated at the U.S Department of Energy's (DOE) Rock Flats Plant (RFP) as part of the U.S. Environmental Protection Agency's (EPA) Superfund and Innovative Technology Evaluation (SITE) program. ...

  4. Technical Safety Appraisal of the Rocky Flats Plant

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Blake P.

    1989-01-01

    This report provides the results of a Technical Safety Appraisal (TSA) of the Rocky Flats Plant (RFP) conducted November 14 to 18 and November 28 to December 9, 1988. This appraisal covered the effectiveness and improvements in the RFP safety program across the site, evaluating progress to date against standards of accepted practice. The appraisal included coverage of the timeliness and effectiveness of actions taken in response to the recommendations/concerns in three previous Technical Safety Appraisals (TSAs) of RFP Bldg. 707 conducted in July 1986, Bldgs. 771/774 conducted in October/November 1986, and Bldgs. 776/777 conducted in January/February 1988. Results of this appraisal are given in Section IV for each of 14 technical safety areas at RFP. These results include a discussion, conclusions and any new safety concerns for each technical safety area. Appendix A contains a description of the system for categorizing concerns, and the concerns are tabulated in Appendix B. Appendix C reports on the evaluation of the contractor's actions and the current status of each of the 230 recommendations and concerns contained in the three previous TSA reports.

  5. SMC Specifications and Standards Program

    Science.gov (United States)

    2011-08-01

    Common technical language • Common RFP/Contract Tools • Increased visibility and understanding of industry practices • Including span of...systems initially aquired before 2004 SMC/SES 2004.0 Use on launch systems aquired after 2004 The current version 0 is acquisi·tion reform

  6. Advanced Stellar Compass - Alenia Mars Express

    DEFF Research Database (Denmark)

    Kilsgaard, Søren; Betto, Maurizio; Jørgensen, John Leif;

    1998-01-01

    This document, submitted in reply to an Alenia R.f.P., is a proposal to implement the Advanced Stellar Compass (ASC) in the Mars Express mission.The Mars Express is an ESA dedicated mission to Mars scientific investigation.The ASC is a very advanced instrument designed by the Space Instrumentation...

  7. Advanced Stellar Compass - ROCSAT 2 - Proposal

    DEFF Research Database (Denmark)

    Riis, Troels; Betto, Maurizio; Jørgensen, John Leif;

    1998-01-01

    This proposal is the DTU response to the Dornier DSS (DSS) request for proposal (r.f.p.) for the ROCSAT-2 star tracker.ROCSAT-2 is jointly developed by DSS with the taiwanese Space Agency (NSPO) and consists mainly of an optical camera as well as a customer furnished scientific instrument. ROCSAT...

  8. Advanced Stellar Compass - Proposal for the LunARSat project

    DEFF Research Database (Denmark)

    Betto, Maurizio; Jørgensen, John Leif; Kilsgaard, Søren;

    1998-01-01

    request for proposal (r.f.p.) for the LunARSat star tracker.The Advanced Stellar Compass (ASC) is a highly advanced and autonomous Stellar Reference Unit designed, developed and produced by the Space Instrumentation Group of the Department of Automation of the Technical University of Denmark.The document...

  9. A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

    Science.gov (United States)

    Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

    2015-01-01

    Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

  10. Young Children's Reading for Pleasure with Digital Books: Six Key Facets of Engagement

    Science.gov (United States)

    Kucirkova, Natalia; Littleton, Karen; Cremin, Teresa

    2017-01-01

    This paper offers a new characterisation of young children's (2-8 years) reading for pleasure (RfP) with digital books. This characterisation is rooted in a re-contextualisation of Anna Craft's conceptualisation of twenty-first century childhoods in "Creativity and education futures" (Stoke on Trent, Trentham, 2011) and a review of the…

  11. Two-plasmid vector system for independently controlled expression of green and red fluorescent fusion proteins in Staphylococcus aureus.

    Science.gov (United States)

    Brzoska, Anthony J; Firth, Neville

    2013-05-01

    We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

  12. 75 FR 958 - Approval and Promulgation of Air Quality Implementation Plans; Maryland; 2002 Base Year Emission...

    Science.gov (United States)

    2010-01-07

    ... emissions modeling software, MOBILE6. The FMVCP and RVP emission reductions are then removed from the base... and/or VOC) accounting for any growth that occurs during the six year period following the baseline... source emissions accounting for all mobile control measures. The MVEBs for the 2008 RFP are shown...

  13. 75 FR 953 - Approval and Promulgation of Air Quality Implementation Plans; Maryland; 2002 Base Year Emission...

    Science.gov (United States)

    2010-01-07

    ... reductions are determined by the State using EPA's on-road mobile source emissions modeling software, MOBILE6... SIP revision must provide for a 15 percent emission reduction (either NO X and/or VOC) accounting for... the 2008 RFP is based on the projected 2008 mobile source emissions accounting for all mobile...

  14. Man-made respirable-sized organic fibers: what do we know about their toxicological profiles?

    Science.gov (United States)

    Warheit, D B; Reed, K L; Webb, T R

    2001-04-01

    Man-made organic fibers (MMOFs) have been manufactured for over 50 years. Until recently, there have been few concerns raised regarding the safety of organic fiber dusts. This is due, in large part, to the perception that the dimensions of most, if not all, of these products were too large to be inhaled into the distal lungs of workers, i.e., were considered to be nonrespirable. A brief review of some of the issues related to organic fiber toxicology is presented herein. Some of the organic fiber-types used in commerce are identified and some fundamental tenets of fiber toxicology are discussed. In addition, the European Union, in their recent consideration for banning chrysotile asbestos fibers, evaluated some organic fiber substitutes and compared them to the hazards of asbestos. A brief review of their conclusions is described below. Finally, the results of some recent studies assessing the mechanisms of biodegradability of para-aramid respirable-sized, fiber-shaped particulates (RFP) are presented. Para-aramid (p-aramid) RFP are the most extensively-studied respirable organic fiber-type and RFP is the new term which describes respirable-sized organic fibers (ECETOC, 1996) (1). The results of these studies provide clues regarding the mechanism(s) of p-aramid RFP shortening in the lungs of exposed animals, and may be relevant for humans.

  15. 76 FR 17711 - Notice of Availability of Calendar Year 2012 Competitive Grant Funds

    Science.gov (United States)

    2011-03-30

    ...) will be available beginning April 11, 2011. Applicants must file a Notice of Intent to Compete (NIC) to participate in the competitive grants process. Applicants must file the NIC by May 13, 2011, 5 p.m. E.D.T... officials. The RFP, containing the NIC and grant application, guidelines, proposal content...

  16. 77 FR 4564 - Request for Information Regarding the Reinsurance Program Under the Affordable Care Act

    Science.gov (United States)

    2012-01-30

    ... more future Requests for Proposals (RFP). This RFI solicits information about entities that could... to issue a grant. The purpose of this RFI is to inform one or more Requests for Proposals, not to... validating requests for reinsurance payments; Remitting reinsurance payments; Reconciling and...

  17. Observation of Electron Bernstein Wave Heating in the MST Reversed Field Pinch

    Science.gov (United States)

    Seltzman, Andrew; Anderson, Jay; Dubois, Ami; Almagri, Abdulgader; Nonn, Paul; McCollam, Karsten; Chapman, Brett; Goetz, John; Forest, Cary

    2016-10-01

    We report the first observation of electron Bernstein wave heating in the MST RFP. Similar to a high density stellarator, the RFP is inaccessible to electromagnetic ECRH. The plasma current and |B|operating range of MST allows a 5.5 GHz RF source (100kW, 4ms pulse) to heat on the fundamental and up to 4th harmonic EC resonances. With an x-ray diagnostic most sensitive to edge electrons located +12 degrees toroidally from the antenna, the measured emission is a strong function of predicted heating inside versus outside the Bt =0 reversal layer of the RFP. Measured during a scan of plasma current, distinct edges in a plot of emissivity versus predicted deposition layer align with the deposition layers crossing of this reversal layer and confirm EBW heating on the fundamental through 4th EC harmonic. Additional confirmation of the absorption location has been demonstrated by using auxiliary poloidal current drive to reduce electron diffusion rates and sweep the location of the Bt =0 surface across a static RF absorption location in RFP discharges. In these discharges EBW enhancement of the 15-40keV x-ray energies has been observed. Work supported by USDOE.

  18. 7 CFR 277.18 - Establishment of an Automated Data Processing (ADP) and Information Retrieval System.

    Science.gov (United States)

    2010-01-01

    ... activities and expenditures in relation to the approved Planning APD or Implementation APD. An APDU may also... for Proposal or RFP means the document used for public solicitations of competitive proposals from... resources and availability of those resources, and the assignments of roles and responsibilities for project...

  19. Library Signage: Doing It Right.

    Science.gov (United States)

    Wismer, Donald

    This paper describes and provides documentation for the development of a signage system for the Maine State Library. A brief description of the process is accompanied by copies of the Request for Proposal (RFP); a score sheet used to evaluate the offerings of various companies bidding for the contract and the results of the evaluation; a copy of…

  20. A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

    Science.gov (United States)

    Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

    2015-01-01

    Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

  1. Report of the Defense Science Board on Use of Commercial Components in Military Equipment

    Science.gov (United States)

    1989-06-01

    services adopt the open systems architecture concept, endorse the interna - tional ISO/OSI standards and protocols, comply with GOSIP, and make optimum...and logistica support that uws short. concise. easy to understand and streamlined. A-33 CHS RFP OVERVIEW THE CHS SOLICITATION IS: * UNIQUE

  2. [Transient expression in microplasmodia of Physarum polycephalum].

    Science.gov (United States)

    Liu, Shide; Cheng, Caixia; Lin, Ziyang; Zhang, Jianhua; Li, Minghua; Zhou, Zhuolong; Tian, Shengli; Xing, Miao

    2009-06-01

    The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRedl-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 micros electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.

  3. Utility and infrastructure needs for private tank waste processing

    Energy Technology Data Exchange (ETDEWEB)

    Reynolds, B.A.

    1996-05-01

    This document supports the development of the Draft TWRS Privatization RFP. The document provides summaries of a wide variety of utility infrastructure and support services that are available at the Hanford Site. The needs of the privatization contractors are estimated and compared to the existing infrastructure. Recommendations are presented on the preferred and alternate routes of supplying the identifies requirements.

  4. 78 FR 73769 - Approval and Promulgation of Air Quality Implementation Plans; West Virginia; Approval of the...

    Science.gov (United States)

    2013-12-09

    ..., RFP, emissions inventories, and contingency measures. \\3\\ PM 10 refers to particulates nominally 10... emissions inventory for the West Virginia portion of the Area for the 2006 24-hour PM 2.5 NAAQS. In this... established on July 16, 1997 (62 FR 38652, July 18, 1997). EPA promulgated an annual standard at a level of 15...

  5. 77 FR 62159 - Approval and Promulgation of Implementation Plans; North Carolina Portion of the Charlotte...

    Science.gov (United States)

    2012-10-12

    ... action. B. Adjusted Base Year Inventory and 2008 RFP Target Levels The process for determining the... calculating the target level of emissions in 2008. The adjusted VOC inventory for calculating the target level... requirement. The actual projected 2008 inventory for all sources with all control measures in place, including...

  6. 78 FR 53113 - Approval and Promulgation of Implementation Plans; California; San Joaquin Valley; Contingency...

    Science.gov (United States)

    2013-08-28

    ...). With respect to the level of emission reductions associated with contingency measures, EPA has... contingency measures for 2012 are, therefore, no longer needed. The emission inventory used in the RFP... measuring progress for the annual PM 2.5 standard. The inventory in the 2011 Progress Report, used in the...

  7. Experiment list: SRX346921 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available air, by a process called melanogenesis. Coloration can be altered by the number of melanocytes or the amount...e control; Homo sapiens; ChIP-Seq source_name=Primary human melanocytes infected with a lentivirus for RFP (

  8. RFX-mod: A multi-configuration fusion facility for three-dimensional physics studies

    Energy Technology Data Exchange (ETDEWEB)

    Piovesan, P.; Bonfiglio, D.; Auriemma, F.; Bonomo, F.; Carraro, L.; Cavazzana, R.; De Masi, G.; Fassina, A.; Franz, P.; Gobbin, M.; Marrelli, L.; Martin, P.; Martines, E.; Momo, B.; Piron, L.; Valisa, M.; Veranda, M.; Vianello, N.; Zaniol, B.; Agostini, M. [Consorzio RFX, EURATOM-ENEA Association, Corso Stati Uniti 4, 35127 Padova (Italy); and others

    2013-05-15

    RFX-mod [Sonato et al., Fusion Eng. Des. 66, 161 (2003)] exploits its 192 active coils in both reversed-field pinch (RFP) and tokamak configurations with varying degrees of 3D shaping, providing also a test bed for validating stellarator codes and 3D nonlinear magnetohydrodynamic codes. This makes RFX-mod a unique and flexible facility for comparative studies on 3D shaping and control. The paper discusses how 3D fields allow access to RFP and tokamak advanced regimes. 3D fields are used to feedback control Single Helicity (SH) RFP equilibria with 1/7 helicity up to ∼2 MA. They also allow accessing SH regimes with higher density (Greenwald fraction up to 0.5), presently inaccessible in spontaneous SH regimes. Feedback on the 2/1 resistive-wall mode in RFX-mod tokamak plasmas allows for safe operation at q(a)<2, an almost unexplored promising regime. Forcing the 2/1 mode to saturate at finite but small level, a helical tokamak equilibrium with significant n = 1 modulation is produced and a new way to tailor sawteeth is found. The effects of different levels of 3D shaping on momentum transport in both RFP and tokamak helical states are discussed.

  9. [Methodological issues of sentinel lymph nodes biopsy in patients with breast cancer].

    Science.gov (United States)

    Kanaev, S V; Novikov, S N; Krivorot'ko, P V; Semiglazov, V F; Zhukova, L A; Krzhivitskiĭ, P I

    2013-01-01

    Radionuclide imaging of sentinel lymph nodes (SLN) was performed in 122 breast cancer patients, which before the biopsy of lymph nodes it was performed intratumoral injection of colloidal radiopharmaceuticals (RFP): in 89 patients--nanocolloidal (NC) and in 33--colloidal with particle size from 200 to 1000 nm. After the introduction of NC the SLN image was obtained in 83 of 89 women. (93.3%). After the introduction of large colloids (200-1000 nm or more) SLN visualization in this group was achieved in 27 of 33 patients, i.e., in 81.8% of cases (p lymph nodes of the second and higher orders in axillary as well as under-and supraclavicular regions. On the contrary while using larger colloids, RFP accumulated only in SLN of axillary region in 85.1%. These differences in the topography of the absorption of various diameters radiocolloids were reliable (p = 0.01). Using the NC RFN compared with colloidal RFP of larger diameter can reliably improve SLN visualization till 98.9% however leads to a concomitant accumulation of RFP in lymph nodes of the second order in 55.8% of patients.

  10. Radiation Biomarker Research Using Mass Spectrometry

    Science.gov (United States)

    2007-07-01

    34. Heynick Louis, N.; Johnston Sheila, A.; Mason Patrick, A., Radio frequency electromagnetic fields: cancer, mutagenesis, and genotoxicity ...their possible carcinogenic, mutagenic, and genotoxic effects. This project found, however, that according to the majority of papers published RF...P. A., Effects of blood flow on skin heating induced by millimeter wave irradiation in humans. Health Physics 2004, 86, (2), 115-120

  11. Computational Omics - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the NVIDIA Foundation are pleased to announce funding opportunities in the fight against cancer. Each organization has launched a request for proposals (RFP) that will collectively fund up to $2 million to help to develop a new generation of data-intensive scientific tools to find new ways to treat cancer.

  12. An (125)I-labeled octavalent peptide fluorescent nanoprobe for tumor-homing imaging in vivo.

    Science.gov (United States)

    Luo, Haiming; Shi, Jiyun; Jin, Honglin; Fan, Di; Lu, Lisen; Wang, Fan; Zhang, Zhihong

    2012-06-01

    Targeting radiopeptides are promising agents for radio-theranostics. However, in vivo evaluation of their targeting specificity is often obscured by their short biologic half-lives and low binding affinities. Here, we report an approach to efficiently examine targeting radiopeptides with a new class of octavalent peptide fluorescent nanoprobe (Octa-FNP) platform, which is composed of candidate targeting peptides and a tetrameric far-red fluorescent protein (tfRFP) scaffold. To shed light on this process, (125)I-Octa-FNP, (125)I-tfRFP and (125)I-peptide were synthesized, and their targeting functionalities were compared. Both fluorescence imaging and radioactive quantification results confirmed that (125)I-Octa-FNP had a significantly higher cellular binding capability than (125)I-tfRFP. In vivo biodistribution studies show that at 6 h post-injection, (125)I-Octa-FNP had 2-fold and 30-fold higher tumor uptake than that of (125)I-tfRFP and (125)I-peptide, respectively. Moreover, γ-imaging at 24 h post-injection revealed a remarkable accumulation of (125)I-Octa-FNP in the tumor while maintaining an extremely low background contrast, which was further confirmed by immunofluorescence analysis. These data suggested that, as an engineered and multivalent platform, Octa-FNP could enhance the tumor targeting of a designed peptide and provide excellent contrast radioimaging, making it a valuable tool for the evaluation of the targeting ability of specifically designed radiopeptides for cancer theranostics.

  13. 40 CFR 51.1000 - Definitions.

    Science.gov (United States)

    2010-07-01

    ... organic carbon, directly emitted sulfate, directly emitted nitrate, and other inorganic particles... emissions that contribute to the formation of PM2.5. PM2.5 precursors include S02, NOX, volatile organic compounds, and ammonia. Reasonable further progress (RFP) means the incremental emissions reductions toward...

  14. 76 FR 3917 - National Human Genome Research Institute; Notice of Closed Meeting

    Science.gov (United States)

    2011-01-21

    ... HUMAN SERVICES National Institutes of Health National Human Genome Research Institute; Notice of Closed... privacy. Name of Committee: National Human Genome Research Institute Special Emphasis Panel, TRND--RFP... Person: Rudy O. Pozzatti, PhD, Scientific Review Officer, Scientific Review Branch, National Human...

  15. NMSBA: Sandia Biotech 2016 Report

    Energy Technology Data Exchange (ETDEWEB)

    Ruffing, Anne [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2016-06-01

    The objective of this project is to modify the FluorAbody plasmid previously developed by Sandia Biotech to include a binding site for biotin by introducing the biotin carboxyl carrier protein (BCCP)and a gold binding protein (GBP) into a loop of the red fluorescent protein (mRFP).

  16. 48 CFR 15.203 - Requests for proposals.

    Science.gov (United States)

    2010-10-01

    ... information regarding cost comparisons between Government and contractor performance. (c) Electronic commerce... RFP shall specify the electronic commerce method(s) that offerors may use (see subpart 4.5). (d... and suitability of electronic commerce methods; and (iv) Adequacy of administrative procedures...

  17. 50 CFR 648.207 - Herring Research Set-Aside (RSA).

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Herring Research Set-Aside (RSA). 648.207... during the interim years and decides to publish a second RFP. Proposals to fund research that would start..., researchers must provide the Council and NMFS with a report of research findings, which must include:...

  18. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    Science.gov (United States)

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  19. GenBank blastx search result: AK060683 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060683 001-029-F09 U43202.1 RFP provirus of Murine leukemia virus gag precursor protein, pol precurso...r protein, env precursor protein genes, complete cds.|VRL VRL 1e-12 +2 ...

  20. Evolved Expendable Launch Vehicle (EELV)

    Science.gov (United States)

    2015-12-15

    2015. When the draft RFP was posted a Space and Missile Systems Center (SMC) internal Comment Resolution Matrix ( CRM ) was inadvertently exposed for...competitiveness; and benefit the U.S. economy. The overall goal of the EELV December 2015 SAR March 14, 2016 16:05:14 UNCLASSIFIED 8 launch system investment is

  1. 76 FR 66169 - Office of Advocacy and Outreach Federal Financial Assistance Programs

    Science.gov (United States)

    2011-10-26

    ... narrative or any other attachment included in the proposal that contains such information: ``The following... RFP. At OAO's discretion, either written or oral feedback will be provided to unsuccessful applicants... transfer system restricted or placed on ``manual review,'' which restricts the awardee's ability to...

  2. 77 FR 50595 - Approval and Promulgation of Air Quality Implementation Plans; Connecticut, Massachusetts, and...

    Science.gov (United States)

    2012-08-22

    ... from mobile on- road and non-road sources, such that complete emission inventories are available to... within our September 20, 2010 proposal occurred within step 6 of Table 3d, where the detailed RFP target..., area, non-road mobile, on-road mobile and biogenic sources. The inventories were submitted as...

  3. Rio 2016 golf Olympic course: technical brief

    OpenAIRE

    2015-01-01

    Through the RFP, Rio 2016™ seeks to identify the best concept design for the Olympic golf course, for both women’s and men’s competition at the Rio 2016™ Olympic Games as well as the best legacy solution for the future.

  4. 76 FR 1642 - Notice of Availability of Calendar Year 2011 Competitive Grant Funds

    Science.gov (United States)

    2011-01-11

    ... (NIC; RFP Form- H) to participate in the competitive grants process. The deadline for filing the NIC is.... The dates shown in this notice for filing the NIC and the grant proposals supersede the dates in the... Applicants who may submit a NIC and a grant proposal to participate in the competitive grants process:...

  5. 75 FR 12802 - Notice of Availability of Calendar Year 2011 Competitive Grant Funds

    Science.gov (United States)

    2010-03-17

    ...) will be available April 8 2010. Applicants must file a Notice of Intent to Compete (NIC) to participate in the competitive grants process. Applicants must file the NIC by May 13 2010, 5 p.m. E.D.T. The due... governing boards are controlled by locally elected officials. The RFP, containing the NIC and...

  6. 48 CFR 2415.304 - Evaluation factors.

    Science.gov (United States)

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Evaluation factors. 2415.304 Section 2415.304 Federal Acquisition Regulations System DEPARTMENT OF HOUSING AND URBAN... assigned a numerical weight (except for pass-fail factors) which shall appear in the RFP. When using...

  7. Engineered Human Ferritin Nanoparticles for Direct Delivery of Tumor Antigens to Lymph Node and Cancer Immunotherapy

    Science.gov (United States)

    Lee, Bo-Ram; Ko, Ho Kyung; Ryu, Ju Hee; Ahn, Keum Young; Lee, Young-Ho; Oh, Se Jin; Na, Jin Hee; Kim, Tae Woo; Byun, Youngro; Kwon, Ick Chan; Kim, Kwangmeyung; Lee, Jeewon

    2016-01-01

    Efficient delivery of tumor-specific antigens (TSAs) to lymph nodes (LNs) is essential to eliciting robust immune response for cancer immunotherapy but still remains unsolved. Herein, we evaluated the direct LN-targeting performance of four different protein nanoparticles with different size, shape, and origin [Escherichia coli DNA binding protein (DPS), Thermoplasma acidophilum proteasome (PTS), hepatitis B virus capsid (HBVC), and human ferritin heavy chain (hFTN)] in live mice, using an optical fluorescence imaging system. Based on the imaging results, hFTN that shows rapid LN targeting and prolonged retention in LNs was chosen as a carrier of the model TSA [red fluorescence protein (RFP)], and the flexible surface architecture of hFTN was engineered to densely present RFPs on the hFTN surface through genetic modification of subunit protein of hFTN. The RFP-modified hFTN rapidly targeted LNs, sufficiently exposed RFPs to LN immune cells during prolonged period of retention in LNs, induced strong RFP-specific cytotoxic CD8+ T cell response, and notably inhibited RFP-expressing melanoma tumor growth in live mice. This suggests that the strategy using protein nanoparticles as both TSA-carrying scaffold and anti-cancer vaccine holds promise for clinically effective immunotherapy of cancer. PMID:27725782

  8. Young Children's Reading for Pleasure with Digital Books: Six Key Facets of Engagement

    Science.gov (United States)

    Kucirkova, Natalia; Littleton, Karen; Cremin, Teresa

    2017-01-01

    This paper offers a new characterisation of young children's (2-8 years) reading for pleasure (RfP) with digital books. This characterisation is rooted in a re-contextualisation of Anna Craft's conceptualisation of twenty-first century childhoods in "Creativity and education futures" (Stoke on Trent, Trentham, 2011) and a review of the…

  9. LETTER TO THE EDITOR: Anisotropy of ion temperature in a reversed-field-pinch plasma

    Science.gov (United States)

    Sasaki, K.; Hörling, P.; Fall, T.; Brzozowski, J. H.; Brunsell, P.; Hokin, S.; Tennfors, E.; Sallander, J.; Drake, J. R.; Inoue, N.; Morikawa, J.; Ogawa, Y.; Yoshida, Z.

    1997-03-01

    Anomalous heating of ions has been observed in the EXTRAP-T2 reversed-field-pinch (RFP) plasma. Ions are heated primarily in the parallel direction (with respect to the magnetic field), resulting in an appreciable anisotropy of the ion temperature. This observation suggests that the magnetohydrodynamic fluctuations are dissipated primarily by the ion viscosity.

  10. Numerical studies of the reversed-field pinch at high aspect ratio

    Science.gov (United States)

    Sätherblom, H.-E.; Drake, J. R.

    1998-10-01

    The reversed field pinch (RFP) configuration at an aspect ratio of 8.8 is studied numerically by means of the three-dimensional magnetohydrodynamic code DEBS [D. D. Schnack et al., J. Comput. Phys. 70, 330 (1987)]. This aspect ratio is equal to that of the Extrap T1 experiment [S. Mazur et al., Nucl. Fusion 34, 427 (1994)]. A numerical study of a RFP with this level of aspect ratio requires extensive computer achievements and has hitherto not been performed. The results are compared with previous studies [Y. L. Ho et al., Phys. Plasmas 2, 3407 (1995)] of lower aspect ratio RFP configurations. In particular, an evaluation of the extrapolation to the aspect ratio of 8.8 made in this previous study shows that the extrapolation of the spectral spread, as well as most of the other findings, are confirmed. An important exception, however, is the magnetic diffusion coefficient, which is found to decrease with aspect ratio. Furthermore, an aspect ratio dependence of the magnetic energy and of the helicity of the RFP is found.

  11. 50 CFR 648.56 - Scallop research.

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Scallop research. 648.56 Section 648.56... Scallop Fishery § 648.56 Scallop research. (a) Annually, the Council and NMFS shall prepare and issue a Request for Proposals (RFP) that identifies research priorities for projects to be conducted by...

  12. Modeling of Resistive Wall Modes in Tokamak and Reversed Field Pinch Configurations of KTX

    Science.gov (United States)

    Han, Rui; Zhu, Ping; Bai, Wei; Lan, Tao; Liu, Wandong

    2016-10-01

    Resistive wall mode is believed to be one of the leading causes for macroscopic degradation of plasma confinement in tokamaks and reversed field pinches (RFP). In this study, we evaluate the linear RWM instability of Keda Torus eXperiment (KTX) in both tokamak and RFP configurations. For the tokamak configuration, the extended MHD code NIMROD is employed for calculating the dependence of the RWM growth rate on the position and conductivity of the vacuum wall for a model tokamak equilibrium of KTX in the large aspect-ratio approximation. For the RFP configuration, the standard formulation of dispersion relation for RWM based on the MHD energy principle has been evaluated for a cylindrical α- Θ model of KTX plasma equilibrium, in an effort to investigate the effects of thin wall on the RWM in KTX. Full MHD calculations of RWM in the RFP configuration of KTX using the NIMROD code are also being developed. Supported by National Magnetic Confinement Fusion Science Program of China Grant Nos. 2014GB124002, 2015GB101004, 2011GB106000, and 2011GB106003.

  13. 77 FR 21690 - Approval and Promulgation of Air Quality Implementation Plan for 1997 8-Hour Ozone Standard; Arizona

    Science.gov (United States)

    2012-04-11

    ... Preprocessor System (EPS3.0), based on the 2002 Periodic Emission Inventory for the three ozone episodes... 5-3 and 5-4 of the 2007 Ozone Plan. The 2002 inventory was projected to 2008 by accounting for...), reasonable further progress (RFP), emission inventories, transportation conformity motor vehicle...

  14. Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells

    DEFF Research Database (Denmark)

    Egerod, Kristoffer L; Engelstoft, Maja S; Lund, Mari L;

    2015-01-01

    characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly...

  15. 78 FR 18849 - Disapproval of Implementation Plan Revisions; State of California; South Coast VMT Emissions...

    Science.gov (United States)

    2013-03-28

    ... control strategies and transportation control measures to offset any growth in emissions from growth in..., please schedule an appointment during normal business hours with the contact listed in the FOR FURTHER...., offsetting emissions growth, attainment of the reasonable further progress (RFP) reduction, and attainment...

  16. Use of a dual reporter plasmid to demonstrate Bactofection with an attenuated AroA(- derivative of Pasteurella multocida B:2.

    Directory of Open Access Journals (Sweden)

    Sarah Othman

    Full Text Available A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL cells and an attenuated AroA(- derivative of Pasteurella multocida B:2 (strain JRMT12, harbouring the plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine.

  17. A 3D approach to equilibrium, stability and transport studies in RFX-mod improved regimes

    Energy Technology Data Exchange (ETDEWEB)

    Terranova, D; Bonfiglio, D; Gobbin, M; Lorenzini, R; Marrelli, L; Martines, E; Momo, B; Predebon, I; Spizzo, G; Agostini, M; Alfier, A; Apolloni, L; Auriemma, F; Baruzzo, M; Bolzonella, T [Consorzio RFX, Associazione EURATOM-ENEA sulla Fusione, Padova (Italy); Boozer, A H [Department of Applied Physics and Applied Mathematics, Columbia University, New York, NY (United States); Cooper, A W [EPFL, Association EURATOM-Confederation Suisse, Centre de Recherches en Physique des Plasmas, Lausanne (Switzerland); Hirshman, S P; Sanchez, R [ORNL Fusion Energy Division, Oak Ridge, TN (United States); Pomphrey, N, E-mail: david.terranova@igi.cnr.i [Princeton Plasma Physics Laboratory, Princeton, NJ (United States)

    2010-12-15

    The full three-dimensional (3D) approach is now becoming an important issue for all magnetic confinement configurations. It is a necessary condition for the stellarator but also the tokamak and the reversed field pinch (RFP) now cannot be completely described in an axisymmetric framework. For the RFP the observation of self-sustained helical configurations with improved plasma performances require a better description in order to assess a new view on this configuration. In this new framework plasma configuration studies for RFX-mod have been considered both with tools developed for the RFP as well as considering codes originally developed for the stellarator and adapted to the RFP. These helical states are reached through a transition to a very low/reversed shear configuration leading to internal electron transport barriers. These states are interrupted by MHD reconnection events and the large T{sub e} gradients at the barriers indicate that both current and pressure driven modes are to be considered. Furthermore the typically flat T{sub e} profiles in the helical core have raised the issue of the role of electrostatic and electromagnetic turbulence in these reduced chaos regions, so that a stability analysis in the correct 3D geometry is required to address an optimization of the plasma setup. In this view the VMEC code proved to be an effective way to obtain helical equilibria to be studied in terms of stability and transport with a suite of well tested codes. In this work, the equilibrium reconstruction technique as well as the experimental evidence of 3D effects and their first interpretation in terms of stability and transport are presented using both RFP and stellarator tools.

  18. A 3D approach to equilibrium, stability and transport studies in RFX-mod improved regimes

    Energy Technology Data Exchange (ETDEWEB)

    Terranova, D. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Bonfiglio, D. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Boozer, A. H. [Columbia University; Cooper, W Anthony [CRPP/EPFL, Association Euratom-Suisse, Lausanne, Switzerland; Gobbin, M. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Hirshman, Steven Paul [ORNL; Lorenzini, R. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Marrelli, L. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Martines, E. [RFX, Padova, Italy; Momo, B. [RFX, Padova, Italy; Pomphrey, N. [Princeton Plasma Physics Laboratory (PPPL); Predebon, I. [RFX, Padova, Italy; Sanchez, Raul [ORNL; Spizzo, G. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Agnostini, M. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Alfier, A. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Apolloni, L. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Auriemma, F. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Baruzzo, M. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Bolzonella, T. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Bonomo, F. [Consorzio RFX, Italy; Brombin, M. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Canton, A. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Cappello, S. [Association Euratom ENEA Fusion, Consorzio RFX, Padua; Carraro, L. [Association Euratom ENEA Fusion, Consorzio RFX, Padua

    2010-01-01

    The full three-dimensional (3D) approach is now becoming an important issue for all magnetic confinement configurations. It is a necessary condition for the stellarator but also the tokamak and the reversed field pinch (RFP) now cannot be completely described in an axisymmetric framework. For the RFP the observation of self-sustained helical configurations with improved plasma performances require a better description in order to assess a new view on this configuration. In this new framework plasma configuration studies for RFX-mod have been considered both with tools developed for the RFP as well as considering codes originally developed for the stellarator and adapted to the RFP. These helical states are reached through a transition to a very low/reversed shear configuration leading to internal electron transport barriers. These states are interrupted by MHD reconnection events and the large T(e) gradients at the barriers indicate that both current and pressure driven modes are to be considered. Furthermore the typically flat T(e) profiles in the helical core have raised the issue of the role of electrostatic and electromagnetic turbulence in these reduced chaos regions, so that a stability analysis in the correct 3D geometry is required to address an optimization of the plasma setup. In this view the VMEC code proved to be an effective way to obtain helical equilibria to be studied in terms of stability and transport with a suite of well tested codes. In this work, the equilibrium reconstruction technique as well as the experimental evidence of 3D effects and their first interpretation in terms of stability and transport are presented using both RFP and stellarator tools.

  19. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

    Science.gov (United States)

    Boggs, Kristin; Venkatesan, Nandakumar; Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F; Mistretta, Charlotte M; Mishina, Yuji; Liu, Hong-Xiang

    2016-01-01

    Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

  20. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

    Directory of Open Access Journals (Sweden)

    Kristin Boggs

    Full Text Available Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC. Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5 and young postnatal (P1-10 mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1 P0-Cre/R26-tdTomato (RFP to label NC, NC derived Schwann cells and derivatives; (2 Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3 Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.